This publication is Open Access under the license indicated. Learn More
Review
Single-Virus Tracking: From Imaging Methodologies to Virological ApplicationsClick to copy article linkArticle link copied!
- Shu-Lin LiuShu-Lin LiuState Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. ChinaEngineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430074, P. R. ChinaMore by Shu-Lin Liu
- Zhi-Gang WangZhi-Gang WangState Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. ChinaMore by Zhi-Gang Wang
- Hai-Yan XieHai-Yan XieSchool of Life Science, Beijing Institute of Technology, Beijing 100081, P. R. ChinaMore by Hai-Yan Xie
- An-An LiuAn-An LiuState Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. ChinaMore by An-An Liu
- Don C. LambDon C. LambPhysical Chemistry, Department of Chemistry, Center for Nanoscience (CeNS), and Center for Integrated Protein Science Munich (CIPSM) and Nanosystems Initiative Munich (NIM), Ludwig-Maximilians-Universität, München, 81377, GermanyMore by Don C. Lamb
- Dai-Wen Pang*Dai-Wen Pang*Email:[email protected]State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. ChinaCollege of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P. R. ChinaMore by Dai-Wen Pang
Abstract
Click to copy section linkSection link copied!
Uncovering the mechanisms of virus infection and assembly is crucial for preventing the spread of viruses and treating viral disease. The technique of single-virus tracking (SVT), also known as single-virus tracing, allows one to follow individual viruses at different parts of their life cycle and thereby provides dynamic insights into fundamental processes of viruses occurring in live cells. SVT is typically based on fluorescence imaging and reveals insights into previously unreported infection mechanisms. In this review article, we provide the readers a broad overview of the SVT technique. We first summarize recent advances in SVT, from the choice of fluorescent labels and labeling strategies to imaging implementation and analytical methodologies. We then describe representative applications in detail to elucidate how SVT serves as a valuable tool in virological research. Finally, we present our perspectives regarding the future possibilities and challenges of SVT.
Copyright © 2020 American Chemical Society
Note
This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
1. Introduction
Click to copy section linkSection link copied!
Viruses are intracellular parasites that rely on host cells for completion of their life cycles. Most viruses are composed mainly of nucleic acids (RNA or DNA), structural proteins (e.g., capsid), and a lipid membrane (for enveloped viruses). The primary function of any virus is to reproduce in host cells. For this purpose, viruses should accomplish two major tasks: (i) to break through the barriers that block virus entry and transport into cell cytosol and (ii) to release their genome at the preferred sites within the cells for viral transcription and replication. (1−4) The newly synthesized viral proteins and genomes are assembled in the infected cells to generate progeny viruses, which are then released to the extracellular space by exocytosis or by lysing the host cells. Additionally, viruses may take different pathways to infect host cells, and the complicated infection processes usually include multiple steps and intricate interactions between viral components and cellular structures. (5−7) Thus, it is important to understand the complicated infection mechanisms of viruses in time and space for fighting against virus infection and preventing viral diseases.
Early researchers mainly utilized transmission electron microscopy (TEM) and biochemical experiments to investigate viral infection mechanisms in cells. TEM has played an essential role in studying the infection pathway of viruses, but it can only acquire static images from the scenario of virus infection in live cells. In vitro biochemical experiments commonly use the samples isolated from organisms to conduct ensemble measurements and deduce the effects. Conventional methods lack the ability to acquire dynamic information on individual viruses during the infection process, since the cellular events occur in a stochastic manner across spatial and temporal scales. The biggest challenge is how to realize the visualization of infection processes directly and dynamically in live cells and thereby uncover the mechanisms of infection and proliferation.
Fluorescence microscopy has had a great impact on cell biology ranging from the molecular to the organism scale. Initially, fluorescence was mainly used to visualize the intracellular distribution of proteins in fixed cells via antibodies. (8,9) With improvements in microscopy, it has become possible to measure individual biomolecules as they perform their function in their native environment using single-particle tracking (SPT). (10−17) SPT has successfully solved many basic biological questions and greatly enhances our repertoire of research approaches for investigating, for example, membrane organization, (18−20) protein folding, (21−23) molecular motor dynamics, (24−26) and cell signal transduction. (27−29) Thereinto, single-virus tracking (SVT) allows researchers to follow individual viruses, visualize their transport behaviors, dissect their dynamic interactions with the host cells, and reveal the underlying mechanisms of viral processes. (30−33) In SVT studies, viruses are addressed independently, avoiding ensemble averaging and making it possible to investigate the dynamic behaviors of single viruses in their native, complex surroundings. Thus, time-dependent unsynchronized infection events can be monitored in real time. Hence, the SVT technique is a powerful approach for studying the real-time and in situ dynamics of viral processes in live cells, and it is attracting the attention of researchers. Until now, this method has revealed a variety of complicated infection mechanisms of various viruses including the mechanisms of viral entry, trafficking, and egress. SVT has also been used to follow the uptake and cellular distribution of artificial viruses and drug delivery carriers due to their similar nature.
In this review, we will first describe the historical retrospect of the SVT technique, and then discuss the fluorescent labels used for SVT, discuss the advantages and limitations of each kind of fluorescent labels, and describe how to use the fluorophores for virus labeling. Subsequently, we will elaborate on the various approaches for SVT, the imaging instruments, and data analysis methods for accurately extracting the dynamic information on virus infection from live-cell measurements. We then highlight a couple applications of SVT and finally propose the future possibilities and challenges of the SVT technique.
2. Historical Retrospect of Single-Virus Tracking
Click to copy section linkSection link copied!
Single-virus tracking is a new and growing technique. It originates from single-particle techniques, which have each become a remarkable tool in biological fields. These techniques add new insights beyond conventional ensemble methods by providing dynamic information regarding the biological processes. There are a number of methods used to monitor the mobility of particles including fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single-particle tracking (SPT). FRAP was established in the 1970s to measure the mobility of molecules via the recovery speed of fluorescence intensity after photobleaching a given region. (34,35) In the same decade, FCS was developed to detect and analyze the fluctuations of fluorescence intensity caused by fluorescent molecules entering and leaving the observation volume. (36) The dynamic parameters, such as diffusion coefficient and average residence time, could be extracted from the autocorrelation analysis. (37,38) Strictly speaking, FRAP and FCS measure the average behaviors of hundreds or even thousands of molecules, and the geometry of the photobleached volume or the point-spread-function (PSF) of the FCS excitation beam, respectively, needs to be known in detail. These techniques only provide limited dynamic information and do not truly reflect the kinetics of biological processes in time and space on the single particle level. SPT, however, is capable of monitoring the movements of individual molecules directly by optical microscopy, and it can detect subpopulations and event detection changes in diffusional behavior of a single particle. Hence, the technique is appealing for investigating dynamic events in live cells.
SPT dates back hundreds if not thousands of years. Galileo Galilei tracked the moons of Jupiter and contributed data that resulted in overturning the view of the universe at that time. SPT on the microscale began in the early 1970s, when Howard Berg built a microscope for tracking single bacteria. (39,40) The first subcellular tracking experiments were performed at the beginning of the 1980s, when Barak et al. tracked individual low-density lipoprotein (LDL)-receptor complexes in live cells. (41) These measurements opened up a new avenue for studying the dynamic mechanisms of individual biomolecules (Figure 1). Notably, advances in imaging instruments and algorithms greatly improved the imaging speed and accuracy of the SPT technique, which enabled the investigation of more complex processes with a better spatial-temporal resolution. (42−48) Since then, the applications of SPT had a dramatic increase in the biological field. A major breakthrough in three-dimensional (3D) SPT occurred in 1994. Using a modified epifluorescence microscope where a weak cylindrical lens had been placed in the detection path, Kao et al. successfully tracked individual fluorescent particles and determined z positions from the image shape and orientation with a peak detection algorithm. (49) Inspired by this research, many researchers made efforts to develop imaging methods and analyzing algorithms for 3D SPT. (50−57) In recent years, a number of 3D SPT methods have become available to track the dynamic behaviors of biomolecules in the 3D environment. For recent reviews, we refer the reader to refs (58−61).
Figure 1
Figure 1. Timeline of the key developments of the single-virus tracking technique.
Viruses, due to their small size and the dramatic impact they have on human health, are an important and popular system for SPT experiments. Hence, SPT performed on viruses has come to be known as single-virus tracking (SVT). Already in the 1990s, SVT techniques began to show their talent at probing the dynamic mechanisms of virus infection. Originally, organic dyes were used to label viruses by antigen–antibody interactions in fixed cells. (62,63) With the emergence and application of fluorescence video microscopy, organic dyes could be used to labeled viruses and the budding and fusion events of enveloped viruses monitored in live cells. (64−66) From the early 2000s, the SVT technique started to play a more and more important role in studying infection mechanisms of viruses. (67−75) One milestone in SVT was the experiments performed by the group of Bräuchle where they could follow the entry of adeno associated viruses labeled with a single organic fluorophore. (69) In live-cell measurements, care has to be taken when labeling viruses to ensure that the labeling does not interfere with the function of the virus and a single fluorophore is the ultimate limit for fluorescent labeling. The laboratory of Zhuang also contributed significantly to SVT with beautiful investigations that visualized the infectious behaviors of viruses and systematically dissected the dynamic mechanisms of virus entry, virus transport, and genome release. (72−74)
Almost contemporaneously, fluorescent proteins (FPs) came to the fore as fluorescent labels in the biological field. The key feature of FPs is that they allow specific cellular or viral proteins to be labeled by genetic engineering. Once a fortuitous location had been determined for virus labeling, it no longer became necessary to check after each sample preparation whether the labeling had affected the infectivity of the virus. For these reasons, the use of FPs emerged in virology and contributed immensely to the study of virus–cell interactions. In 1995, the green fluorescent protein (GFP) (76) was first introduced into the expression cassette of the potato virus X. (77) Subsequently, different kinds of viral components, including envelope protein, tegument, and capsid, were genetically labeled with FPs, and many subsequent attempts were made to monitor individual FPs-labeled viruses in host cells using SVT. (78−81) Especially the visualization of the transport behaviors of FPs-labeled viruses helped to accelerate our understanding of virus entry, fusion, and cell-to-cell transmission of human immunodeficiency virus (HIV). (82−86) Moreover, the advances made in SPT were quickly applied to SVT, and real-time 3D tracking of FP-labeled viruses provided more accurate information regarding viral processes in live cells. (87,88)
As an alternative to organic dyes and FPs, quantum dots (QDs) have also become an important tool for SPT and SVT and are heavily utilized in the fields of biology, virology, and medicine. The excellent brightness and superior photostability of QDs enable them to be tracked for extended periods of time with low laser intensity, making them particularly favorable for acquiring time-series images or z-stacks for 3D reconstructions. In the early 2000s, QDs were first utilized to track glycine receptors on the plasma membrane. (89) This stimulated the application of QDs in the SPT field and triggered the further development of imaging algorithms. In particular, special imaging algorithms were developed that overcome the drawbacks of QDs in SPT experiments, such as QDs blinking. (90−92) After that, QDs-based SPT made great advances in the investigation of dynamic processes occurring on the plasma membrane and in intracellular/intercellular environments. (93−97) In 2008, Joo et al. proposed a site-specific strategy to label the surface of lentiviruses with QDs, which pointed out a new way forward for QDs applications in virology. (98) Diverse strategies emerged to label different viral components with QDs, (99−106) and QD-labeled viruses were implemented for the long-term tracking of individual viruses during virus infection. (107−110) Additionally, by combining QD-labeling strategies with 3D SVT, viral behavior could be followed over long time scales in three dimensions and new insights gained regarding virus infection. (111,112) These results again ignited the enthusiasm of researchers to study the infection mechanisms of viruses by SVT. (113−118) There is no denying that SVT has greatly improved our understanding of the infection mechanisms of viruses.
3. Fluorescent Labels for Single-Virus Tracking
Click to copy section linkSection link copied!
The first step necessary for performing SVT experiments is to label viral components with fluorescent labels. Viruses are typically densely packed structures, and there can be limitations on the size and location of the tags that can be used as to not inhibit the functionality or even assembly of the virus. Hence, particular care needs to be taken in choosing the correct labeling approach and location on the virus, and proper controls need to be performed to verify that the functionality and infectivity of the virus is not hampered. Also, the assembly of viruses occurs directly in the living host cells such that in cellular labeling approaches are needed to visualize the early stages of assembly. These limitations make the labeling of viruses particularly challenging in comparison to the labeling of other objects such as nanoparticles used for therapeutic applications. In addition, one wishes to acquire image sequences with high spatiotemporal resolution and high signal-to-background ratio, which depends on the number and type of fluorescent labels and the imaging instrument. There is an intricate relationship among the optical characteristics of fluorescent labels, the duration of imaging and the spatial resolution and the accessible temporal sampling of imaging instruments. The brighter the labels are, the faster the time resolution can be and the higher the spatial resolution that can be achieved. Moreover, the more photostable the label is, the longer the virus can be tracked. A fluorescent label is evaluated by the relevant spectroscopic features. High brightness is one of the important properties for any fluorescent label, which means that a good fluorescent label should possess a strong ability to capture photons, such as a large molar absorption coefficient and a high fluorescence quantum yield. Meanwhile, the high photostability is the essential property belonging to the good fluorescent labels, which can endure many excitation–deexcitation cycles prior to photobleaching. This is the principal criterion for fluorescent labels used in the SVT field. It should be noted, however, that in some cases, viruses have a large number of components that can be labeled without deleterious effects. For example, the Gag protein of HIV can be labeled with FPs in a ratio of 1:1 without significantly altering its structure and infectivity. (119) As HIV contains approximately 2400 Gag proteins, ∼1000 FPs are coupled to a single viral particle and the lower photophysical properties of the FP are compensated for by the sheer number of fluorophores. (120)
There are several types of fluorophores that have emerged for labeling viral structures in the SVT field, including organic dyes, FPs, and nanoparticles (Figure 2). Each type of fluorophore has its advantages and drawbacks that the researchers need to balance according to the requirements of SVT experiments. (121) The focus of this section is to describe the recent developments and features of existing fluorescent labels and to highlight the prospective applications for future research in the SVT field.
Figure 2
Figure 2. Comparison of the size scales of fluorescent labels and the spatial resolutions of biological imaging techniques.
3.1. Organic Dyes
As small fluorescent labels (<1 kDa), organic dyes have proven indispensable for fluorescence labeling of biological systems. (122) The fluorescence properties of organic dyes are governed by both fluorophore structure and chemical environment and can be fine-tuned by elaborate design strategies. Beyond their small size, the major advantages of most organic dyes are their good photophysical properties, commercial availability, the availability of a multitude of reactive groups for various labeling strategies, and the wide spectral range of options. (123) Compared with FPs, organic dyes have several excellent properties, such as higher brightness, smaller size, better photostability, and a wider color palette, (10,122,124) which have been broadly used for single-virus imaging. (31,125−127) According to their inherent nature and characteristics, organic dyes can be classified into three categories for virus labeling: covalent labeling dyes, lipophilic dyes, and intercalating dyes.
3.1.1. Covalent Labeling Dyes
Many organic dyes can be used to bind viral components covalently. During the development of the SVT technique, several families of fluorescent dyes have been used for labeling viruses. The photophysical properties of these dyes for single-virus imaging vary widely. More detailed information regarding the properties of the covalent labeling dyes is shown in Table 1.
Table 1. Covalent Labeling Dyes for Virus Imaging
a
The color of emitted light.
b
Maximum excitation wavelength.
c
Maximum emission wavelength.
d
Extinction coefficient.
e
Fluorescence quantum yield.
f
Fluorescence lifetime.
g
Viral ribonucleoprotein.
h
Influenza virus.
i
Adeno-associated virus.
j
Seneca valley virus.
k
Poliovirus.
l
Rabies virus.
m
Semliki forest virus.
n
Vesicular stomatitis virus.
o
Simian virus 40.
p
Human papillomavirus.
q
Foot-and-mouth disease virus.
r
Murine polyoma virus.
s
Canine parvovirus.
t
Reovirus.
u
Uukuniemi virus.
v
Human adenovirus.
w
Not determined.
x
Information from Thermo Fisher.
y
Information from GE Healthcare.
z
Information from Atto-TEC.
Cyanine dyes have received wide attention for biomolecular labeling applications due to their high absorption cross sections, leading to high brightness and photostability. They consist of two quaternized heteroaromatic bases joined by a polymethine bridge. Their emission profiles extend from about 450 to 1000 nm, which can be tuned by the length of the polymethine bridge. (147−149) Cy3 and Cy5 are the most popular cyanine dyes for SVT. (119,129−133) It is worth pointing out that an epoch-making progress happened in 2001, (69,150) when adeno-associated viruses (AAVs) labeled with a single Cy5 dye were tracked in real time to dissect the entry pathway of individual viruses in living cells (Figure 3). The detailed observation and quantitative description of viral behaviors paved a new road to illuminate virus–host cell interactions at single-virus level. Thereafter, the field paid more attention to the use of organic dyes to label various components of viruses for tracking. One also began to use environment-sensitive fluorophores, such as CypHer5, which is a pH-sensitive cyanine dye that has low fluorescence at basic pH and high fluorescence at acidic pH. Based on this property, it was applied to monitor the viral movements from the plasma membrane to acidic endosomes. The double labeling of viruses with Cy3 and CypHer5 made it possible to simultaneously monitor the transport and acidification processes of viruses. (68,128)
Figure 3
Figure 3. Uptake of Cy5-labeled adeno-associated virus (AAV) by a live HeLa cell. (a) Representative trajectories of AAV particles in cells at different stages of the infection. (b) Zoom in of trajectory 2 showing several membrane interactions of the AAV at the cell surface. (c) Mean number of consecutive cell interactions derived for viruses that did not dock. (d–e) Distribution of adsorption times for (d) 137 nondocking and (e) 42 membrane penetrating trajectories. Adapted with permission from ref (69). Copyright 2001 The American Association for the Advancement of Science.
The Alexa Fluor family of organic dyes are synthesized though the sulfonation and modification of certain well-known dye classes such as rhodamine, fluorescein, and cyanine dyes. Due to the sulfonation, Alexa Fluor dyes are normally negatively charged and more hydrophilic than their precursors. With the aid of additional modifications, Alexa Fluor dyes are more photostable and less pH-sensitive than the original dyes. (151,152) In the meanwhile, the emission spectra of the Alexa Fluor series span the visible spectrum and extend into the near-infrared region. These properties make them ideal for investigating the cellular uptake and endosomal transport of viruses. Thus, Alexa Fluor derivatives have been popularly applied to label viruses, including murine polyoma virus (MPV), (139) canine parvovirus (CPV), (142) vesicular stomatitis virus (VSV), (134) simian virus 40 (SV40), (135,141) human papillomavirus (HPV), (137) foot-and-mouth disease virus (FMDV), (138) AAV, (136) and reovirus (RV). (143) For example, by labeling RV with Alexa Fluor 647, Kirchhausen et al. found that individual RV particles were captured and internalized by clathrin-coated pits and vesicles, illustrating RV required access to endosomes for successful infection. (143) Multiple-color imaging of Alexa Fluor-labeled VSV and a shorter, defective interfering particle indicated that the elongated shape of a VSV particle triggered the recruitment of actin filaments to complete the viral internalization process, and the cargo geometry was important for specifying the entry modes of the viruses (Figure 4). (140)
Figure 4
Figure 4. Clathrin structures capture vesicular stomatitis viruses (VSVs) and defective interfering particles (DI-T) with similar kinetics. (a) Schematic of the clathrin-dependent virus internalization pathway. (b) VSVs and DI-T particles captured by clathrin structures in the same cell. BSC1 cells stably expressing s2-eGFP (green) were inoculated with Alexa Fluor 647-labeled DI-T (blue, blue arrowheads) and Alexa Fluor 568-labeled VSV (red, red arrowheads). Adapted with permission from ref (140). Copyright 2010 Public Library of Science.
There are other organic dyes used for labeling viruses such as fluorescein, Atto dyes, and Texas Red. Fluorescein, as a classical fluorescent reagent in biological research, possesses relatively high brightness, strong pH sensitivity, and poor photostability. Owing to the pH-sensitivity of fluorescein, the fluorescence of labeled viruses is quenched and undetectable under acidic conditions. Using the pH sensitivity, investigators could distinguish internalized fluorescein-labeled viruses from the extracellular viruses by changing the culture medium to pH 4.0. (137,139,144) Texas Red is a conventional red fluorescent dye, and its derivative, Texas Red-X (TRX) succinimidyl ester, is commercially available and readily reactive for conjugating to viruses. (67,145,146) Monitoring of TRX-labeled SV40 found that SV40 was internalized into cells by caveolae and transported to the endoplasmic reticulum by caveosomes. (67) Atto dyes have enhanced photostability and longer fluorescence lifetime than either fluorescein or most cyanine dyes, and the emission profiles cover the visible and near-infrared wavelengths. These dyes have been used as fluorescent labels in a wide range of biological imaging experiments including SVT. For example, the unequivocal images of Atto 647N-labeled capsids of CPV demonstrated that CPV capsids had a relatively short residence time on the cell surface, which limited the efficiency of virus internalization. (142)
3.1.2. Lipophilic Dyes
The lipid membrane of enveloped viruses is derived from the plasma membrane or intracellular membrane of the host cells. Hence, virus labeling represents a significant application area for fluorescent membrane probes. Membrane probes include lipophilic organic dyes and fluorescent analogs of natural lipids. While some lipophilic dyes are particularly useful for SVT experiments, other lipid probes are scarcely used to label viruses. Lipophilic dyes are able to incorporate into the envelope of viruses by hydrophobic–lipophilic interactions (Table 2). For lipophilic dyes-based single-virus imaging, these dyes have an additional advantage in that they self-quench when they are incorporated into viral particles at high concentrations. At low pH levels or upon fusion where the viral and cellular membranes mix leading to a decrease in concentration, the lipophilic dyes dequench leading to a striking increase in fluorescence. Thus, the lipophilic dyes can detect the genome-release events of viruses, since the dequenching of the fluorescence signal could be considered as the sign of the occurrence of virus-endosome or virus-plasma membrane fusion. As one kind of early applied lipophilic dyes, rhodamine derivatives were used to study the kinetics of the virus-cell membrane fusion events on the membrane surface. (65,153,154,161) For example, R110-labeled influenza viruses were used to investigate the real-time hemifusion and the pore formation of influenza viruses on a lipid bilayer. The occurrence of the hemifusion was indicated by the transient brightening of individual viruses caused by the fluorescence dequenching of R110. (154)
Table 2. Lipophilic Dyes and Intercalating Dyes for Single-Virus Imaging
a
The color of emitted light.
b
Maximum excitation wavelength.
c
Maximum emission wavelength.
d
Extinction coefficient.
e
Fluorescence quantum yield.
f
Fluorescence lifetime.
g
Not determined.
h
Influenza virus.
i
Uukuniemi virus.
j
Human immunodeficiency viruses.
k
Ebolavirus.
l
Hepatitis B virus.
m
Vesicular stomatitis virus.
n
Dengue virus.
o
Hepatitis C virus.
p
Avian sarcoma and leukosis virus.
q
Chikungunya virus.
r
Poliovirus.
s
Human rhinovirus.
t
Information from Thermo Fisher.
With the emergence and development of fluorescence microscopy, researchers began to observe individual fluorescent viruses for obtaining more intuitive information about virus infection. Long-chain dialkylcarbocyanines (e.g., DiD, DiI, and DiO) with varying fluorescent excitations and emissions were widely adopted to label individual viruses for SVT. These dyes possess high extinction coefficients, moderate quantum yields, and short lifetimes in a hydrophobic environment. Their fluorescence is only detectable when they insert into lipid membranes. Owing to the strong autofluorescence of the cells (toward the blue end of the visible spectrum), the deep-red lipophilic dye (DiD) has been used extensively to track the infection behaviors and dissect the infection pathways of enveloped viruses, including influenza virus, (68,72,74,128) dengue virus (DENV), (73,75) hepatitis C virus (HCV), (71) avian sarcoma and leukosis virus (ASLV), (155) chikungunya virus (CHIKV), (156) and HIV. (82,83) The analogues of DiD, DiO (green) and DiI (red), have also been used to monitor the transport behaviors of viruses, such as HIV, (157) ebola virus (EBOV), (158) hepatitis B virus (HBV), (159) and VSV. (160) By quantitatively measuring the fluorescence intensity of individual viruses, the virus–endosome fusion events could be detected in real time (Figure 5). (68) However, detection of the actual release of the interior content of the virus, as a prerequisite for virus infection, still requires more accurate approaches. In addition, owing to the self-quenching of lipophilic dyes in viruses, the number of viral particles that can be fluorescently detected is very low. (73) When a large number of viruses bind to the cell surface, only 2% of viruses are indicated by DiD signals. This makes it challenging to efficiently and globally monitor the behavior of viruses in individual cells. (99)
Figure 5
Figure 5. Tracking the transport and fusion of individual influenza viruses. (a) Trajectory of a DiD-labeled virus inside a cell. (b) Time trajectories of the velocity (black) and the DiD fluorescence intensity (blue) of a virus. (c–e) Histogram of the viral velocity in each stage. (Inset) Shown is the measured average mean square displacement (⟨Δr2⟩) vs time (Δt) for a virus. Adapted with permission from ref (68). Copyright 2003 National Academy of Sciences, U.S.A.
3.1.3. Intercalating Dyes
Generally, the viral genome is encapsulated into the intact virus particle, which is not accessible for dye attachment. However, several intercalating dyes can penetrate the outer components of viruses to label the viral genomes to a certain extent (Table 2). Ribogreen is an intercalating dye with little fluorescence and negligible absorbance. The dye is fluorogenic, meaning that its fluorescence intensity amplifies by several orders of magnitude when it binds to nucleic acids. This dye has been used for detecting and quantifying both RNA and DNA. By incubating ribogreen with the human rhinovirus (HRV), this dye contacted and bound with the viral genome during “capsid breathing”. (166−168) Later, a metabolic labeling strategy was developed to label the viral genome during virus replication. For instance, acridine orange was incorporated into developing poliovirus (PV) to label the viral RNA. (163−165) However, these dyes could rapidly inactivate the viral RNA upon illumination. SYTO dyes are a kind of cell-permeable nucleic acid dye, which binds to nucleic acids by passive diffusion through the plasma membrane. Each of these dyes possesses different characteristics including optical properties, nucleic acid binding preferences, cell permeability, and DNA/RNA selectivity and can be used to stain DNA and RNA in both live and dead eukaryotic cells. (169) As an orange fluorescent nucleic acid binding dye, SYTO 82 has been successfully used to label the viral genome of RNA viruses, including PV and influenza virus. (99,131,170) [Ru(phen)2(dppz)]2+ showed its potential to label the viral genomes of DNA viruses during viral self-assembly. (171,172)
3.2. Fluorescent Proteins
Green fluorescent protein (GFP) was discovered and purified from Aequorea victoria by Osamu Shimomura in the early 1960s. It began to be utilized as a tool for molecular biologists when the nucleotide sequence of GFP was reported and expressed in Escherichia coli and Caenorhabditis in 1994. (173,174) The application of GFP as a genetically encoded fluorescence marker heralded a new era in cell biology. (175) Thereafter, a broad range of genetic variants of fluorescent protein were developed by mutagenesis, and the diversity of excitation–emission spectra was further extended. (76,176) In 2008, the Nobel Prize was awarded “for the discovery and development of the green fluorescent protein, GFP” to recognize the achievements of GFP labeling technology in the medical and biological sciences. More recently, GFP-like proteins from other species have been discovered with new optical properties, resulting in a further expansion of the color palette. (177,178) Nowadays, there are more than 1000 FP variants reported, which cover the color range from blue to near-infrared spectrum (Figure 6). (179−182) A representative list of FPs and their optical properties are given in Table 3.
Figure 6
Figure 6. Characterization of near-infrared FPs. (a–c) Normalized excitation (a), emission (b), and full absorption spectra of different iRFPs. (d) Schematic representation of directed molecular evolution that led to iRFPs with distinct spectral properties. (e) Brightness of HeLa cells transiently transfected with iRFPs, normalized to the value for iRFP713-expressing cells. Adapted with permission from ref (181). Copyright 2013 Springer Nature.
Table 3. Optical Properties of Representative FPs
| Protein | λexa(nm) | λemb(nm) | εabsc(M–1 cm–1) | Φfd(%) | pKa | Relative Brightnesse(% of EGFP) | refs |
|---|---|---|---|---|---|---|---|
| Sirius | 355 | 424 | 15,000 | 24 | <3.0 | 11 | (183) |
| EBFP2 | 383 | 448 | 32,000 | 56 | 4.5 | 53 | (184,185) |
| TagBFP | 402 | 457 | 52,000 | 63 | 2.7 | 98 | (185) |
| mTurquoise | 434 | 474 | 30,000 | 84 | 4.5 | 75 | (186) |
| ECFP | 434 | 475 | 32,500 | 41 | 4.7 | 40 | (187) |
| TagCFP | 458 | 480 | 37,000 | 57 | 4.7 | 63 | (188) |
| mTFP1 | 462 | 492 | 64,000 | 85 | 4.3 | 162 | (187) |
| EGFP | 488 | 507 | 56,000 | 60 | 6.0 | 100 | (187) |
| mWasabi | 493 | 509 | 70,000 | 80 | 6.5 | 167 | (189) |
| mNeonGreen | 506 | 517 | 116,000 | 80 | 5.7 | 276 | (190) |
| EYFP | 514 | 527 | 84,000 | 61 | 6.5 | 153 | (191) |
| Citrine | 516 | 529 | 77,000 | 76 | 5.7 | 174 | (192) |
| mOrange | 548 | 562 | 71,000 | 69 | 6.5 | 146 | (178) |
| mKO2 | 551 | 565 | 63,800 | 57 | 5.5 | 108 | (193) |
| TagRFP | 555 | 584 | 100,000 | 48 | <4.0 | 143 | (194) |
| mRuby2 | 559 | 600 | 113,000 | 38 | 5.3 | 128 | (195) |
| mCherry | 587 | 610 | 72,000 | 22 | <4.5 | 47 | (178) |
| mKate2 | 588 | 633 | 62,500 | 40 | 5.4 | 74 | (196) |
| mNeptune | 600 | 650 | 67,000 | 20 | 5.4 | 40 | (197) |
| iRFP670 | 643 | 670 | 114,000 | 11 | 4.0 | 38 | (181) |
| TagRFP675 | 598 | 675 | 46,000 | 8 | 5.7 | 11 | (198) |
| iRFP702 | 673 | 702 | 93,000 | 8 | 4.5 | 23 | (181) |
| iRFP713 | 690 | 713 | 98,000 | 6 | 4.5 | 18 | (181) |
| iRFP720 | 702 | 720 | 96,000 | 6 | 4.5 | 17 | (181) |
| pH-sensitive FPs | |||||||
| Ecliptic pHluorin | 495 | 511 | NDf | ND | 7.1 | ND | (199) |
| Super-Ecliptic pHluorin | 495 | 512 | ND | ND | 7.2 | ND | (200) |
| pHuji | 566 | 598 | 31,000 | 22 | 7.7 | 20 | (200) |
| pHoran4 | 547 | 561 | 83,000 | 66 | 7.5 | 163 | (200) |
a
Maximum excitation wavelength.
b
Maximum emission wavelength.
c
Extinction coefficient.
d
Fluorescence quantum yield.
e
The relative brightness values were calculated from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP.
f
Not determined.
FPs are genetically encodable such that the cells and organisms can label themselves. (201) Therefore, this method avoids additional procedures for purifying, tagging, and introducing labeled proteins into cells. However, the fluorescent intensity of FPs in live cells is not only relevant to the molecular brightness in themselves, but also to the number of FP molecules in their functional form. For GFP-like proteins, the fluorescence only can be observed after the chromophore has matured and the polypeptide chain has folded. Thus, the expression level is related to many factors, such as transcription and transfection efficiency, protein stability and folding, and chromophore maturation. (202,203) Thus, although the labeling efficiency is near “100%”, the effective labeling can be much less. GFP and GFP derived proteins tend to have a high maturation efficiency (>85%), whereas red fluorescent proteins can be significantly lower, for example 40% for the case of mCherry. (204) For SVT experiments, FPs are a convenient tag for labeling the relevant cellular structures and viral components, especially the internal components of viruses. However, viruses are very dense structures, and the relatively large size of FPs can make it a problem when using them to label viral proteins. Hence, the correct location for adding the FP to the viral genome needs to be found. Often, it is beneficial to spike the sample with a mixture of labeled and unlabeled components. In the case of adding a FP to the Gag protein of HIV-1, spiking with a ratio of 1:1 already restores wild-type like infectivity. (119)
To obtain FPs-labeled viruses, the recombinant gene technology should be used to fuse viral protein genes with FPs genes. (177,205) When the recombinant cDNA clone was transfected into live cells, the viruses could be detected via their fluorescence. For single-virus labeling, FPs can be divided into three classes: autofluorescent proteins, pH-sensitive FPs, and phototransformable FPs.
3.2.1. Autofluorescent Proteins
The main feature of GFP and GFP-like proteins is that its fluorescence is encoded in the sequence and is typically preserved when fused with other proteins. This is a major breakthrough for specific fluorescent tagging of proteins in live cells using simple molecular biology. This discovery aroused the enthusiasm of many researchers to create GFP mutants with better brightness, faster folding, less propensity to oligomerize, or different excitation and emission wavelengths. Enhanced green fluorescent protein (EGFP) was one of the first enhanced variants, exhibiting more desirable characteristics for the practical use in mammalian cells. Wild-type GFP has unsatisfactory properties with respect to brightness (due to the chromophore being often in a dark, protonated state), folding properties, and excitation spectrum. Numerous GFP variants always emit fluorescence in the magic range of 442 to 529 nm. (206) To break this limitation, efforts were devoted to cloning similar FPs from other organisms. So far, the whole palette of autofluorescent proteins spans the emission wavelength from blue to near-infrared spectrum. Both the spectral range as well as photophysical properties of FPs are being continuously expanded.
One of the most important applications of FPs is site-specific labeling of viral components for single-virus imaging in live cells, including the envelope, (207−209) capsid, (209−212) matrix, (213) ribonucleoprotein (vRNP), (207) and other components. (82,84,214−216) For example, a GFP-labeled influenza virus was generated by carrying a GFP reporter in the NS segment to visualize the dynamics of infection progression (Figure 7). (217) Tracking double-labeled rabies viruses (RABV) comprising a RFP-labeled envelope and an EGFP-labeled vRNP found that RABV was transported as a cargo in neurites of neuroblastoma cells in the retrograde direction. (207)
Figure 7
Figure 7. Generation of recombinant influenza viruses carrying a GFP reporter. (a) Schematic representation of the NS segment of WT PR8 virus and NS1-GFP virus. (b) A549 cells were infected with recombinant PR8 virus carrying NS1-GFP. At 10 h postinfection, cells were fixed and stained for NP. NP staining is shown in red and NS1-GFP is shown in green. (c) Fluorescent micrographs of NS1-GFP virus plaques taken at 20× magnification. Adapted with permission from ref (217). Copyright 2010 National Academy of Sciences, U.S.A.
3.2.2. pH-Sensitive Fluorescent Proteins
By mutagenesis, researchers discovered many extremely useful mutants of wide-type FPs, which have various optical properties and environmental sensitivities. (176,182) As protonation affects the photoproperties of the wild-type GFP chromophore, variants could be produced that were sensitive to the physiological pH in the cell and detect changes in pH. The optical parameters of representative pH-sensitive FPs are shown in Table 3. These proteins always display high fluorescence under neutral conditions, while the fluorescence signal markedly decreased under acidic conditions. Thus, they exhibit unique advantages for studying the transport mechanisms of endocytosis and exocytosis in live cells. (218) Since many viruses need to hijack the endocytic pathway of host cells to realize the genome release for virus replication, targeted expression of pH-sensitive FPs to viruses allows for the real-time analysis of the virus-endosome fusion events in live cells. Thus, the pH-sensitive FPs are valuable fluorophores for investigating the virus infection mechanisms. (214,215,219) For example, pHluorin, a pH-sensitive GFP variant, has been used to label HIV. By labeling the Gag protein and altering the external pH of the medium, the fission of newly assembled HIV particles could be detected. (220) By labeling the surface of HIV, the fluorescent signal was used to monitor the uptake and delivery of HIV into acidic endosomes. (214) Meanwhile, Hogue et al. captured the earliest viral exocytosis events and elucidated the intracellular transport pathways and egress mechanisms of alpha herpesvirus. (221) Additionally, pHluorin and mKate2 (a pH-resistant FPs) were introduced to label the envelope and internal content of ASLV simultaneously. Live-cell imaging of infectious dual-labeled ASLV demonstrated the transport and fusion behaviors of ASLV were closely associated with early and intermediate endosomes (Figure 8). (215)
Figure 8
Figure 8. Single ASLV-A entry into acidic endosomes and virus-endosome fusion. (a) Schematic diagram illustrating virus labeling and how the endosomal pH drops and subsequent ASLV-A fusion is visualized. (b) ASLV-A (yellow) fusion with TVA950 cells transiently expressing mKO-Rab5 (blue). Pseudoviruses were labeled with EcpH-ICAM (green) and Gag-mKate2 (red). The right top image panels show consecutive snapshots of the boxed region showing the virus prior to internalization (left), immediately after entry into acidic Rab5-positive endosomes (middle), and after fusion with early endosomes (right). The graph in panel (c) shows the fluorescence intensities of mKO-Rab5 and the viral EcpH-ICAM (green) and Gag-mKate2 (red) signals as a function of time. Adapted with permission from ref (215). Copyright 2014 BioMed Central Ltd.
3.2.3. Phototransformable Fluorescent Proteins
Another extremely powerful functionality of some FPs is that they can be activated or spectrally shifted using light. (222,223) Phototransformable fluorescent proteins (PtFPs) have already attracted worldwide attention with their skyrocketing popularity for super-resolution microscopy in recent years. (222,224) In 2002, Kaede, a GFP homologue, was discovered that was photoconvertible from green-to-red fluorescence emission under UV illumination. (225) In the same year, using mutagenesis, a photoactivatable GFP variant (paGFP) was developed, which was initially irradiated by 413 nm light and then emitted strong fluorescence when excited with 488 nm light. (226) Nowadays, a wide range of PtFPs have been developed to satisfy the requirements for different colors and modes of conversion, including photoactivatable, photoconvertible, and photoswitchable proteins. These proteins have been used for tracking the dynamics of cellular components in live cells (Figure 9). (224,227,228) For example, photoactivated-localization microscopy (PALM), as a kind of single molecule-localization super-resolution microscopy, (229) mainly relies on the amazing photophysical behaviors of PtFPs. This technique uses sequential activation of fluorophores and time-resolved localization to acquire high-resolution images, (180,230,231) which has facilitated the investigation of the mechanisms of virus infection. (232−234) Along with the development of SPT and SVT, which have provided subdiffraction resolution already in the 1980s, and superresolution microscopy, the combination of SPT and PALM (SPT-PALM) has been shown to be capable of visualizing multiple trajectories of viral proteins at high density in live cells. (235)
Figure 9
Figure 9. Use of PtFPs for investigating focal adhesions. (a) Protocol for dual-label super resolution imaging by PALM. (b) Dual-color PALM super resolution image overlay of paxillin (green) and zyxin (red). (c) Diffraction-limited, summed molecule, dual-color TIRF image. (d) DIC image. Adapted with permission from ref (228). Copyright 2007 National Academy of Sciences, U.S.A.
3.3. Fluorescent Nanoparticles
A major challenge in SVT is the development of fluorescent labels that combine small size with high brightness and photostability. To address this need and to enhance the imaging capabilities in SVT, researchers have pursued the development of biocompatible fluorescent nanoparticles for single-virus labeling. Compared with organic dyes and FPs, fluorescent nanoparticles usually show unique chemical and optical properties, such as higher brightness and photostability, which are highly preferable for single-virus tracking. Below, we will discuss commonly used nanoparticles: quantum dots and metal nanoparticles.
3.3.1. Quantum Dots
QDs, as a kind of semiconductor nanoparticles, have already attracted tremendous attention in biological applications. (236−240) This is mainly due to the excellent optical properties of QDs, such as high quantum yield, photostability, and size-tunable narrow emission spectra. (241−243) The spectral emission range of QDs covers the UV to infrared and can be adjusted by changing the size, shape, and composition of QDs. (123,244−246) The high brightness of QDs (10–100 times higher than organic dyes or FPs) facilitates the detection sensitivity and allows high-contrast images to be obtained. (123,247) In addition, the excellent photostability of QDs (100–1000 times higher than organic dyes or FPs) makes it possible to track single viruses over several hours with high temporal resolution. (248) Moreover, QDs possess a wide excitation spectrum and a narrow emission spectrum, which makes them well suited for multicolor imaging where viral components and cellular structures can be tracked simultaneously. (249,250) Based on the prominent properties mentioned above, QDs are broadly used in biolabeling, (251−253) in bioimaging, (254,255) and subsequently for single-particle tracking. (256−259)
In SVT experiments, the most available QDs are made of CdSe cores coated with a ZnS shell, (260) which are usually prepared in the organic phase and covered with hydrophobic organic ligands (Figure 10). For biological applications, the solubilization and biofunctionalization of QDs are essential steps owing to the hydrophobic surface of QDs. A substantial amount of pioneering research has been performed regarding these steps, which has led to a strong boost in the number and variety of applications of QDs in the fields of biology and biophysics. (237,243,260−263) So far, uniform, high-quality, biofunctionalized QDs are readily available via experimental synthesis or can be purchased commercially. In 2003, QDs were first applied to track individual glycine receptors on the neuronal membrane, which laid the cornerstone for exploring the dynamics of biomolecules in live cells by using QDs-based SPT. (89) After that, a huge number of QDs-labeled biomolecules of interest were tracked dynamically in a wide variety of biological systems. (94,238,264−267) In 2008, QDs were first successfully exploited to specifically label the envelope of viruses without significant effect on the infectivity of the virus, thereby allowing the uptake mechanism to be investigated in detail. (98) This was considered a watershed moment for SVT. With the further development of labeling strategies, different components of viruses could be labeled with QDs. (268) The detailed labeling strategies are described in detail in the section entitled Labeling Strategies for Nongenetically Encoded Fluorophores.
Figure 10
Figure 10. Properties of QDs. (a) Schematic drawing of a core–shell QD and fluorescent images of QDs of different sizes under UV light. (b) Absorption (left) and emission (right) spectra of CdSe/ZnS QDs. All QD samples and data were obtained in the group of Pang.
At present, QDs-based SVT techniques have acquired spectacular achievements and researchers have elucidated multifarious infection mechanisms of viruses. For example, Pang et al. utilized this technique to analyze the infection behavior of influenza viruses and dissect the transport mechanism of influenza virus trafficking at different stages of infection. The results indicated that influenza viruses underwent a previously unknown five-stage process from the cytomembrane to the perinuclear region along microfilaments and microtubules. A “driver switchover” mechanism was proposed to answer the question of how the transport of influenza viruses switches from microfilaments to microtubules. (108,110,114,115,118) In another example, Cui et al. designed QD-labeled transcription activator-like effectors to specifically target HIV proviral DNA sequences, and they identified single gene loci in the cell nucleus. (116) By encapsulating QD-conjugated RNAs into influenza viruses, they monitored the uncoating process of individual viruses and revealed the mechanisms of uncoating and vRNP trafficking of influenza viruses. (113)
3.3.2. Metal Nanoparticles
In the middle of the 1980s, Brabander and colleagues visualized the movements of gold nanoparticles (AuNPs) with a size of 40 nm on the surface of live cells by using video-enhanced differential interference contrast microscopy. (42,46) This is the first experiment using AuNPs-based SPT in live cells. Over the years, numerous researchers dedicated their efforts to develop suitable methods for data processing and modeling, and then AuNPs-based SPT was increasingly utilized to study the dynamic organization and heterogeneity of the cell membrane. (269,270) One major advantage of AuNPs in SPT experiments is their high stability, because they have no photobleaching and less biodegradation. AuNPs possess unique optical properties due to the surface plasmon resonance and strong light scattering. The scattering light signal of AuNPs requires dark field microscopy or similar optical setup to be detected. The signal intensity is coupled to the illumination intensity, and thus good image contrast and high temporal resolution could be obtained. Experiments with AuNPs-labeled respiratory syncytial viruses (RSVs) demonstrated that the RSVs still maintain their virulence and could be successfully tracked over extended periods of time. (271) However, the ability to detect multiple species via scattering light is very challenging and the limited availability of multiple labels restricted their application for SVT.
Metal nanoclusters (e.g., Au, Ag) are composed of a small number of atoms and typically have sizes below 2 nm. The emission wavelength of fluorescent metal nanoclusters covers the visible to near-infrared region of the electromagnetic spectrum. (273,274) The fluorescence of metal nanoclusters can be tuned by a number of factors, such as size, composition, ligands, aggregation state, ionic strength, and pH value. Due to the unusual physicochemical and good optical properties, fluorescent metal nanoclusters have attracted increasing attention for biological and biomedical applications. (274−276) To date, there are a number of applications in virus detection, (277−279) but very few reports about virus labeling with nanoclusters. Marjomäki et al. developed site-specific protocols to target the enterovirus capsid with monodisperse gold nanoclusters (Figure 11). The end point dilution assay implied that the binding of gold nanoclusters to the viral surface did not lower the infectivity of the virus. (272) Thus, site-specific labeling of enteroviruses with nanoclusters could facilitate the future structural studies of virus uncoating and become an important new tool for future SVT applications.
Figure 11
Figure 11. Gold nanocluster labeling of enteroviruses. (a) Synthesis of the maleimide functionalized Au102(pMBA)44 clusters and their site-specific conjugation to enteroviruses. (b–c) TEM images of CVB3 viruses treated with functionalized gold clusters. (c) Control TEM image with conventional negative staining of a virus sample incubated with nonfunctionalized clusters. Adapted with permission from ref (272). Copyright 2014 National Academy of Sciences, U.S.A.
4. Labeling Strategies for Nongenetically Encoded Fluorophores
Click to copy section linkSection link copied!
Organic dyes and nanoparticles, as nongenetically encoded fluorophores, can be advantageous over FPs owing to their superior brightness and photostability. However, most of these fluorophores can be used to target the viral components by direct chemical reactions or noncovalent interactions, but it is difficult to label viruses site-specifically. For many questions of interest, it is only necessary to label the viral particles and specific labeling of the virus is not important. Other questions require the labeling of specific viral components. For the following discussion of the several strategies that have been developed for attaching nongenetically encoded fluorophores to viral components, we will divide them into nonspecific and site-specific labeling approaches (Figure 12).
Figure 12
Figure 12. Labeling strategies and labeling sites for fluorescent labels in SVT.
4.1. Nonspecific Labeling
In virtue of their intrinsic properties, it is possible to use hydrophobic–lipophilic interactions or intercalation for some organic dyes to label the viral envelope and genome, which have been described in detail in the previous section. For labeling the external components of viruses (including the envelope of enveloped viruses and the capsid of the nonenveloped viruses), many chemical labeling methods have been adopted for organic dyes and nanoparticles, such as cross-linking, click chemistry, and biotin–streptavidin interactions.
4.1.1. Cross-linking Reaction
Cross-linking reaction is the simplest and versatile method for labeling viral components with organic dyes or nanoparticles, which mainly conjugate proteins and biomolecules with fluorophores by a covalent bond. Many cross-linking reagents (called cross-linkers) have been characterized and can be synthesized to combine two or more reactive groups within one molecule. The reactive elements can then chemically attach to specific functional groups of proteins and other molecules on the viral surface.
Proteins are complex structures composed of a linear sequence constructed from 20 different amino acids. However, for labeling, there are typically only four functional groups of proteins that are targeted: primary amines (−NH2), which exist at the N-terminus of each peptide and the side chain of lysine; carboxyls (−COOH), which exist at the C-terminal of each peptide and the side chain of glutamic acid and aspartic acid; sulfhydryl (−SH), which exists in the side chain of cysteine; carbonyls (−CHO), which can exist in oxidized glycoproteins. (280) Many chemical species can react with the four kinds of functional groups to form chemical bonds including isothiocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, and imidoesters (Figure 13). A wide variety of cross-linkers are commercially available, which can be easily conjugated to proteins or other functional group-containing compounds based on the commercial protocols. Thus, any kind of viruses can be covalently labeled with organic dyes or nanoparticles by cross-linking techniques. Especially for nonenveloped viruses, where the capsid is exposed to the outside, covalent labeling is a desirable choice for labeling. For example, AAV was conjugated with QDs using an amino-carboxyl cross-linking reaction (Figure 14). (136) A mild and clickable reaction between hydrazine and aldehyde was also used to label envelope proteins of viruses with QDs. (101,281) The direct chemical labeling methods are flexible and easy to apply, by which QD-virus conjugates or organic dye-labeled viral particles are readily obtained for SVT experiments.
Figure 13
Figure 13. Cross-linking reactions for conjugating fluorescent labels to viruses.
Figure 14
Figure 14. Covalent attachment of QDs to AAV and characterization of the QD-AAV conjugates. (a) QD-AAV networks are generated by an amide bond formation between the carboxylic source on QDs and the primary amines from lysine residues on the AAV capsid via carbodiimide chemistry. (b) Transmission electron microscope (TEM) images of (left) unconjugated QD525, (middle) AAV only, and (right) QD525-labeled AAV. Adapted with permission from ref (136). Copyright 2011 American Chemical Society.
4.1.2. Click Reaction
“Click Chemistry” is a type of biocompatible small molecule reaction that is commonly used in bioconjugation with fast reaction rates, mild reaction conditions, high yields, simple procedure, and high selectivity. In 1963, Huisgen first discovered the unactivated azide–alkyne cycloaddition reaction, but this reaction was ignored for many years owing to the harsh experimental conditions. (282) The term “Click Chemistry” was first put forward by Kolb et al. in 1998 and fully described in 2001, where they also introduced Cu(I) as a catalyst to realize the azide–alkyne cycloaddition reaction at room temperature with high chemical yield. (283,284) The copper-catalyzed azide–alkyne cycloaddition reaction (CuAAC) is the most universal click reaction, but it is often incompatible with living systems due to the potential toxicity of Cu(I). Nowadays, there are several kinds of copper-free azide–alkyne cycloaddition reactions reported, such as the strain-promoted azide–alkyne cycloaddition (SPAAC), (285,286) thiol–ene reaction, (287) and strain-promoted inverse-electron-demand Diels–Alder cycloaddition (SPIEDAC). (288) The copper-free approaches opened up the possibility of labeling particular biomolecules in living systems with click reactions. For instance, to detect DNA synthesis in vivo, a thymidine analogue 5-ethynyl-2′-deoxyuridine was incorporated into DNA during DNA replication, and the terminal alkyne group was labeled with a fluorescent azide by a click reaction. (289) Similarly, RNA synthesis was detected by using a click reaction between a uridine analog 5-ethynyluridine and fluorescent azide. (290) Researchers have also developed an unnatural sugar-based click labeling strategy. (291−293) The azido-sugars were added into the cell medium for cell culture. After metabolism, the azido groups were incorporated into the glycans on the cell surface, which could be used to bind with fluorophores by click chemistry.
The viral surface has potential targets for biofunctional modification, which can be functionalized to add clickable groups for virus labeling. (105,106,294,295) For example, Zhang et al. modified baculoviruses with a clickable group (4-dibenzocyclooctynols) and then labeled viruses with azido-derivatized multidentate-imidazole polymer ligands-modified QDs by copper-free click chemistry. (106) Lin et al. established a site-specific click labeling strategy on the surface of hepatitis D virus (HDV). By incorporation of pyrrolysine analogues carrying various functional groups onto the envelope proteins of live HDV, they successfully exploited the subsequent click reaction to attach a biotin molecule onto the HDV surface (Figure 15). (296) Additionally, by introducing azide and vinyl groups into proteins and the genome of viruses respectively, the envelope and genome of viruses could be labeled simultaneously via copper-free click chemistry and alkene-tetrazine ligation reactions. (297) Ethynyl-modified nucleosides have also been utilized to label newly synthesized DNA of vaccinia virus, adenovirus, and herpes virus by copper(I)-catalyzed click reactions for tracking the viral genome in host cells at the single-molecule level. (298)
Figure 15
Figure 15. Engineering the hepatitis D virus (HDV) assembly process for site-specific incorporation of unnatural amino acids (UAAs) into its surface envelope proteins. (a) Two-step procedure for the assembly of intact HDV carrying site-specifically incorporated UAA-recognized non-natural amino acids in human hepatocytes Huh-7 cells. (b) Structures of five Pyl analogues used in this study: PenK (Ne-pent-4-ynyloxycarbonyl-l-lysine), ACPK (Ne-((1R,2R)-2-azido-cyclopentyl oxy-carbonyl)-l-lysine), BCN (bicyclo[6.1.0]non-4-yn-9-ylmethanol), DiZPK (3-(3-methyl-3H-diazirine-3-yl)-propaminocarbonyl-Ne-l-lysine), and ONBK (o-nitrobenzyloxy carbonyl-Ne-lysine). Adapted with permission from ref (296). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
4.1.3. Biotin–Streptavidin Interaction
The biotin-(strept)avidin system is one of the strongest noncovalent biological interactions present in nature, which also shows high selectivity, fast reaction speed, and good resistance to extremes of temperature or pH. (299) So far, this system has been used extensively in biomolecule detection, protein purification, and biological labeling, and there are a lot of biotinylation reagents available for developing different labeling methods. Therefore, the versatile approach is to covalently attach biotin to the biomolecule of interest and subsequently bind avidin, streptavidin, or neutravidin reagents. To label viruses with fluorophores, the viral components should be biotinylated chemically or enzymatically. Chemical biotinylation utilizes cross-linking reactions to generate nonspecific biotinylation of carboxylates, sulfhydryls, amines, and carbohydrates. Enzymatic biotinylation generates biotins in a specific lysine within a certain sequence of the viruses by a bacterial biotin ligase. (98,300) Details will be described in the following Site-Specific Labeling section. Based on the biotin–streptavidin interaction, two strategies have been developed for labeling viruses.
A one-step labeling approach to virus labeling is to directly obtain fluorescent-labeled virus conjugates by the interaction between biotinylated viruses and streptavidin-modified fluorophores. The conjugates could aid researchers in investigating the interactions between viruses and the plasma membrane in the early stage of virus infection. However, these one-step labeling methods require tedious purification procedures to remove free fluorophores, such as size exclusion and ultrafiltration. These cumbersome steps usually bring about a significant loss in virus and have deleterious effects on the integrity and activity of the viruses. Especially QDs-labeled viruses cannot be preserved for a long time, since viruses can be degraded at room temperature and QDs are unstable or precipitate below freezing. Thus, the method is not well suited for performing many parallel experiments for gathering statistics.
A so-called two-step labeling approach initially incubates the host cells with biotinylated viruses and then the streptavidin-modified fluorophores are added. For QDs labeling, the two-step labeling strategy has been broadly adopted for SVT experiments. After biotinylation, the viruses are added to cells at low temperature and allowed to bind to the viral receptors and then incubated with streptavidin-QDs to fluorescently label the viruses (Figure 16). (108,301,302) The whole process takes less than a half an hour and avoids tedious purification processes. A colocalization analysis indicated that nearly all viral envelopes could be labeled with QDs, and the infectivity of QDs-labeled viruses was still 86% of that of the native viruses. (99) Thus, this method is easy to perform with high efficiency and low damage to the virus infectivity and has been widely used for studying the infection mechanisms of viruses, including influenza viruses and infectious hematopoietic necrosis virus (IHNV). (99,107)
Figure 16
Figure 16. Schematic of a two-step labeling strategy of labeling virus with QDs via the biotin–streptavidin interaction. Adapted with permission from ref (108). Copyright 2012 American Chemical Society.
4.2. Site-Specific Labeling
The primary goal of viruses is to deliver their genome to the proper cellular compartment for transcription and replication. Only labeling external components is insufficient for following the whole infection pathway of a virus because the external components will be dissociated in the process of virus infection. Labeling the internal components of viruses is much more difficult, since fluorophores, especially fluorescent nanoparticles, are difficult to penetrate the external components of the viruses to enter the interior. This is where the use of FPs for fluorescence labeling has great advantages; when the appropriate location can be found that tolerates the tag, the viral genome can handle the increase in genomic size and there are enough copies to allow tracking over reasonable time periods. However, sometimes it would be great to combine the benefits of genetically encoded labels with the brightness and photostability of synthetic dyes or QDs. Thus, one big question is how to encapsulate nanoparticles or organic dyes into viruses and can it be done specifically.
In early 2006, Dixit and co-workers incorporated QDs into the capsids of the brome mosaic virus by self-assembly, and the generated virus-like particles (VLPs) had a similar size to the native virus. (303) Later, combining with site-specific labeling strategies, such as peptide tag-mediated labeling and oligonucleotide-guided labeling, host cell-assisted methods have been extensively developed in recent years, which are an alternative approach to label the internal and external components of viruses during virus assembly. (304−306) For virus labeling, the distinct advantage of host cell-assisted methods is that it is possible to avoid modification of the viral surface, thus minimizing the influence of labeling on the virus–receptor interactions. To date, host cell-assisted methods have played an important role in visualizing the infection behaviors in different infection stages of a variety of viruses including SV40, HIV, and PV. (131,290,307,308)
4.2.1. Peptide Tag-Mediated Labeling
Fluorescent proteins, as genetically encoded fluorescent labels, can be incorporated with high specificity but still suffer from relatively low brightness and poor photostability. (309) Organic dyes and nanoparticles typically have much better photophysical attributes but cannot realize site-specific labeling in live cells. To combine the best of both worlds, another class of genetically encoded proteins has been developed that can catalyze the autoattachment of fluorescent ligands inside living cells. (310) The proteins themselves are nonfluorescent but can be specifically labeled with fluorescent ligands. (311) These tags can be much brighter, more stable, color-tunable, and more chromatically diverse (312) and, similar to FPs, can also possess some specific properties such as environmental sensitivity and photoswitching but with better photophysical properties. (313,314) For imaging experiments, the labeling is controllable in space and time, and the color of the target protein can be selected according to the experimental requirements. (315) We will discuss two major site-specific approaches for labeling viruses with organic dyes or nanoparticles in live cells: self-labeling fusion tags and enzyme-targeted peptide tags (Table 4). (316)
Table 4. Tag-Mediated Site-Specific Labeling Methods
| Tag | Size (amino acids) | Labeling reaction | Fluorophores demonstrated | Virus | Ref for Tag |
|---|---|---|---|---|---|
| Self-labeling fusion tags | |||||
| SNAP tag | 182 | Covalently binding with benzylguanine derivatives | TMRa, SiRb | HIVc, (317,318) | (319−321) |
| CLIP tag | 182 | Covalently binding with benzylcytosine derivatives | TMR | HCVd, (322) | (323) |
| Halo tag | 296 | Covalently binding with haloalkane derivatives | TMR | PrVe, (324) | (325) |
| TMP tag | 157 | Engineered Escherichia coli dihydrofolate reductase noncovalently binding with trimethoprim (TMP)-fluorophore conjugates | Fluorescein, Atto dye | NDf | (326−329) |
| TC tag | 6–10 | Covalently binding with fluorogenic biarsenical compounds | FLAsH, ReAsH | HIV, (330) VSV,g, (331) IVh, (332) | (333,334) |
| His tag | 6 | Noncovalently binding with Ni-NTA-functionalized fluorophores | QDj | Prion, (103) RSVi, (335) | (336) |
| Enzyme-targeted peptide tags | |||||
| AP tag | 15 | Covalently binding with biotin or ketone analogs of biotin | QD, Fluorescein, Alexa Fluoro | HIV, (98,116) Baculovirus (300) | (337,338) |
| LAP tag | 13–22 | Covalently binding with lipoic acid derivatives | QD, Cy3, Alexa Fluoro | HIV (116) | (336,339,340) |
| Tub tag | 14 | Covalently binding with tyrosine derivative | Coumarin | ND | (341,342) |
| S6/A1 tag | 11 | Covalently binding with coenzyme A derivatives | Texas red, Alexa Fluoro, Cy3 | ND | (343,344) |
a
Tetramethylrhodamine.
b
Silicon rhodamine.
c
Human immunodeficiency virus.
d
Hepatitis C virus.
e
Pseudorabies virus.
f
Not determined.
g
Vesicular stomatitis virus.
h
Influenza virus.
i
Respiratory syncytial virus.
j
Quantum dot.
4.2.1.1. Self-Labeling Fusion Tags
Self-labeling fusion tags have recognition domains that offer the specific attachment of fluorescent ligands to the target proteins in live cells. In this approach, the target protein binds with a self-labeling fusion peptide or protein sequence. The protein is expressed in live cells and the specific fluorescent ligands are added for protein labeling. As the self-labeling fusion peptide or protein sequence is small, it is easy to create with a wide variety of fluorescent ligands, which can be optimized for the imaging instrumentation.
The SNAP tag, a 182 residues polypeptide (19.4 kDa), is an engineered version of human O6-alkylguanine-DNA alkyltransferase (hAGT). (345) This protein catalyzes the attachment of O6-alkylguanine or O6-benzylguanine to one of its cysteines. To label the SNAP-tagged protein in live cells, the fluorescent membrane permeable O6-alkylguanine substrates are added into cells having expressed proteins fused with SNAP-tag. (319,320) CLIP tag is a new variant of hAGT, which specifically reacts with O6-benzylcytosine substrates. (323) The orthogonal relationship between the SNAP tag and the CLIP tag can be simultaneously exploited for dual-color labeling. (346)
The Halo tag is a modified haloalkane dehalogenase, which specifically binds the reactive primary alkyl halides and covalently attaches a modified fluorescent ligand to the active-site residue. (325,347) The trimethoprim (TMP) tag was developed on the basis of the strong interaction between the folate analogue TMP and the Escherichia coli dihydrofolate reductase (eDHFR). The protein of interest is fused with eDHFR, expressed in live cells, and then the fluorophore-modified TMP is added into the cells and binds with eDHFR with high affinity and selectivity. (326,348) So far, these tags have been widely used for protein imaging and trafficking in live cells. Based on reverse genetic technology, the halo-tag protein has been fused with the smallest pseudorabies virus (PrV) capsid protein VP26. The recombinant PrV was easily harvested and used directly for the virus tracking without further modification (Figure 17). (324) These labeling systems have several obvious advantages. Fluorescence can be turned on when the fluorescent ligands are added into the cells and turned off using available blocking reagents. Alternatively, a ligand with a second dye can be added at a later time point such that the newly produced proteins of interest are labeled with a different color of dye.
Figure 17
Figure 17. A schematic diagram for the generation of Halo tag-labeled pseudorabies viruses. Adapted with permission from ref (324). Copyright 2016 American Chemical Society.
One of the shortest peptide tags available is the tetracysteine (TC) motif (most commonly CCPGCC). The biarsenical dyes FlAsH (green fluorescence) and ReAsH (red fluorescence) specifically bind the TC tag in live cells. The TC tag is only 6 amino acids, and the protein of interest will fluoresce when the biarsenical dye binds. The smaller size and self-labeling capacity of the TC tag make it a very attractive tool for virus labeling. For example, Rudner et al. inserted a TC tag to the C terminus of HIV Gag and investigated the dynamic process of HIV by two-color imaging analysis. (330) By fusing M protein with TC tags and P protein with EGFP, recombinant VSVs could be dually fluorescent-labeled. Time-sequence images confirmed the adsorption of VSV at the plasma membrane and illustrated that the entry and uncoating of VSV in the infected cells occurred with a half-life of approximately 28 min after virus adsorption. (331) However, due to the interactions with other thiol-containing proteins, the background signal is much higher, and time-consuming washing procedures are required before imaging.
The His tag consists of at least six histidine (His) residues and shows a high affinity and selectivity for Ni2+. The (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system has been widely utilized in protein purification. With the development of fluorescent Ni-NTA-based probes, the His tag has already been used for live-cell imaging. (103,109,349) For example, PEG-interspersed Ni-NTA-functionalized QDs were developed to label prion proteins expressed on cell surfaces. (103) Time-lapse imaging first demonstrated that the entire process of prion internalization could be divided into four discrete but connected stages and that lipid rafts played an important role in prion localization and internalization. (109) Further, by conjugating the viral surface with specific polypeptide containing histidine residues, Huang et al. developed a Ni-NTA based progeny virus labeling strategy, which is noninvasive and can be used to label other enveloped viruses budding from the plasma membrane (Figure 18). (335)
Figure 18
Figure 18. Scheme of the general strategy for in situ virus labeling during progeny virus assembly. The labeling procedure includes (1) infection of the host cells with the virus, (2) after 2 days’ cultivation, the proteins on the host cell surface were conjugated with polypeptides containing his-tags and carboxyl groups, (3) progeny viruses assemble and are released from the cell surface, incorporating the his-tags in the process, and (4) the progeny viruses are further tagged with Ni2+-NTA modified QDs. Adapted with permission from ref (335). Copyright 2016 The Royal Society of Chemistry.
4.2.1.2. Enzyme-Targeted Peptide Tags
An alternative method for site-specific labeling is the use of enzyme-targeted peptide tags. The enzymes can catalyze the attachment of a specific peptide sequence to a fluorescent substrate. Thus, the enzymes could help to realize the modification of the peptides with high site selectivity. There are several kinds of enzymes used for protein labeling, such as ligases and transferases.
Ligases, including biotin ligase, tubulin tyrosine ligase, and lipoic acid ligase, can bind specific peptide tags to a recognizable sequence. (350) For example, the biotin acceptor peptide (AP)-tag, a 15 amino acids long peptide that is a substrate for biotin ligase (BirA), enables the conjugation of a biotin to a lysine side chain on the AP tag. (351) After the BirA-catalyzed biotinylation, fluorescent labeling of the protein of interest can be achieved using the biotin–(strept)avidin interaction. By taking advantage of this site-specific labeling strategy, the viral envelope incorporated an AP tag and subsequently, streptavidin-conjugated QDs were attached to the surface of virion after the biotinylation of the AP tag happened. (98,300) For example, the capsid protein VP39 of baculoviruses could be specifically labeled with streptavidin-modified QDs by modifying the protein with biotin by a genetic recombination technique during viral assembly in host cells. (102) It is worth noting that the various components of viruses will disassociate at different stages of the infection process. To dissect their whole infection pathway, it is a prerequisite for SVT to be able to simultaneously follow the related external and internal components of viruses. Pang et al. developed a cell-assisted strategy to simultaneously label the envelope, capsid, and genome of baculoviruses with QDs, EGFP, and SYTO 82, respectively. Such a triple-labeled virus makes it possible to visualize the dissociation process of key viral components in real time. (170)
Similarly, a short lipoic acid ligase (LplA) acceptor peptide (LAP) tag is a 22 amino acids long peptide, which can be catalyzed by LplA to conjugate with lipoic acid derivatives in an ATP-dependent manner. (352) The protein of interest can be fused with the LAP tag and labeled with fluorescent lipoic acid derivatives. The tub tag is a 14 amino acid hydrophilic recognition sequence. Using tubulin tyrosine ligase (TTL), it is possible to conjugate a tyrosine with the fluorescent tub tag. (353) Some of these tags have been utilized to label viral components during virus infection. For example, Cui et al. used a pair of QD-labeled transcription activator-like effectors (TALEs) to image single genomic loci of HIV-1 provirus in the cell nucleus. One of the TALEs was fused with LAP tag, and labeled by tetrazine-conjugated red QDs via Diels–Alder cycloaddition chemistry. The other TALE was fused with an AP tag, biotinylated, and labeled with streptavidin-modified green QDs. The fluorescence colocalization of the two QD-TALEs demonstrates that there is a single HIV-1 provirus loci in live cells (Figure 19). (116)
Figure 19
Figure 19. Labeling the HIV-1 proviral loci. Within the cytosol of a live cell, the TALEs fused with a short LplA acceptor peptide (LAP) are decorated with trans-cyclooctene and subsequently labeled with tetrazine-conjugated red QDs via Diels–Alder cycloaddition chemistry. The TALEs fused with an AP tag are biotinylated and labeled with streptavidin-conjugated green QDs. The two QD-TALEs bind to the target HIV-1 proviral DNA sequences, and their fluorescence colocalization demonstrates a single-copy HIV-1 provirus loci in the human chromosomes. Adapted with permission from ref (116). Copyright 2017 Springer Nature.
Transferases can transfer functional groups from a donor substrate to a specific amino acid in a peptide sequence. Phosphopantetheinyl transferases (PPTase), such as Sfp and AcpS, can catalyze the transfer of a phosphopantetheinyl group from coenzyme A (CoA) to a conserved serine. Sfp preferentially recognizes the S6-tag, and AcpS preferentially recognizes the A1-tag. (353,354)
4.2.2. Oligonucleotide-Guided Labeling
The base-pairing propensity of DNA and RNA oligonucleotides can be used to detect the presence of specific target sequences (i.e., complementary nucleic acid sequences) by hybridization. Short oligonucleotides can be synthesized and several different types of groups incorporated for fluorescently labeling the oligonucleotides. Organic dye- or QD-labeled oligonucleotides have been designed and transfected into infected host cells to bind the viral genome of interest in live cells. During virus assembly, fluorophores with oligonucleotides can be assembled into the interior of viruses. A paradigm is that QDs were conjugated with QD-labeled guide RNAs containing the sequence of a packing signal of the viral genome, and then the functionalized QDs would be encapsulated into the capsid of VSV glycoprotein pseudotyped lentivirus (PTLV) in living cells (Figure 20). (355)
Figure 20
Figure 20. Working principle of encapsulating SA-QDs into HIV-based lentivirus in living cells. Adapted with permission from ref (355). Copyright 2013 American Chemical Society.
By labeling the viral oligonucleotides, it is possible to use SVT to follow the fate of the viral genome and where replication occurs. Several strategies have been developed to track the transport process of the viral genome in host cells. Fluorescence in situ hybridization (FISH) is a conventional nucleic acid recognition technique, which can be used to detect and localize endogenous or engineered DNA or RNA through Watson–Crick base pairing. However, FISH is performed in fixed specimens leading to a loss of dynamical information in comparison to tracking nucleic acids in live cells. For live-cell imaging, fluorescently labeled linear oligonucleotides were developed to visualize nucleic acids and allow investigations of trafficking or transient interactions. Using this hybridization approach, Molenaar et al. detected various endogenous nuclear RNAs in live cells without interfering with cell vitality. (356) Often, hybridization techniques suffer from a high level of background fluorescence due to the lack of effective approaches to remove unbounded probes. Here, using nucleic hairpin structures with a fluorescence quencher has been developed to minimize the background fluorescence from nonbound beacons. Upon binding to the target nucleic acid sequence, the hairpin opens and the fluorescence signal increases. One such approach uses fluorescence resonance energy transfer (FRET) to quench the fluorescence signal of the donor and thus to significantly reduce the background fluorescence. Using FRET, a series of QD-labeled molecular beacons were developed with controllable QD valency. A nanobeacon with one conjugated DNA was favorable for labeling and imaging single RNA in live cells and was used for ultrasensitive detection of single HIV RNAs in HIV integrated cells. (357)
5. Optical Implementations for Single-Virus Tracking
Click to copy section linkSection link copied!
Many viruses are on the order of 100–200 nm in size, just slightly smaller than the diffraction limit of a high-end microscope. Hence, it is also potentially possible to monitor some viruses directly without the need for labeling them. One label-free approach that has been applied successfully for tracking HIV particles is interferometric scattering microscopy (iSCAT). (358) By using the interference of light reflected from the surface of the coverslip and that coming from the virus particle itself, it becomes possible to visualize the position of the virus and perform SVT with both high spatial and temporal resolution. Kukura and colleagues combined SPT with iSCAT to follow the diffusion of single Simian Virus 40 particles on supported bilayers with 8 ms temporal resolution and nanometer spatial resolution. By labeling the viruses with QDs, we could also measure the orientation of the virus and thus monitor the tumbling of the virus as it slides along the surface. (359)
Once viruses and/or viral components have been successfully labeled fluorescently, SVT measurements can be performed to investigate the various processes in the lifecycle of the virus. For this, fluorescence microscopy has become an indispensable tool. The basic function of a fluorescence microscope is to excite a sample with a specific band of wavelengths, separate the emitted fluorescence from the excitation wavelengths, and detect the fluorescence emission with high sensitivity. As only the emitted light reaches the observer’s eye or the detectors, high-contrast fluorescence images can be recorded on a dark background. In the early part of the 20th century, the first fluorescence microscopes were built by the companies of Carl Zeiss and Carl Reichert. (360) Subsequently, Ellinger and Hirt developed “intravital microscopes” to visualize living organisms. They utilized UV light to excite the sample and put filters between the samples and eyes that would reflect the excitation light and transmit the emitted light. This provided the basic principle of modern fluorescence microscopy. (361) Nowadays, lasers are typically used for excitation of the sample instead of lamps. When usage continues with wave lasers or nonfemtosecond pulsed lasers, the excitation wavelength can be limited to a very narrow region of the spectra and emission filters can be produced to efficiency block that specific wavelength while not blocking much of the fluorescence emission. In addition, many lasers provide a high-quality beam profile that simplifies the construction of the excitation pathway. The recent developments in lasers, optics, and detectors as well as the broad spectrum of available fluorophores and various labeling methods have resulted in a revolution in fluorescence microscopy and the worldwide use of fluorescence microscopes in various fields including cell biology, virology, and biophysics. (33,362,363) There are several commonly used imaging modalities for single-virus tracking, which we will present below. (364,365)
5.1. Wide-Field Microscopy
The simplest optical implementation for SVT experiments relies on wide-field illumination and a highly sensitive camera. In wide-field microscopy, a large field of view is fully illuminated by the excitation light and the fluorescent structures of the sample can be captured quickly and easily by a single camera. (366) To acquire a high amount of signal, objectives with an NA > 1.2 are typically used along with sharp fluorescence filters having a high transmission (>80%). For detection, a fast frame transfer, high-quantum yield camera is essential for SVT. Conventional charge coupled device (CCD)-based cameras have high sensitivity but slow read-out speeds whereas complementary metal oxide semiconductor (CMOS) cameras offer very fast frame rates but a narrow dynamic range. An electron-multiplied charge coupled device (EMCCD) detector performs the electron amplification on chip before read-out to decrease noise and combines fast read-out speed with high quantum yield and optimum resolution. A newly developed scientific complementary metal-oxide semiconductor (sCMOS) camera is based on a CMOS image sensor design and can also offer low noise, rapid frame rates, and high quantum yield. Both cameras are very suitable for quantitative measurements and dynamic imaging. For live-cell studies, an EMCCD camera is capable of obtaining discernible images with a lower number of photons. However, when fast, full-frame transfer rates are essential, sCMOS cameras are superior for fast imaging at speeds approaching or exceeding 1000 fps. (367−369)
The advantages of wide-field microscopy are the large excitation depth, large depth of focus, and low signal loss allowing the tracking of individual viruses in a large volume. However, there is no discrimination against out of focus light. For experiments with low autofluorescence and sparsely distributed fluorescence viruses, this approach works well. However, for experiments with a high background, such as investigations of the HIV assembly using FP-labeled Gag protein, the out-of-focus signals result in the low-contrast images. (370) In addition, owing to the large depth of field, this microscopy modality is rarely used to acquire 3D data over a certain depth of the sample. Thus, wide-field microscopy is mostly limited to 2D SVT tracking. (68)
5.2. TIRF Microscopy
In 1956, E. J. Ambrose first proposed the idea to illuminate cells that contact a glass surface by using total internal reflection. Total internal reflection fluorescence (TIRF) microscopy was first realized by Daniel Axelrod at the beginning of the 1980s. (371) The basic principle of TIRF microscopy is to use an evanescent wave to excite the sample near the glass–liquid interface. An evanescent wave is generated at the glass–liquid interface where the excitation light impinges on the glass-sample interface above the critical angle, at which point the light cannot physically propagate into the sample (typically buffer) and is thus totally reflected. The evanescent wave is a nonpropagating wave whose intensity decays exponentially with distance from the interface. (372−374) There are two main approaches to producing TIR excitation at the glass–sample interface (Figure 21). The original approach, prism-based TIRF, utilizes an optical prism to couple the excitation light to the upper surface of the glass (typically quartz)–sample interface. Upon the development of objectives with a numerical aperture above 1.4, it became possible to focus light onto the coverslip–sample interface such that the incident beam reaches the glass–sample interface above the critical angle, creating an evanescent field. (375) When using an inverted microscope, objective-type TIRF leaves the sample above the objective free and is thus more convenient and easier to use. Objective-type TIRF is the common modality used for live-cell imaging. (376−379)
Figure 21
Figure 21. Schematic drawing of an objective-based TIRF and a prism-based TIRF.
Due to the exponentially decaying intensity of the evanescent wave, TIRF microscopy is only suitable for exciting fluorescent samples within a depth of ∼200 nm from the interface. (371,377) Thus, TIRF largely reduces the thickness of the excitation plane and improves the image contrast significantly with respect to wide-field imaging (Figure 22). Combined with ultrasensitive and fast detectors, TIRF microscopy has empowered researchers to visualize biological events occurring on the plasma membrane of living cells. (380) It is also possible to adjust the excitation beam such that it impinges on the sample just below the critical angle. The excitation beam then propagates into the sample at an inclined angle, which has the rough effect of only exciting a thin plane in the sample. (381) This technique, known as highly inclined and laminated optical sheet microscopy (HILO) or affectionately as poor man’s TIRF, allows TIRF-like measurements to be performed internally in the cell.
Figure 22
Figure 22. Live cell imaging of individual HIV-1 assembly sites. HeLa cell transfected with a mixture of pCHIV and pCHIVeGFP imaged at 25 h post transfection (a) in WF and (b) in TIRF mode. The scale bar represents 5 μm. Adapted with permission from ref (370). Copyright 2009 Public Library of Science.
Nowadays, most common SVT set-ups perform TIRF excitation by means of objective-based TIRF. In this configuration, it is easy to switch between wide-field to TIRF illumination modalities, even on a frame by frame basis. (370) TIRF microscopy is powerful for investigating dynamics at the plasma membrane, such as clathrin-mediated endocytosis of VSV (134) and the fusion event between viral and cellular membranes. (382) A combination of TIRF microscopy and SVT identified different motional modes of MPV-like particles (VLPs) on the cell surface, and the particle trajectories provided new insights into the lateral mobility of membrane components and the heterogeneity of the plasma membrane. (139)
5.3. Confocal Microscopy
Marvin Minsky developed and patented the concept of confocal microscopy in the late 1950s with the aim of performing microscopy in thick tissues such as the brain. The basic principle of confocal microscopy is to use point illumination and a pinhole before the detector to eliminate the out-of-focus signals. For details regarding confocal microscopy, we refer the readers to ref (383). The excitation pinhole and the detection pinhole are both mounted in image planes of the microscope, thus “con-focal”. In this way, only the fluorescence from the focal plane reaches the detector. This microscopy modality has a thinner depth of field and higher resolution compared to wide-field microscopy. One of the demands that Minsky had on confocal microscopy is that it could image in real time, which, according to Minsky, may have significantly delayed the acceptance of confocal microscopy. (384) Shortly after the development of the confocal microscope, lasers were invented. Lasers had high enough energy densities to perform confocal microscopy and, as light coming from the laser is collimated, an excitation pinhole was no longer needed. In addition to the laser, the development of computers and imaging software contributed to the rapid development of confocal microscopy and became commercially available in the late 1980s. There are two types of confocal microscopes (Figure 23). (385) The laser-scanning confocal microscope (LSCM) scans the sample point-by-point. The sample can be scanned, typically using a piezo stage, or the excitation beam can be scanned using galvanometer mirrors (and nowadays also with resonant scanners) that can be used to increase the scanning speed. In either case, data is collected one pixel at a time and, even when the scanners have sufficient speed for imaging, one is often limited by the number of photons available per pixel. Thus, the typical image speed of a LSCM is limited to a couple of images per second and not suitable to track the dynamic behavior of quickly moving biomolecules in live cells. The second approach is to scan multiple volumes simultaneously, and a first apparatus was already built in the late 1960s. (386) This is done using a Nipkow disk and is referred to as spinning-disk confocal microscopy (SDCM). (385) The sample can be excited by multiple points simultaneously in the focal plane and an area detector such as an EMCCD can be used to collect the detected signals. SDCM and similar approaches can significantly improve the imaging speed. Thus, SDCM has become an indispensable tool for investigating the dynamic events of biomolecules in live cells. Furthermore, combining SDCM with a fast piezo z-scan device allows recording of fast three-dimensional images and has been used for 3D single-virus tracking in live cells. (387,388)
Figure 23
Figure 23. Schematic drawing of (a) a laser scanning confocal microscope and (b) a spinning disk confocal microscope.
5.4. Light Sheet Microscopy
To obtain cellular resolution imaging in intact or in vivo tissues, imaging methods must achieve optical sections of the tissue. Since single-particle experiments are extremely sensitive to background fluorescence and photobleaching, optical sectioning schemes such as TIRF and confocal microscopy have already been widely used for single-particle tracking in live cells. Owing to the limited imaging depth of TIRF, it is not suitable for single-particle tracking in extended 3D samples. Confocal microscopy has limitations in speed when used for 3D volumetric imaging. In addition, a good portion of the sample is always illuminated during confocal microscopy leading to photobleaching of the sample even out of the focal plane. To avoid this difficulty and to allow SVT in large biological objects in three dimensions, light sheet microscopy has been introduced. In light sheet microscopy (LSM), a focused thin plan of light is used to illuminate the sample and the emission light is collected orthogonally with a standard microscope objective and imaged. (389−391) The 3D volumetric image can be obtained by moving the coaligned excitation and detection plane relative to the sample. (392) Thus, compared with confocal microscopy, LSM is able to image thicker tissues (>1 cm) and has already successfully been utilized to image complex organisms at single-cell resolution in three dimensions, e.g. embryos of C. elegans, zebrafish, Drosophila, and even mouse embryos. (393−396) In addition, the plane that is illuminated by the light sheet is in the focal plane for detection and out-of-focus regions are not illuminated.
As the infection of viruses occurs in intricate 3D environments, the viral infection behavior exhibits diversified patterns in vivo, and consequently, the advent of light sheet microscopy will provide new insights regarding virus infection in living biological specimens. (397) Recently, inspired by LSM, Bosse and colleagues developed what they called a “Ring light sheet” and used it to reveal the remodeling of the nuclear architecture upon infection with herpes simplex virus allowing the nuclear herpes virus capsids to reach the nuclear membranes by diffusion. (398) Benefiting from the high spatiotemporal resolution provided by LSM, the tracking of rapid nuclear particle motility could be realized. The findings settled the question of how herpes virus capsids egress from the nucleus and illustrated a pathway for very large nuclear particles to cross the nuclear space by remodeling of the nuclear structure. Hoyer et al. developed a plane-scanning reversible saturable/switchable optical transitions light-sheet nanoscope (RESOLFT), which circumvents the diffraction limit of the light sheet fluorescence microscope in the axial dimension. (399) It is believed that this charming microscopy will spark enormous interest notably in virus research because it is suitable for long-term three-dimensional imaging at high spatiotemporal resolution in living biological specimens.
5.5. SPT-PALM
In this review article, we have not discussed much about super-resolution microscopy in respect to SVT studies. One the one hand, viruses, being often slightly smaller than the diffraction limit, are excellent objects that profit greatly from the increase produced in resolution by super-resolution techniques. As such, super-resolution methods will play an important role for viral studies. For example, PALM (229)/stochastical optical reconstruction microscopy (STORM) (400) microscopies were utilized to define spherical assembly sites of HIV (∼130 nm) in fixed cells, which is consistent with the size of mature virion particles (232,401−403) and to investigate the structure of the endosomal sorting complex required for transport (ESCRT) complex involved in the release of HIV. (404,405) On the other hand, SVT is, in a sense, a super-resolution technique as we are following a single object with nanometer spatial resolution. Thus, traditional SVT methods are more than sufficient for “super-resolution” tracking of individual viruses without the complications of the various super resolution methods, which typically have a low temporal resolution, have a high optical toxicity, and require heavy postacquisition processing. However, it should be noted that in recent years, super resolution techniques have already expanded to allow multicolor, three-dimensional single-particle tracking experiments in live cells. (406,407)
The one super-resolution approach that is of interest for SVT is SPT-PALM. By combining SPT and PALM, individual molecules present at high density can be tracked in live cells. In SPT-PALM, a small number of molecules are photoactivated in a live cell and followed using SPT. As the molecules are only sparsely activated, they can be easily tracked. After the molecules photobleach, new molecules can be photoactivated and tracked. SPT-PALM provides a large quantity of single-particle trajectories, though they are often very short due to the limited photostability of the photoactivatable FPs used for SPT-PALM. This technique has been successfully utilized to characterize the dynamics of biomolecules in a wide range of biological systems. (408−410) With respect to SVT, SPT-PALM has been used to track VSVG tagged with EosFP, providing several orders of magnitude more trajectories per cells as compared to traditional SVT methods. Thereby the authors could generate a spatially resolved map of single-molecule motility within the cell provided information regarding the heterogeneity of the cellular environment (Figure 24). (235)
Figure 24
Figure 24. SPT-PALM imaging of VSVG in COS7cells. (left) PALM image of VSVG tagged with EosFP. (middle) All SPT-PALM trajectories of localized VSVG molecules. (right) Diffusion map of individual EosFP-VSVG molecules in the cell. Adapted with permission from ref (235). Copyright 2008 Springer Nature.
Unfortunately, owing to the poor photophysical properties of autofluorescent proteins, the trajectories of molecules are very short and the localization accuracy is much lower in SPT-PALM experiments. The acquisition time also has a strong influence on the localization accuracy of particles, which is associated with several factors such as imaging modality, brightness of the tags, detector sensitivity and speed, etc. Thus, this technique is much more suited for detecting slow-diffusion events.
5.6. Orbital Tracking
Up until now, all tracking methods that we have been discussing involve ex post facto tracking. Movies are recorded in 2 or 3D and the tracking is performed afterward. These methods are mainly based on modified standard microscopes and achieve subpixel localization accuracy by 2D Gaussian fitting algorithm and 3D resolution using, for example, biplane (411) or multifocal plane detection, (54−56) a double-helix point spread function, (412−414) or astigmatic imaging. (415,416) Thanks to the emergence of spinning-disk confocal microscopy with high temporal resolution, it is also easy to implement z-stack imaging on a commercial confocal microscope. (417) A distinct advantage of the image-based approaches is that many fluorescent particles can be tracked at the same time. However, these methods have a lower temporal resolution.
There is a second class of SPT methods that track single particles in real time using a feedback loop. This includes arranging four detection volumes in a tetrahedral geometry for locating the particle, championed by Werner, (418) a guided confocal microscope using prisms (for lateral tracking) and a pinhole (for axial tracking) from Yang, (419) or the newly developed MinFlux approach from Hell. (420) We utilize the orbital tracking approach proposed originally by J. Enderlein in 2000 to monitor fluorescent molecules motilities within a 2D membrane (421) and first realized in 2D and 3D by Gratton et al. in 2003. (57) In the orbital tracking approach, the laser beam is orbited in a circular path around the fluorescent particle. When the particle is in the center of the orbit, the fluorescence intensity will stay constant throughout the orbit. When the molecule moves from the center of the orbit, the fluorescence signal will modulate with the orbit and the x-y position can be obtained from the phase and the modulation of the periodic fluorescence signal using a fast Fourier transform. (422) For determining the z-position of the fluorescent particle, the difference is taken of the fluorescence intensity between two different z-planes separated by approximately half the axial width of the point-spread function. The localization method is fast and can be utilized in a fast feedback mechanism where the orbit is recentered on the fluorescent particle and the location of the particle is written to disk. Thus, this approach allows us to track individual particles in three dimensions in real time. (58,423,424) The orbiting tracking system was used to monitor the assembly and egress of the matrix protein of Ebola virus. The researchers found that the actin coordinates the movement and assembly of VP40, a critical step in viral egress. (213)
Recently, Lamb et al. utilized a 3D orbital tracking microscope to track individual mitochondria in zebrafish embryos with nanometer precision and millisecond temporal resolution (Figure 25). (425) This demonstrates the possibility of following individual objects with high precision in living organisms. With respect to SVT, the uptake and transport of artificial viruses has also been followed using orbital tracking. (426) Orbital tracking exhibits several distinct advantages compared with image-based approaches. First of all, it has a very high spatial and temporal resolution (2–50 nm and 1–32 ms). Second, the fast Fourier transform as the localization algorithm is not sensitive to background noise and a single particle can be tracked in a scattering environment, although with a slightly lower precision. The main drawback of the orbital tracking approach is that it is not suitable to simultaneously tracking multiple particles in live cells. When particles are moving slowly, multiplexing can be performed, but in general, the approach only tracks one particle at one time. Thus, this approach is not convenient for collecting a high amount of statistics for rare events. On the other hand, as one is following the particle, one has the ability to measure various properties of that particle (e.g., spectra or lifetime (427)) or to photoactivate or manipulate that particle. In addition, the trajectory is written directly to disk in real time, which allows one to skip analysis steps 6.1 and 6.2 in the following section.
Figure 25
Figure 25. 3D orbital tracking. (a) Light microscopy transmission image of a zebrafish embryo and a zoom in on the tail with a typical Rohon–Beard neuron labeled by a membrane-targeted fluorescent protein (shown in yellow). (scale bar, 200 μm). (b) Schematic of the custom-built 3D real-time orbital tracking microscope consisting of a laser scanning confocal modality for tracking and a wide-field modality for simultaneous environmental observation. (c) (Upper image) Confocal reconstruction of a sensory neuron where both the membrane and the individual mitochondria are fluorescently labeled (scale bar, 100 μm). (Lower Image) Photoactivation of a single PA-GFP-labeled axonal mitochondrion (in yellow) (scale bar, 5 mm). (d) Schematic representation of the 3D orbital tracking approach. Different particle locations are indicated through spheres of varying color. Depending on the location of the particle, the phase and modulation of the signal vary. (e) Trajectory of an anterograde moving mitochondrion (100 Hz, 20,000 data points). (f) Autocorrelation carpet (top) of the angle between consecutive orbits and segregation of the trajectory into regions of directed transport (green) and stationary phases (red). Adapted with permission from ref (425). Copyright 2019 ELife Sciences Publications.
6. Data Analysis
Click to copy section linkSection link copied!
The goal of SVT experiments is to quantitatively assess the dynamic properties of viruses during the infection process. After being able to fluorescently label the molecules and follow them using various SVT methods, the crucial procedure becomes how to extract the dynamic information from a time-series of diffraction-limited images of viruses acquired by fluorescence microscopy. For data analysis, the essential steps are to obtain the localization of each particle with subdiffraction resolution and then reconstruct single-virus trajectories by connecting the particle positions in each frame (Figure 26). (428−430) In this part, several advanced algorithms have been developed to perform precise and accurate localization and unbiased tracking. (365,431,432)
Figure 26
Figure 26. Schematic representation of single-virus tracking. (a) Time-series of images acquired using fluorescence microscopy. The optical spatial resolution is about 250 nm in the lateral direction and about 500 nm in the axial direction, although the localization of the particle can be determined with much higher precision. (b) Steps in SVT analysis. From the collected images, the location of the different particles is first determined, and then the same particle needs to be linked through the different frames. Once the trajectories have been determined, various analyses can be performed such as a mean-squared-displacement analysis.
Once the trajectories have been determined, various analyses can be performed to uncover the underlying biological mechanisms in live cells. Generally, to interpret and classify the dynamics of viral behavior, the mean-square displacement (MSD) curves are calculated and fitted to models according to different types of movements. (270,433) The dynamic parameters obtained from curve fitting can be used to classify the types of the motional behaviors. As the type of motional behavior can also change, the instantaneous dynamic parameters (such as instantaneous speed and intensity) are also considered. (269)
6.1. Particle Detection
In SVT experiments, the raw data obtained by the various microscopy methods is typically a time series of diffraction-limited images containing fluorescent particles. In each frame of the series, the blurred spots denote fluorescent particles. Upon detection of the individual particles, we would like to determine the location of the particle with high precision. With SPT, we typically know that the detected signal is coming from a single object. Hence, it is sufficient to locate the center of the diffracted limited spot or PSF, which can be done to a much higher precision than the width of PSF or diffraction limited resolution. To locate the center position, it is first important to understand the intensity profile of the PSF. The intensity of each particle is given by the convolution of the particle shape with the PSF. The 3D diffraction pattern of single particles can be described as a 3D PSF from the Born–Wolf model. (434)
where 
, 
, 
, a/f = NA/n, ρ = r/a. a, f, and r are the radius of the exit pupil, the focal distance of the objective, and the radial coordinate, respectively, n is the refractive index of the medium, λ is the wavelength of light, NA is the numerical aperture, J0(vρ) is the Bessel function of zero order, and A is the amplitude. (435) Various algorithms have been developed to detect the particle center, which can be divided into two categories: geometric-based methods and PSF fitting methods.
, , , a/f = NA/n, ρ = r/a. a, f, and r are the radius of the exit pupil, the focal distance of the objective, and the radial coordinate, respectively, n is the refractive index of the medium, λ is the wavelength of light, NA is the numerical aperture, J0(vρ) is the Bessel function of zero order, and A is the amplitude. (435) Various algorithms have been developed to detect the particle center, which can be divided into two categories: geometric-based methods and PSF fitting methods.The simple and most straightforward method is the centroid method, which finds the particle positions by calculating the intensity centroid of the bright region. It can be written as (50)
where xjik is the coordinate of the pixel in the x direction and Iijk is the intensity at the pixel (xijk, yijk, zijk). The formulas for the y and z centroid positions are analogous.
This method has the advantage that it is fast and easy to program. However, the obvious disadvantage of this method is that the localization accuracy is very sensitive to the background noise and the area selected region for the calculation.
Since the intensity of an object taken by a microscope can be described by PSF, PSF-fitting methods are widely used for particle localization. Hereinto, the Gaussian model is commonly used, since the Airy disk can be well approximated by the Gaussian distribution. In SVT, the most common fitting method is the Gaussian nonlinear least-squares fitting method. This method utilizes the iterative fitting to search for the parameters, which can minimize the weighted square error between the fit and the data. (59)
where (xf,yf, zf) is the actual position of the particle. Sx, Sy and Sz are the widths of PSF in the x, y, and z directions, respectively. A and B are the amplitude and background noise, respectively. (436)
The Gaussian nonlinear least-squares fitting method possesses very high localization accuracy and has been widely used in localization-based super-resolution techniques such as STORM and PALM. (432,437) However, this approach is very computationally expensive and not well suited for the large data volumes analyzed in 3D SPT. In addition, initial values are required to be set before Gaussian fitting, which can be very tedious, and inappropriate values can lead to a local minimum or, in the worst case, cause the calculation to crash.
The radial symmetry approach, as a new-type geometric-based method, has been proposed for localizing particle centers with subpixel resolution. (435,438) This method utilizes the fact that the gradient line at each pixel surrounding the signal from a single particle in a 2D or 3D image should theoretically intersect the true center of the fluorescent particle. Considering the influence of background noise and optical aberrations, the gradient lines do not intersect and the center of the particle is estimated by selecting the point that minimizes the distance to all gradient lines. This method has no need for iterative, numerical fitting steps, so the computation speed of the method is about 2 orders of magnitude faster than that of the Gaussian fitting method and similar to that of the centroid method. Also, the radial symmetry approach has low edge sensitivity and subpixel accuracy, similar to that of the Gaussian nonlinear least-squares fitting method (Figure 27). (435) These features make our algorithm an intensely competitive method for 3D SPT applications.
Figure 27
Figure 27. Detection of the accurate position of particles with subdiffraction resolution. (a) Image generated by sampling the point spread function (PSF) of wide-field microscopy on a 3D grid with a lattice size of 20 nm. (b) 3D CCD image simulated from the PSF image (a) with a signal-to-noise ratio of 20. The blue crosses indicate the true center of the 3D particle. (c) 3D scatter plots of the errors illustrating the error ranges of the centroid (green), Gaussian fitting (blue), and radial symmetry algorithm (red), respectively. Adapted with permission from ref (435). Copyright 2013 Springer Nature.
6.2. Particle Linking
Once particles have been detected in the individual frames of a movie, the next step is to connect the localizations coming from the same particle from frame to frame and thereby reconstruct the particle trajectory. For data collected at low particle density, the nearest-neighbor algorithm is frequently used to identify localizations coming from the same particle in different frames. For each particle, the distances to all positions in the next frame are calculated and the minimum distance is taken as the most likely position for the same particle in the successive frame. These two positions are linked together. This procedure is repeated to link positions for as many particles in as many frames as possible and thereby obtain the entire trajectories for all the particles. (439) However, to achieve this last step, the tracking algorithm has to overcome a lot of difficulties such as temporary particle disappearance, particle merging, and particle splitting. Thus, the results acquired from this algorithm are not very reliable and usually require additional input. (440,441) In addition, it is always advisible to visually check the calculated trajectories and to manually correct them if necessary.
At high particle density, the occurrence of the interactions between particles greatly increases the difficulty of particle linking and makes the nearing-neighbor algorithms unsuitable for automatically reconstructing trajectories. Many algorithms have been developed to address tracking at high density and deal with the challenges caused by particle disappearance, merging, splitting, etc. Multiple-hypothesis tracking (MHT) is one of the most accurate methods to solve these problems. (442) All the trajectories are simultaneously constructed for the entire movie. An optimization strategy is used to select the largest nonconflicting ensemble of trajectories where no two trajectories share the same position in any frame. This method is globally optimal in both time and space, but algorithms with high computational efficiency are required to just track a few tens of particles in a few tens of frames simultaneously. Therefore, several schemes have been developed to approximate the MHT solution. (53,443−445) It is worth mentioning that Jaqaman et al. have provided a robust tracking algorithm for SPT under high-density conditions that can solve all the challenges mentioned above with high accuracy and computational feasibility. Based on one mathematical framework, this method initially links the detected positions in successive frames and then connects track segments to close the gap and detect merge and split events (Figure 28). (446)
Figure 28
Figure 28. CD36 receptor aggregation activity depending on motion type. (a) Epifluorescence image of a macrophage where CD36 has been immunolabeled using a primary Fab fragment followed by a Cy3-conjugated secondary Fab fragment. (b) CD36 tracks in a control macrophage. (c and d) CD36 tracks in (c) a blebbistatin-treated and (d) a nocodazole-treated macrophage. Scale bars, 1 μm. (e) Bar plot showing the fraction of particles undergoing linear motion. (f) x coordinate, y coordinate, and amplitude of two sample trajectories as a function of time where merging events (green ovals), splitting events (purple ovals), and closed gaps (orange ovals) have been highlighted. Adapted with permission from ref (446). Copyright 2008 Springer Nature.
6.3. Trajectory Analysis
Once the individual trajectories have been collected, one can begin to extract the wealth of information such measurements provided. Some analyses are straightforward such as looking at colocalization and interactions between different cellular or viral components, or measuring the kinetics of viral assembly via the increase and saturation in fluorescence intensity. As mention in section 3.1.2, fluorescence intensity can also be used as a reporter of membrane fusion, especially when lipophilic dye-labeled viruses are used. Thus, the intensity vs time plot displays a distinct increase after fusion events. (68)
Other analyses can be performed to gain information with respect to the motional behavior of the viruses or viral components. The diffusion coefficient is determined by analyzing the mean square displacement (MSD), which is a measure of the spatial extent of random motion. The MSD is defined as
where r(Δt) is the particle position at the time point of Δt, Δt is the acquisition time of each frame, N is the total frames for the particle trajectory, and n and i are integers. (269,447,448)
How the MSD depends on the time lag (nΔt) depends on the motional behavior of the particle. For Brownian motion, the MSD depends linearly on the time lag: (12,449)
where d is the space dimensionality and D is the diffusion coefficient. Hence, the diffusion coefficient can be determined from the slope of the MSD plot.
Pure random or Brownian motion is the simplest type of stochastic process. However, the cell is a crowded heterogeneous environment where many interactions can potentially happen. (12,17) In such a heterogeneous environment, particles often display anomalous diffusion. The relationship between MSD and nΔt for anomalous diffusion is given by
where α is anomalous diffusion exponent (α < 1).
Another type of motion that is often observed in live cells is confined diffusion. In confined diffusion, there is Brownian motion inside an enclosed environment from which the particle cannot easily escape. The dependence of MSD vs nΔt is related to the shape of the confined region and the spatial dimensionality. The formula of this type of motion in 2D for confinement in a square is given by
where L is the length of the side of the square. (269,450,451)
In live cells, viruses are often transported along the cytoskeleton. The cytoskeleton, such as microfilaments and microtubules, is the highway of the cells. Several molecular motors walk along the cytoskeletal filaments and transport their cargo. For directed motion with a diffusional component, the MSD is given by
where V is the constant velocity for directed transport. (59) By fitting the MSD, the type of diffusional behavior can be determined as well other relevant information such as the diffusion coefficient, the velocity of directed transport, the size of confinement, or the degree of deviation from Brownian motion.
The MSD analysis is a statistical analysis and thereby very powerful but assumes the motional behavior is constant during the measurement. Often, viruses change their motional behavior, for example from 2D diffusion on the cell membrane to directed transport and then to 3D anomalous diffusion within the cytosol. In cases like this, the instantaneous speed of the particle is a useful way to characterize the transport behavior of the particle. It can clearly identify the obvious periods of directed transport of the particle and provide information on the types of motors involved in the transport process. Research has reported that the movements related to actin filaments have lower instantaneous speed in the range of 0.1–0.4 μm/s, and the instantaneous speeds associated with microtubules are one to several μm/s in live cells. (97,130,452,453) The particles may experience several different motional modes in one trajectory. By means of the dependence of instantaneous speed vs time, the different motions from a trajectory can be distinguished and analyzed separately.
7. Viral Infection Mechanisms Revealed by Single-Virus Tracking
Click to copy section linkSection link copied!
In the section below, we describe the current state of various applications of SVT in virology and elaborate the representative studies in detail to illustrate what information can be acquired and how SVT can be applied experimentally. The lifecycle of viruses, as obligatory intracellular parasites, is strongly coupled to interactions with the host cell. There are a number of processes that are needed for viral replication. The first encounter between viruses and host cells usually occurs via the attachment factors or receptors (such as a protein, an oligosaccharide, or a glycolipid, etc.) exposed on the cell surface. To overcome the barrier of the plasma membrane, numerous viruses hijack the endocytic pathway of host cells for internalization. Viruses are then trapped into membrane vesicles and transported to special regions for genome release. After the replication of viral components, new viruses are assembly and eventually egress to look for new cells to invade. (454,455) There is an extensive amount of excellent work in the literature, and it is not possible to highlight all articles that have made significant contributions to the field or deserve special attention.
7.1. Virus-Receptor Interactions
For virus infection, viruses primarily binding nonspecifically to attachment factors on the cell surface and migrate along the cell surface until they recognize and bind to the specific receptors. The virus-receptor binding will activate the downstream signaling and trigger the internalization of viruses. (3,5,454,456) The detailed processes of how viruses move on the cell surface to bind specific receptors is difficult to explore by conventional biochemical approaches. SVT can directly provide detailed kinetic information on this process and such studies have revolutionized our understanding of virus–receptor interactions. (59,457)
A recent study has highlighted the vital role of filopodia during virus infection. (458) Filopodia are actin-rich bundles protruding from the plasma membranes and participate in probing the extracellular environment, promote cell motility, and facilitate cell-to-cell interactions. More and more pieces of research reported that filopodia were exploited during the initial step of virus infection and visualized movements of different viruses along filopodia. (71,132,137,139,208,212,216) By tracking individual murine leukemia viruses (MLVs), the researchers found that MLV initially attached to the filopodia and underwent a rapid and actin-dependent lateral movement along the filopodia toward the cell body (Figure 29). (208) Meanwhile, by monitoring the lateral motility of HPV on the cell surface, four modes of HPV mobility were discovered, and only the directed movement along actin protrusions (such as filopodia or retraction fibers) enhanced HPV infection. (137) All these findings support the fact that viruses must bind to cellular receptors for promoting internalization into the cell body.
Figure 29
Figure 29. Virus cell surfing along filopodia. (a) Individual murine leukemia virus (MLV) labeled with YFP (red) surfing along the filopodium of a HEK 293 cell transduced with mCAT-1-CFP (green). The time in the upper righthand corner is given in seconds, and the motion from two particles is summarized as white arrows in the right-most panel. (b) Image summarizing the overall movement of selected particles on filopodia of a single HEK 293 cell. (c) Thirty-one frames from a recorded movie superimposed to show the transport and photobleaching of MLVs during the SVT experiment (moving particles are highlighted in white). (d) To quantify virus cell surfing, the motility of 85 individual MLV particles was plotted over time where time point 0 represents the moment the virus attaches to a filopodium. Adapted with permission from ref (208). Copyright 2005 The Rockefeller University Press.
In addition, recent studies implied that viruses have different receptors for viral entry depending on cell types. The simultaneous tracking of ASLV and different receptors revealed that the infection efficiency was closely associated with the type of receptors the viruses bound to. Binding to lipid-anchored receptors may result in virus–cell membrane fusion where the genome is released into the cytosol. However, the transmembrane receptor accelerated virus uptake and provided a delay of the virus–endosome fusion event. (155) As another example, sialic acids act as receptors for many viruses, including human and avian influenza viruses. Human and avian influenza viruses preferentially bind to sialic acid linked to galactose by α-2,6 linkage and α-2,3 linkage, respectively. (459,460) The binding specificity of sialic acid receptors is an important barrier in cross-species transmission. SVT was utilized to explore the dynamic mechanisms of different receptors in live cells. These observations indicated that the infection behavior of viruses was determined by the transport behavior of the sialic acid receptors. (112)
7.2. Virus Internalization
To infect and replicate, viruses must enter the intracellular environment of the host cells. After viruses bind to the receptors on the cell surface, there are mainly two strategies for virus internalization: endocytosis-independent and endocytosis-dependent internalization. In the first strategy, viruses bind to the receptors on the cell surface and then directly fuse with the plasma membrane to enter the cell. It has been known that certain enveloped viruses, such as herpes simplex virus (HSV) and Sendai virus, are internalized into cells by fusing with the plasma membrane directly at neutral pH. (454,461,462) It is considered an effective way for enveloped viruses to deliver viral genome into the cytosol. In this process, viruses first bind the specific receptors on the cell surface, which then promote the virus–membrane fusion event. On the other hand, endocytosis is hijacked by most viruses to enter the cells, which may occur via several different mechanisms, such as clathrin-mediated, caveolae-mediated, clathrin- and caveolae-independent endocytosis, and macropinocytosis. (456,463,464)
In the past, researchers investigated the endocytosis-dependent uptake of viruses by using conventional techniques such as TEM and inhibition experiments. A detailed understanding of the entry mechanisms and the dynamic processes involved in viral internalization is still lacking for most viruses. SVT has greatly contributed to our understanding of viral entry mechanisms in particular when combined with multicolor live-cell imaging. For example, by visualizing the interaction between the capsid of CPV and transferrin receptors on the cell surfaces, it became apparent that the interactions facilitate trapping of the viral capsid into clathrin-mediated endocytic structures by a rapid diffusion-based mechanism (Figure 30). (142) By tracking the infection behaviors of influenza virus in living cells, the entry of influenza virus followed two distinct pathways: a clathrin-mediated pathway, and a clathrin- and caveolae-independent pathway. (72) Simultaneous tracking of viruses and clathrin-coated pits indicate that most viruses promote the de novo formation of clathrin-coated pits around the viruses and exploit the clathrin-mediated pathway for virus internalization. In parallel, the remaining viruses enter the host cells by a clathrin- and caveolin-independent endocytic pathway with similar efficiency. Through the application of QD-based SVT and multicolor imaging, Pang’s lab revealed that dynamin is involved in the clathrin-mediated pathway of influenza virus uptake and participates in the maturation and membrane fission of clathrin-coated pits in this pathway. (114) Macropinocytosis is usually considered as a nonspecific mechanism for virus entry. Several kinds of viruses have been reported to utilize macropinocytosis to infect the cells such as VV, HIV, and HSV. (462)
Figure 30
Figure 30. Real-time imaging of clathrin-dependent CPV internalization. (a) Example of clathrin-dependent CPV endocytosis. Left panels, CRFK σ2-eGFP cells (green, AP-2) were inoculated with fluorescent capsids (red) and imaged as before. Right panel, diffusion path of the capsid shown at left. A color-coded line trace of the capsid diffusion path is overlaid onto the t = 51-s image. (b) Plot of the background-corrected AP-2 (green) and capsid (red) fluorescence intensities with respect to time for the event in panel a. For frames prior to pit initiation, the AP-2 fluorescence intensity was quantified at the eventual site of pit initiation. (c) Efficiency of clathrin-dependent CPV entry. (d) Examples of CPV dissociation from the cell surface. Time-lapse images showing the attachment (downward-facing arrows) of two capsids (red; no. 1, no. 2) and subsequent capsid dissociation (upward-facing arrows). (e) Residence time of CPV capsids that dissociated from CRFK cells. Adapted with permission from ref (142). Copyright 2012 American Society for Microbiology.
7.3. Virus Transport
As many viruses enter cells via an endocytosis pathway, they find themselves in intracellular vesicles and are transported to specific sites in the cell for genome release and replication. Endosomal transport along the cytoskeleton is assisted by several molecular motors including myosin, dynein, and kinesin. As a result, viral transport is a complex and multistep process, and viruses often follow several different pathways in the cytosol. With SVT, it is possible to monitor individual viruses in real time and dissect the infection behavior of each virus in living cells and thereby provide new mechanistic insights into the infection pathway of viruses. HIV has long been assumed to fuse at the plasma membrane and release the genome directly into the cytosol. However, by monitoring the early phase of viral infection, new evidence arose suggesting that the virus fused with endosomes in the cytosol of cells after receptor-mediated internalization (Figure 31). (83) Furthermore, Cui et al. demonstrated that HIV was endocytosed and translocated into endosomes in a clathrin- and actin-dependent manner in macrophages and the viral core was released into the cytoplasm by endosomal fusion. (157)
Figure 31
Figure 31. Identification of HIV-1 fusion sites by single-virus imaging. (a) Schematic presentation of redistribution of viral lipid and content markers upon fusion with a plasma membrane (left) and with an endosome (right). Viruses colabeled with membrane (red) and content (green) markers are pseudocolored yellow. (b and c) Partial fusion of JRFL with the plasma membrane of TZM-bl cells. The time from the beginning of imaging is shown. The two-dimensional projection of the particle’s trajectory (cyan) is overlaid on the last image. Changes in fluorescence intensities (in arbitrary units) of membrane (red) and content (green) markers, as well as the instantaneous velocity (blue trace) of the particle, are shown. Adapted with permission from ref (83). Copyright 2009 Elsevier.
There is plenty of evidence from SVT experiments that many viruses are transported in the cytosol in an actin filament- and microtubule-dependent manner, and microtubules assist viruses to move from the plasma membrane to the perinuclear region. This phenomenon has been reported for several kinds of viruses, including influenza virus, (68,108) DENV, (75,162) and adenovirus. (70,465) Viral transportation is a highly regulated process in live cells. For example, SVT results indicated that influenza viruses follow a five-stage process during trafficking inside the cell: viruses attach to the cell surface initially, move slowly along the actin filaments in the cell periphery, and travel rapidly toward the cell nucleus in a microtubule-dependent manner, followed by interacting and fusing with late endosomes for genome release. (108) Furthermore, the transport behavior of influenza viruses in the cytosol could be influenced by the microtubule structures. The microtubule configuration influences the destiny of individual viruses during viral transport in living cells. (110) QDs-based SVT and multicolor imaging illustrated that the retrograde motor proteins, myosin VI and dynein, were responsible for the seamless transport of viruses from microfilaments to microtubules during virus transport. (118) 3D tracking of influenza virus demonstrated that the distinct transport behaviors of viruses were associated with early and late endosomes, and the transition from early to late endosomes occurred in the perinuclear region. (111) Multicolor tracking of individual viruses and autophagosome provided an unambiguous dissection of the autophagic trafficking of viruses. Roughly one-fifth of the viruses found to enter the host cells did so through the autophagic pathway. The results provided dynamic and distinct insights into the relationship between autophagy and virus entry. (115)
7.4. Fusion and Genome Delivery of Viruses
After viruses are internalized into cells, the viral genome needs to be released into the cytosol for replication. Enveloped viruses usually enter cells via endocytosis, which has the advantage that they can easily be transported toward the nucleus, but they need to find a way to escape the endosomes. The fusion proteins of most viruses are commonly activated under acidic pH. For instance, in late endosomes, the acidic environment induces a conformational change in the hemagglutinin, a fusion protein of influenza viruses, which mediates virus–endosome fusion for genome release. (466) Of course, not all viruses undergo pH-dependent release; nonenveloped viruses can escape the endosome for genome release through a fusion-independent manner and even for enveloped viruses. There are several pH-independent pathways and the molecular mechanisms underlying this portion of the virus life cycle remain only partially understood.
Some viruses fuse directly at the plasma membrane or can fuse either with the plasma membrane or with endosomes. Prototype foamy virus is thought to enter via both pathways. Using a dual-labeled variant of PFV, Lindemann and co-workers could visualize the fusion event in real time. Here, the envelop protein was labeled with mCherry and the capsid with eGFP. Before fusion, the virus was observable in both the eGFP and mCherry channels. Upon fusion, the eGFP from the capsid was observed to separate from the mCherry signal and was then transported into the cytoplasm (Figure 32). (467)
Figure 32
Figure 32. Fusion of PFV. (a) Bright field image of a cell overlaid with the trajectory of a dual-color PFV virus during fusion at or near the plasma membrane. The dual-color signal is shown in yellow and the single-labeled capsid in green. (b) Tracking image correlation (TrIC) analysis of the fusion event observed in panel (a). (upper panel) The fluorescence intensity of the Gag-eGFP signal (green) and mCherry-Env (red) signal, (second panel) instantaneous velocity, (third panel) cross-correlation amplitude (blue) and randomized cross-correlation signal (black), and (lower panel) relative distance between the Gag-eGFP and mCherry-Env signals plotted as a function of time. (c) 3D relative trajectory of the Env-labeled envelop with respect to the Gag-capsid showing movement in the order of hundreds of nanometers between the two labels before the completion of the fusion process. Adapted with permission from ref (467). Copyright 2013 Elsevier.
By virtue of multicolor labeled HIV viruses, Melikyan et al. found that complete viral fusion occurred in endosomes and not at the plasma membrane. (83) By labeling HIV integrase, Charneau and co-workers showed that cytosolic HIV complexes moved directly toward the cell nucleus in a microtubule and actin-dependent manner, then docked near the nuclear membrane, and eventually diffused inside the nucleus. (87) In the case of PV, time-lapse imaging analysis indicated that the virus enters the host cell via a tyrosine- and actin-dependent endocytic pathway, and the genome release of PV occurs in the endosomes near the plasma membrane. (131) This result settled a long-lasting debate regarding where PV releases its genome.
SVT measurements of the dynamic uncoating of individual influenza viruses indicated that approximately 30% of influenza virus particles undergo uncoating through fusion with late endosomes. After viral fusion and uncoating, vRNPs are released separately into the cytosol and, following a three-stage transport process, reach the vicinity of the cell nucleus. Visualization of the intranuclear dynamic behavior of the vRNPs revealed two diffusion patterns within the nucleus. Thus, SVT significantly contributed to our understanding of the uncoating and vRNP trafficking mechanisms of influenza virus (Figure 33) (113) in particular and is helping to disperse the enigma of genome delivery in general.
Figure 33
Figure 33. Real-time imaging of vRNP of influenza A viruses (IAV) release from a Rab7-positive endosome. (a) A fluorescence movie of a cell containing QD625-labeled influenza viruses (red) and Rab7-ECFP labeled endosomes (cyan) was recorded and SVT was performed. (b) Trajectories of the QD-labeled influenza virus colocalizing with the fluorescently labeled endosome from panel a. (c) Fluorescence image of an infected cell treated with NH4Cl. (d) Trajectories of the QD625 and ECFP fluorescent signals in NH4Cl-treated cells. (e) Model for IAV uncoating and vRNP dynamics. An IAV virion enters the host cell via endocytosis. Individual vRNPs finally undergo a three-stage active transport process to arrive at the cell nucleus and display two diffusion patterns within the nucleus. Adapted with permission from ref (113). Copyright 2019 National Academy of Sciences, U.S.A.
Furthermore, the viral genome of influenza virus is generally replicated in the cell nucleus and exported to the cytosol as vRNPs and transported to the plasma membrane for virus egress with an uncertainty. (468,469) Live-cell tracking of fluorescent vRNPs showed that the cytoplasmic vRNPs accumulated in the recycling endosome vesicles with a Rab11 and microtubule-dependent manner (470,471) and in the vRNP/Rab11 hotspots.
7.5. Assembly and Egress of Viruses
After genome replication and protein synthesis, the goal of virus infection is to assemble and release progeny virus particles. The newly synthesized components are transported to a specific site for virus assembly, and the progeny viruses can be released from the host cells by a variety of mechanisms such as exocytosis, lysis of the cell, or budding from the plasma membrane. Compared to virus entry and transport processes, it is more challenging to investigate the dynamic mechanisms of virus assembly and egress since it is hard to label newly synthesized viral components with fluorescence. Here, FP-labeling of individual components is powerful and, with labeled constructs, fluorescence fluctuation spectroscopy can play an important role where the motional behavior of a small number of proteins can be analyzed to gain insights into mobility and interactions between viral components or between virus and cellular components. Hendrix and co-workers used a number of correlation methods to elucidate the early cytosolic steps in HIV assembly. (472) Nevertheless, there are also situations where SVT can be used to elucidate the molecular location, dynamics, and mechanisms during viral assembly and egress. (473)
Many enveloped viruses such as HIV are assembled and released from the plasma membrane of the host cell by the abscission of the viral envelope. (474) The Gag proteins of HIV are necessary for the assembly and release of virus particles. Time-lapse imaging showed that HIV Gag is initially distributed in the perinuclear region of the cytosol and then travels to the plasma membrane. (475) Using the photon counting histogram approach as well as a quantitative PALM analysis, the size and density of the HIV-Gag cluster during the assembly of virions could be calculated. (476−478) To investigate the dynamic process of HIV genome packaging, HIV RNA was labeled with a photoconvertible Eos protein. TIRF imaging results indicated that the presence of the Gag protein distinctly increased the dwell time of HIV RNA near the plasma membrane. (479) Furthermore, simultaneous measurement of F-actin and HIV particles revealed that there is no characteristic pattern nor transient recruitment of F-actin during the viral budding process. The result demonstrated that the actin filaments-dependent transport pathway was dispensable for HIV Gag assembly on the plasma membrane. (480) By combining wide-field and TIRF microscopy, the assembly of the HIV protein shell was observed within ∼8–9 min after nucleation of an assembly site and virus particles were formed individually. HIV release occurred ∼25 min after nucleation of the viral assembly site. (370) Moreover, the transient recruitment of the ATPase VPS4 (vacuolar protein sorting 4) to nascent HIV particles at HIV budding sites on the host cell plasma membrane was observed before viral release, indicating that VPS4A played a distinct role in membrane scission for HIV-1 release (Figure 34). (481)
Figure 34
Figure 34. Recruitment of VPS4A to HIV assembly sites. (a) Wide-field image and time projections (5,926 s) from a TIRFM image series exemplifying frequent colocalization of eGFP-VPS4A bursts (green) with nascent HIV particles (magenta). (b) TIRFM images of the assembly and release of a HIV particle (top panel, arrows) and the corresponding eGFP-VPS4A channel (bottom panel, arrows). (c) Number of VPS4A bursts detected within 528 s (200 frames) in the presence of the indicated HIV derivatives (left) and number of HIV budding sites detected (right). (d) Wide-field image and time-projected TIRFM image of cells coexpressing eGFP-VPS4A (green) and the nonbudding HIV late minus mutant (magenta). All scale bars, 800 nm. Adapted with permission from ref (481). Copyright 2011 Springer Nature.
7.6. Cell-to-Cell Transmission of Viruses
Upon release of new viruses, the life-cycle continues with the infection of a new host cell. One mechanism for infection is cell-to-cell transmission. Nascent viruses reach a new host cell through direct cell–cell contacts, a pathway for infection that has been estimated to be 100–1000 times more efficient than the spread by cell-free dissemination. (482,483) Significantly, this spread pathway is less susceptible to the external surroundings (e.g., neutralizing antibodies, antivirus drugs, etc.). Virological synapses, (86,205,484) cell filopodia, (208,212,458,485,486) and membrane nanotubes (487−490) are three typical physical connections between cells which dramatically augment cell-to-cell transmission of many viruses. By means of SVT, the cell-to-cell transmission process can be visualized and the underlying mechanisms further uncovered by the dynamic evidence.
For instance, many viruses including HIV, HSV, and human T-lymphotropic virus (HTLV) tend to establish virological synapses between infected donor cells and uninfected target cells through cell-to-cell transmission. (491−494) The direct translocation of HIV across the virological synapse was captured by continuous time-lapse monitoring of HIV. The dynamic information provided by quantitative, high-speed three-dimensional video microscopy indicated that HIVs dissemination between cells may be enhanced by virological synapse-mediated cell adhesion coupled with viral endocytosis. (486) Additionally, retroviruses could establish filopodial bridges by connecting infected cells with noninfected cells, and virus particles budding from infected cells would move along the outer surface of the filopodial bridge toward noninfected cells. (485) The whole process of transmission was visualized by SVT and clearly demonstrated that the cell filopodia could be exploited by retroviruses for the purpose of viral spread. Membrane nanotubes, as recently discovered membranous tethers between cells, facilitate direct intercellular communication, signaling, and the spread of pathogens. (96,489,495,496) Compared with filopodial bridges, membrane nanotubes usually span over longer distances, which means that these structures can aid the spread of virus rapidly and efficiently. The movements of GFP-fused recombinant HIVs on nanotubes were recorded, indicating that the viruses moved along the nanotubes from the infected toward uninfected cell. The process of HIVs transported along membrane nanotubes was receptor-dependent with a speed of 0.08 ± 0.03 μm/s. The results showed that membrane nanotubes presented a novel route for the rapid spread of HIV between T cells (Figure 35). (489)
Figure 35
Figure 35. Membrane nanotubes present a novel route for HIV-1 to spread between T cells. (a) Membrane nanotubes were formed after intercellular contact between an infected Jurkat T cell (red) and an uninfected Jurkat T cell. (b) The frequency of membrane nanotubes formed between uninfected and infected Jurkat T cells or between two populations of uninfected Jurkat T cells. (c) Time-lapse imaging of Gag-GFP (green), expressed in the context of the fully infectious virus, along a membrane nanotube connecting infected with uninfected Jurkat T cells (red). (d) The arrow indicates Gag-GFP within the cytoplasm of the initially uninfected T cell. (e) The position of Gag-GFP is plotted against time showing generally directed movement from uninfected to infected cells. Adapted with permission from ref (489). Copyright 2008 Springer Nature.
8. Conclusions and Perspectives
Click to copy section linkSection link copied!
Single-virus tracking is a powerful tool for investigating complex dynamic processes of viruses and has become a standard lab technique now in the field of virology. The evolution and development of SVT in the past decades shed light on the mechanisms involved in the life-cycle of a variety of viruses including virus internalization, virus transport, genome delivery, and virus assembly. As an interdisciplinary technique, SVT required consecutive efforts from experts in chemistry, biology, virology, and physics. With the development of new fluorescent tags, better labeling strategies, and novel optical instruments, SVT is continuously undergoing rapid expansion. In this review, we have given practical considerations of how to perform SVT experiments in live cells, including the choice of fluorescent tags, labeling strategies, imaging instrumentation, and data analysis, and have demonstrated the power of SVT by mentioning a few applications.
SVT has been established nearly two decades ago, and this booming technique shows no signs of slowing down. However, there are still several issues that need to be ironed out for SVT in the future. First of all, the sizes of different kinds of viruses (10–300 nm) vary largely and fluorescent tags may impair the virus infectivity in varying degrees. To reduce the influence of fluorescent labeling, only a limited amount of fluorescent tags can be used to attach to the viral components. New organic dyes and FPs with better photophysical properties are being developed and will contribute to improve SVT. Even so, monitoring the viral infection processes continuously with high temporal resolution over the entire infection process remains a big challenge. QDs, with their excellent optical brightness and photostability, are a great option and have the potential of tracking the whole infection process of individual viruses in living cells. However, QDs also have some limitations such as their larger size, photoblinking, and potential interference with the functionality of the virus. Fortunately, in many cases, QDs have been shown to have minimal influence on the virus characteristics and do not affect the viral infectivity, although to what extent QDs labeling may affect the real behavior of viruses in living cells is unclear. SVT will certainly profit from the development of new-types of QDs with small size and nonblinking to circumvent these limitations. (497−499) Smaller QDs will also most likely minimize any deleterious effects of the labeling process.
Second, the process of virus infection involves a vast number of interactions between host cells and viruses. Interactions often occurred between a small number of viral and cellular molecules. One of the future challenges will be visualizing these interactions. Here, multicolor labeling will be necessary. One possibility, as discussed above, is fluorescence fluctuation spectroscopy. A second approach is super-resolution imaging, which has developed extensively over the past ten years and allows researchers to delineate the cellular processes on the nanoscale. (500) Super-resolution methods are commercially available and have pushed the optical resolution into the 20–100 nm range. (230,501,502) This opens the avenue for investigating the virus infection mechanisms within a subcellular environment and with subviral resolution thereby making it possible to uncover the underlying mechanisms of virus infection. New imaging techniques are still in demand to combine single-virus tracking and super-resolution microscopy for capturing and quantitative understanding of the fundamental processes involved in virus infection at nanometer spatial resolution.
Lastly, single-virus tracking is currently only limited to investigations of the infection mechanism of viruses in cultured cells, which is a simplified model system. In vivo experiments are needed to fully comprehend the mechanisms of virus infection. By tracking individual viruses in live tissue and animals, it may be possible to dissect the processes involved in the cell-to-cell transmission of viruses and learn how viruses break through the defense barriers of the host. To date, several groups reported the noninvasive visualization of viruses in mice, (503,504) but the real-time tracking of individual viruses in vivo still remains a challenge because of the limitations of current bioimaging techniques. Although optical microscopy has been a fundamental tool for biological researchers for more than three centuries, in vivo imaging is still restricted by light scattering. Recent advances in optical imaging techniques and in vivo microscopy allow images to be acquired at high resolution and unprecedented depths. (505,506) Additionally, the use of near-infrared fluorescent tags allows researchers to enhance tissue penetration of light and minimize the interference of tissue autofluorescence in vivo. (507−509) A series of near-infrared QDs were recently synthesized with an emission range from 800 to 1600 nm. (244,510,511,512) Of particular interest are the ultrasmall near-infrared QDs with a size of ∼2 nm and low toxicity generated via quasi biosynthesis, for example by utilizing the GSH enzymatic pathway. (245,246) Thus, enabled with these novel tools, new opportunities are becoming available for SVT to probe the dynamic interactions of viruses in vivo and thereby elucidate the mysteries of the infection processes.
Author Information
Click to copy section linkSection link copied!
- Dai-Wen Pang - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. China; College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P. R. China;
http://orcid.org/0000-0002-7017-5725;
- Shu-Lin Liu - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. China; Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430074, P. R. China;http://orcid.org/0000-0002-1043-4238
- Zhi-Gang Wang - State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, and School of Medicine, Nankai University, Tianjin 300071, P. R. China;http://orcid.org/0000-0002-3461-4014
- Hai-Yan Xie - School of Life Science, Beijing Institute of Technology, Beijing 100081, P. R. China;http://orcid.org/0000-0002-6330-7929
- Don C. Lamb - Physical Chemistry, Department of Chemistry, Center for Nanoscience (CeNS), and Center for Integrated Protein Science Munich (CIPSM) and Nanosystems Initiative Munich (NIM), Ludwig-Maximilians-Universität, München, 81377, Germany;http://orcid.org/0000-0002-0232-1903
S.-L.L. and Z.-G.W. contributed equally to this work.
Biographies
Click to copy section linkSection link copied!
Shu-Lin Liu
Shu-Lin Liu obtained her B.S. degree from Zhengzhou University (2007) and obtained her Ph.D. degree (2013) in Analytical Chemistry from Wuhan University. She worked as a postdoctoral researcher at University of Illinois at Chicago (2013–2017). After that, she worked as a professor at China University of Geosciences and now is a professor in Chemistry Department of Nankai University. Her research interests are mainly focused on single-virus tracking, lipid-mediated cell signaling, and developing new techniques for biological applications.
Zhi-Gang Wang
Zhi-Gang Wang received his B.S. degree (2005) and M.S. degree (2008) from the Department of Chemistry at Zhengzhou University. He obtained his Ph.D. degree (2014) in Analytical Chemistry from Wuhan University and finished his postdoctoral research at University of Illinois at Chicago during 2014–2017. Currently, he is an associate professor at School of Medicine, Nankai University. His main research interests include bioapplication of nanoparticles, single molecule/particle tracking, and signaling pathway and biosynthesis of QDs.
Hai-Yan Xie
Hai-Yan Xie is a professor in the School of Life Sciences, Beijing Institute of Technology, China. She received her Ph.D. in Analytical Chemistry from Wuhan University in 2004. From 2013 to 2014, she worked as a visiting scholar in Stanford University. Her research focuses on the interdisciplinary work on biorthogonal chemistry, nanotechnology, and biomimetic biology for disease diagnosis and treatments.
An-An Liu
An-An Liu received her bachelor’s degree from Nankai University in 2008 and her Ph.D. in analytical chemistry from Wuhan University in 2016. Then she carried out postdoctoral research on single molecule imaging at Kyoto University and Okinawa Institute of Science and Technology Graduate University in Japan. She joined Nankai University in 2019 as an assistant professor. Her research focuses on developing nanomaterials and nanoprobes for bioimaging.
Don C. Lamb
Don C. Lamb is Professor for Biophysical Chemistry at the LMU Munich. He obtained his Ph.D. degree from the University of Illinois at Urbana–Champaign and was a research fellow at the Harvard Medical School, an Alexander von Humboldt Research Fellow at the TU Munich, a member of the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana–Champaign, and a visiting scientist at the University of Ulm. His research focused on ultrasensitive fluorescence methods, advanced microscopy methods, protein function and dynamics, fluorescence fluctuation spectroscopies, live-cell imaging, single particle tracking, single virus tracing, and DNA nanodevices.
Dai-Wen Pang
Dai-Wen Pang graduated with a B.S. degree in chemistry from Wuhan University in 1982 and received his Ph.D. degree in electrochemistry from Wuhan University in 1992. He was a professor at Wuhan University from 1996 to 2018 and now is a distinguished professor at Nankai University. His research interest focuses on biomedical-used quantum dots (BioQDs, especially ultrasmall biocompatible NIR-fluorescent semiconductor nanocrystals), including space-time coupled living-cell synthesis of QDs, quasi-bio synthesis of QDs, single-virus tracking with QDs, photoluminescence mechanism of luminescent nanomaterials, and also backlight display with QDs. He was the Head of the Creative Research Group for Biomedical Probes of the National Natural Science Foundation of China (2006–2012) and 973 Chief Scientist appointed by the Ministry of Science and Technology of China for two projects of the National Key Scientific Program (2006–2015). He is now the Director of the Research Center for Analytical Sciences of Nankai University, member of the National Steering Committee for Nanotechnology, member of the Editorial Advisory Board of Analytical Chemistry, and Associate Editor for New Journal of Chemistry.
Acknowledgments
Click to copy section linkSection link copied!
This work was supported by the National Natural Science Foundation of China (Nos. 21535005, 21877102, 21977054, 91953107, and 21827808) and by the Ludwig-Maximilians-University Munich via the Center for NanoScience and the LMUInnovativ BioImaging Network.
References
Click to copy section linkSection link copied!
This article references 512 other publications.
- 1Smith, A. E.; Helenius, A. How Viruses Enter Animal Cells. Science 2004, 304, 237– 242, DOI: 10.1126/science.1094823Google Scholar1How Viruses Enter Animal CellsSmith, Alicia E.; Helenius, AriScience (Washington, DC, United States) (2004), 304 (5668), 237-242CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Viruses replicate within living cells and use the cellular machinery for the synthesis of their genome and other components. To gain access, they have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into the host cell. Many animal viruses take advantage of endocytic pathways and rely on the cell to guide them through a complex entry and uncoating program. In the dialogue between the cell and the intruder, the cell provides crit. cues that allow the virus to undergo mol. transformations that lead to successful internalization, intracellular transport, and uncoating.
- 2Greber, U. F.; Way, M. A Superhighway to Virus Infection. Cell 2006, 124, 741– 754, DOI: 10.1016/j.cell.2006.02.018Google Scholar2A superhighway to virus infectionGreber, Urs F.; Way, MichaelCell (Cambridge, MA, United States) (2006), 124 (4), 741-754CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Microtubule-mediated transport of macromols. and organelles (also known as "cargo") is essential for cells to function. Deficiencies in cytoplasmic transport are frequently assocd. with severe diseases and syndromes. Cytoplasmic transport also provides viruses with the means to reach their site of replication and is the route for newly assembled progeny to leave the infected cell. This parasitic relationship of viruses with the host cytoskeleton provides an excellent basis for cell biologists to unlock the secrets of cytoplasmic transport and unravel mechanisms of disease. Recent advances in live cell imaging and computational tracking of fluorescently labeled viruses are now revealing how complex the movements of single viruses are in infected cells. This review focuses on microtubule-based motility of viruses and highlights the mechanisms regulating cytoplasmic transport.
- 3Marsh, M.; Helenius, A. Virus Entry: Open Sesame. Cell 2006, 124, 729– 740, DOI: 10.1016/j.cell.2006.02.007Google Scholar3Virus entry: open sesameMarsh, Mark; Helenius, AriCell (Cambridge, MA, United States) (2006), 124 (4), 729-740CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biol. studies, live-cell imaging, and systems biol. have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addn., such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.
- 4Gruenberg, J. Viruses and Endosome Membrane Dynamics. Curr. Opin. Cell Biol. 2009, 21, 582– 588, DOI: 10.1016/j.ceb.2009.03.008Google Scholar4Viruses and endosome membrane dynamicsGruenberg, JeanCurrent Opinion in Cell Biology (2009), 21 (4), 582-588CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)A review. Cell surface mols., ligands, and solutes can be endocytosed into animal cells via several pathways in addn. to clathrin-mediated endocytosis, which all seem to lead to canonical endosomes. It seems that viruses can enter and infect cells through most of, if not all, endocytic routes, having evolved different, sometimes elaborate, strategies to (mis)use cellular machineries to their own benefit during infection. Here, the author discusses recent progress in understanding the pathways followed by animal viruses into cells, and how these studies are also providing novel insights into the understanding of some mol. mechanisms that control endocytic membrane transport.
- 5Sun, E.; He, J.; Zhuang, X. Live Cell Imaging of Viral Entry. Curr. Opin. Virol. 2013, 3, 34– 43, DOI: 10.1016/j.coviro.2013.01.005Google Scholar5Live cell imaging of viral entrySun, Eileen; He, Jiang; Zhuang, XiaoweiCurrent Opinion in Virology (2013), 3 (1), 34-43CODEN: COVUAF; ISSN:1879-6257. (Elsevier B. V.)A review. Viral entry encompasses the initial steps of infection starting from virion host cell attachment to viral genome release. Given the dynamic interactions between the virus and the host, many questions related to viral entry can be directly addressed by live cell imaging. Recent advances in fluorescent labeling of viral and cellular components, fluorescence microscopy with high sensitivity and spatiotemporal resoln., and image anal. enabled studies of a broad spectrum across many viral entry steps, including virus-receptor interactions, internalization, intracellular transport, genomic release, nuclear transport, and cell-to-cell transmission. Collectively, these live cell imaging studies have not only enriched our understandings of the viral entry mechanisms, but also provided novel insights into basic cellular biol. processes.
- 6Pelkmans, L.; Helenius, A. Insider Information: What Viruses Tell Us about Endocytosis. Curr. Opin. Cell Biol. 2003, 15, 414– 422, DOI: 10.1016/S0955-0674(03)00081-4Google Scholar6Insider information: what viruses tell us about endocytosisPelkmans, Lucas; Helenius, AriCurrent Opinion in Cell Biology (2003), 15 (4), 414-422CODEN: COCBE3; ISSN:0955-0674. (Elsevier Science Ltd.)A review. Viruses have long served as tools in mol. and cellular biol. to study a variety of complex cellular processes. Currently, there is a revived interest in virus entry into animal cells because it is evident that incoming viruses make use of numerous endocytic pathways that are otherwise difficult to study. Besides the classical clathrin-mediated uptake route, viruses use caveolae-mediated endocytosis, lipid-raft-mediated endocytic pathways, and macropinocytosis. Some of these are subject to regulation, involve novel endocytic organelles, and some of them connect organelles that were previously not known to communicate by membrane traffic.
- 7Conner, S. D.; Schmid, S. L. Regulated Portals of Entry into the Cell. Nature 2003, 422, 37– 44, DOI: 10.1038/nature01451Google Scholar7Regulated portals of entry into the cellConner, Sean D.; Schmid, Sandra L.Nature (London, United Kingdom) (2003), 422 (6927), 37-44CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)A review. The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. "Endocytosis" encompasses several diverse mechanisms by which cells internalize macromols. and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of mol. interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiol. and, thus, must be viewed in a broader context than simple vesicular trafficking.
- 8Horan, P. K.; Melnicoff, M. J.; Jensen, B. D.; Slezak, S. E. Fluorescent Cell Labeling for in Vivo and in Vitro Cell Tracking. Methods Cell Biol. 1990, 33, 469– 490, DOI: 10.1016/S0091-679X(08)60547-6Google Scholar8Fluorescent cell labeling for in vivo and in vitro cell trackingHoran P K; Melnicoff M J; Jensen B D; Slezak S EMethods in cell biology (1990), 33 (), 469-90 ISSN:0091-679X.There is no expanded citation for this reference.
- 9Giepmans, B. N. G. The Fluorescent Toolbox for Assessing Protein Location and Function. Science 2006, 312, 217– 224, DOI: 10.1126/science.1124618Google Scholar9The Fluorescent Toolbox for Assessing Protein Location and FunctionGiepmans, Ben N. G.; Adams, Stephen R.; Ellisman, Mark H.; Tsien, Roger Y.Science (Washington, DC, United States) (2006), 312 (5771), 217-224CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Advances in mol. biol., org. chem., and materials science have recently created several new classes of fluorescent probes for imaging in cell biol. Here the authors review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live vs. fixed cells: detn. of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small org. fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
- 10Alcor, D.; Gouzer, G.; Triller, A. Single-Particle Tracking Methods for the Study of Membrane Receptors Dynamics. Eur. J. Neurosci. 2009, 30, 987– 997, DOI: 10.1111/j.1460-9568.2009.06927.xGoogle Scholar10Single-particle tracking methods for the study of membrane receptors dynamicsAlcor Damien; Gouzer Geraldine; Triller AntoineThe European journal of neuroscience (2009), 30 (6), 987-97 ISSN:.Single-particle tracking (SPT) applications have been growing rapidly in the field of cell biology, and in particular in neurobiology, as a means of unravelling the involvement of diffusion dynamics of neurotransmitter receptors and other synaptic proteins in the regulation of neuronal activity. Suitable probes and technological improvements make SPT more accessible than it used to be and open up broad applications in cellular biology. In this technical highlight, we give an overview of the experimental approach in SPT. The concepts and results in neurobiology have already been the object of detailed reviews. Here, we focus on a qualitative description of the implementation of SPT, from molecule labelling to acquisition, data treatment and analysis of protein diffusion properties. Constraints, limitations and future developments are discussed.
- 11Cang, H.; Shan Xu, C.; Yang, H. Progress in Single-Molecule Tracking Spectroscopy. Chem. Phys. Lett. 2008, 457, 285– 291, DOI: 10.1016/j.cplett.2008.03.098Google Scholar11Progress in single-molecule tracking spectroscopyCang, Hu; Shan Xu, C.; Yang, HawChemical Physics Letters (2008), 457 (4-6), 285-291CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)A review; a topic of much current interest is the chem. reactivity in complex environments, involving dynamical processes spanning multiple scales in both time and space. While challenging to study using std. ensemble-averaged methods, it is amenable to investigations using single-mol. approaches. Ultimately, one would like to monitor the dynamics of individual mols. as they undergo chem. transformations through time-dependent spectroscopic studies, and to correlate various mol. properties with their immediate phys. and chem. environments. This article provides an overview of the recent advances toward this goal and describes future developments and potential applications.
- 12Levi, V.; Gratton, E. Exploring Dynamics in Living Cells by Tracking Single Particles. Cell Biochem. Biophys. 2007, 48, 1– 15, DOI: 10.1007/s12013-007-0010-0Google Scholar12Exploring dynamics in living cells by tracking single particlesLevi, Valeria; Gratton, EnricoCell Biochemistry and Biophysics (2007), 48 (1), 1-15CODEN: CBBIFV; ISSN:1085-9195. (Humana Press Inc.)A review. In the last years, significant advances in microscopy techniques and the introduction of a novel technol. to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biol. The authors' understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to the actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein mols. by following their way through the cell. In this review, the authors introduce the single particle tracking technol. to new users. The instrumentation is briefly described and some of the algorithms commonly used to locate and track particles explained. Also, the authors present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells.
- 13Shen, H.; Tauzin, L. J.; Baiyasi, R.; Wang, W.; Moringo, N.; Shuang, B.; Landes, C. F. Single Particle Tracking: From Theory to Biophysical Applications. Chem. Rev. 2017, 117, 7331– 7376, DOI: 10.1021/acs.chemrev.6b00815Google Scholar13Single Particle Tracking: From Theory to Biophysical ApplicationsShen, Hao; Tauzin, Lawrence J.; Baiyasi, Rashad; Wang, Wenxiao; Moringo, Nicholas; Shuang, Bo; Landes, Christy F.Chemical Reviews (Washington, DC, United States) (2017), 117 (11), 7331-7376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theor. background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biol. and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.
- 14Xia, T.; Li, N.; Fang, X. H. Single-Molecule Fluorescence Imaging in Living Cells. Annu. Rev. Phys. Chem. 2013, 64, 459– 480, DOI: 10.1146/annurev-physchem-040412-110127Google Scholar14Single-molecule fluorescence imaging in living cellsXia, Tie; Li, Nan; Fang, XiaohongAnnual Review of Physical Chemistry (2013), 64 (), 459-480CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. The transition of single-mol. fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomols. in their native environments and their roles in cellular processes. Here we review recent advances of single-mol. biophys. approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-mol. fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochem. parameters that can be obtained by imaging individually labeled biomols. in cells and what can be learned from such characterizations.
- 15Manzo, C.; Garcia-Parajo, M. F. A Review of Progress in Single Particle Tracking: from Methods to Biophysical Insights. Rep. Prog. Phys. 2015, 78, 124601, DOI: 10.1088/0034-4885/78/12/124601Google Scholar15A review of progress in single particle tracking: from methods to biophysical insightsManzo, Carlo; Garcia-Parajo, Maria F.Reports on Progress in Physics (2015), 78 (12), 124601/1-124601/29CODEN: RPPHAG; ISSN:1361-6633. (IOP Publishing Ltd.)Optical microscopy has for centuries been a key tool to study living cells with min. invasiveness. The advent of single mol. techniques over the past two decades has revolutionized the field of cell biol. by providing a more quant. picture of the complex and highly dynamic organization of living systems. Amongst these techniques, single particle tracking (SPT) has emerged as a powerful approach to study a variety of dynamic processes in life sciences. SPT provides access to single mol. behavior in the natural context of living cells, thereby allowing a complete statistical characterization of the system under study. In this review we describe the foundations of SPT together with novel optical implementations that nowadays allow the investigation of single mol. dynamic events with increasingly high spatiotemporal resoln. using mol. densities closer to physiol. expression levels. We outline some of the algorithms for the faithful reconstruction of SPT trajectories as well as data anal., and highlight biol. examples where the technique has provided novel insights into the role of diffusion regulating cellular function. The last part of the review concs. on different theor. models that describe anomalous transport behavior and ergodicity breaking obsd. from SPT studies in living cells.
- 16Miller, H.; Zhou, Z.; Shepherd, J.; Wollman, A. J. M.; Leake, M. C. Single-Molecule Techniques in Biophysics: A Review of the Progress in Methods and Applications. Rep. Prog. Phys. 2018, 81, 024601 DOI: 10.1088/1361-6633/aa8a02Google Scholar16Single-molecule techniques in biophysics: a review of the progress in methods and applicationsMiller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J. M.; Leake, Mark C.Reports on Progress in Physics (2018), 81 (2), 024601/1-024601/47CODEN: RPPHAG; ISSN:1361-6633. (IOP Publishing Ltd.)A review. Single-mol. biophysics has transformed our understanding of biol., but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equil., covering multiple lengths from the nanoscale of single mols. to up to several orders of magnitude higher in cells, tissues and organisms. Biomols. are often characterized by underlying instability: multiple metastable free energy states exist, sepd. by levels of just a few multiples of the thermal energy scale kBT, where kB is the Boltzmann const. and T abs. temp., implying complex inter-conversion kinetics in the relatively hot, wet environment of active biol. matter. A key benefit of single-mol. biophysics techniques is their ability to probe heterogeneity of free energy states across a mol. population, too challenging in general for conventional ensemble av. approaches. Parallel developments in exptl. and computational techniques have catalyzed the birth of multiplexed, correlative techniques to tackle previously intractable biol. questions. Exptl., progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent expts., including the underpinning mathematics. These methods are broadly divided into tools which detect mols. and those which manipulate them. For the former we discuss the progress of super-resoln. microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mech. perturb mols. We also consider in silico progress of single-mol. computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative at. force microscopy and fluorescence imaging, to probe questions closer to native physiol. behavior. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.
- 17Wieser, S.; Schutz, G. J. Tracking Single Molecules in the Live Cell Plasma Membrane-Do’s and Don’t’s. Methods 2008, 46, 131– 140, DOI: 10.1016/j.ymeth.2008.06.010Google Scholar17Tracking single molecules in the live cell plasma membrane-Do's and Don't'sWieser, Stefan; Schuetz, Gerhard J.Methods (Amsterdam, Netherlands) (2008), 46 (2), 131-140CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)A review. In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomol. within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomols. labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resoln. of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single mols. is state-of-the-art. The advances in the fields of microscopy brought new cell biol. questions into reach. The investigation of a single fluorescent mol.-or simultaneously of an ensemble of individual mols.-provides principally new information, which is generally hidden in ensemble-averaged signals of mols. In this paper we describe strategies how to make use of single mol. trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single mol. tracking. A diffusing membrane constituent-e.g. a protein or a lipid-experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently obsd. as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available anal. models and point out potential pitfalls, which may result in quant. or three even qual. misinterpretations.
- 18Kusumi, A.; Tsunoyama, T. A.; Hirosawa, K. M.; Kasai, R. S.; Fujiwara, T. K. Tracking Single Molecules at Work in Living Cells. Nat. Chem. Biol. 2014, 10, 524– 532, DOI: 10.1038/nchembio.1558Google Scholar18Tracking single molecules at work in living cellsKusumi, Akihiro; Tsunoyama, Taka A.; Hirosawa, Kohichiro M.; Kasai, Rinshi S.; Fujiwara, Takahiro K.Nature Chemical Biology (2014), 10 (7), 524-532CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Methods for imaging and tracking single mols. conjugated with fluorescent probes, called single-mol. tracking (SMT), are now providing researchers with the unprecedented ability to directly observe mol. behaviors and interactions in living cells. Current SMT methods are achieving almost the ultimate spatial precision and time resoln. for tracking single mols., detd. by the currently available dyes. In cells, various mol. interactions and reactions occur as stochastic and probabilistic processes. SMT provides an ideal way to directly track these processes by observing individual mols. at work in living cells, leading to totally new views of the biochem. and mol. processes used by cells whether in signal transduction, gene regulation or formation and disintegration of macromol. complexes. Here we review SMT methods, summarize the recent results obtained by SMT, including related superresoln. microscopy data, and describe the special concerns when SMT applications are shifted from the in vitro paradigms to living cells.
- 19Kusumi, A.; Shirai, Y. M.; Koyama-Honda, I.; Suzuki, K. G. N.; Fujiwara, T. K. Hierarchical Organization of the Plasma Membrane: Investigations by Single-Molecule Tracking vs. Fluorescence Correlation Spectroscopy. FEBS Lett. 2010, 584, 1814– 1823, DOI: 10.1016/j.febslet.2010.02.047Google Scholar19Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopyKusumi, Akihiro; Shirai, Yuki M.; Koyama-Honda, Ikuko; Suzuki, Kenichi G. N.; Fujiwara, Takahiro K.FEBS Letters (2010), 584 (9), 1814-1823CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Single-mol. tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a no. of unprecedented observations, thus fostering a new basic understanding of mol. diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, contg. diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane mols. stay together for limited durations. Mol. interactions occur in the time-dependent inhomogeneous two-dimensional liq. of the plasma membrane, which might be a key for plasma membrane functions.
- 20Kusumi, A.; Fujiwara, T. K.; Chadda, R.; Xie, M.; Tsunoyama, T. A.; Kalay, Z.; Kasai, R. S.; Suzuki, K. G. Dynamic Organizing Principles of the Plasma Membrane That Regulate Signal Transduction: Commemorating the Fortieth Anniversary of Singer and Nicolson’s Fluid-Mosaic Model. Annu. Rev. Cell Dev. Biol. 2012, 28, 215– 250, DOI: 10.1146/annurev-cellbio-100809-151736Google Scholar20Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic modelKusumi, Akihiro; Fujiwara, Takahiro K.; Chadda, Rahul; Xie, Min; Tsunoyama, Taka A.; Kalay, Ziya; Kasai, Rinshi S.; Suzuki, Kenichi G. N.Annual Review of Cell and Developmental Biology (2012), 28 (), 215-250CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)A review. The recent rapid accumulation of knowledge on the dynamics and structure of the plasma membrane has prompted major modifications of the textbook fluid-mosaic model. However, because the new data have been obtained in a variety of research contexts using various biol. paradigms, the impact of the crit. conceptual modifications on biomedical research and development has been limited. In this review, we try to synthesize our current biol., chem., and phys. knowledge about the plasma membrane to provide new fundamental organizing principles of this structure that underlie every mol. mechanism that realizes its functions. Special attention is paid to signal transduction function and the dynamic aspect of the organizing principles. We propose that the cooperative action of the hierarchical three-tiered mesoscale (2-300 nm) domains-actin-membrane-skeleton induced compartments (40-300 nm), raft domains (2-20 nm), and dynamic protein complex domains (3-10 nm)-is crit. for membrane function and distinguishes the plasma membrane from a classical Singer-Nicolson-type model.
- 21Suzuki, K. G.; Kasai, R. S.; Hirosawa, K. M.; Nemoto, Y. L.; Ishibashi, M.; Miwa, Y.; Fujiwara, T. K.; Kusumi, A. Transient GPI-Anchored Protein Homodimers Are Units for Raft Organization and Function. Nat. Chem. Biol. 2012, 8, 774– 783, DOI: 10.1038/nchembio.1028Google Scholar21Transient GPI-anchored protein homodimers are units for raft organization and functionSuzuki, Kenichi G. N.; Kasai, Rinshi S.; Hirosawa, Koichiro M.; Nemoto, Yuri L.; Ishibashi, Munenori; Miwa, Yoshihiro; Fujiwara, Takahiro K.; Kusumi, AkihiroNature Chemical Biology (2012), 8 (9), 774-783CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Advanced single-mol. fluorescent imaging was applied to study the dynamic organization of raft-assocd. glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (∼200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiol. expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts contg. up to four CD59 mols., which triggered intracellular Ca2+ responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains contg. GPI-APs.
- 22Zhang, W.; Jiang, Y.; Wang, Q.; Ma, X.; Xiao, Z.; Zuo, W.; Fang, X.; Chen, Y. G. Single-Molecule Imaging Reveals Transforming Growth Factor-Induced Type II Receptor Dimerization. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 15679– 15683, DOI: 10.1073/pnas.0908279106Google Scholar22Single-molecule imaging reveals transforming growth factor-β-induced type II receptor dimerizationZhang, Wei; Jiang, Yaxin; Wang, Qiang; Ma, Xinyong; Xiao, Zeyu; Zuo, Wei; Fang, Xiaohong; Chen, Ye-GuangProceedings of the National Academy of Sciences of the United States of America (2009), 106 (37), 15679-15683, S15679/1-S15679/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Transforming growth factor-β (TGF-β) elicits its signals through two transmembrane serine/threonine kinase receptors, type II (TOSRII) and type I receptors. It is generally believed that the initial receptor dimerization is an essential event for receptor activation. However, previous studies suggested that TGF-β signals by binding to the preexisting TβRII homodimer. Here, using single mol. microscopy to image green fluorescent protein (GFP)-labeled TβRII on the living cell surface, we demonstrated that the receptor could exist as monomers at the low expression level in resting cells and dimerize upon TGF-β stimulation. This work reveals a model in which the activation of serine-threonine kinase receptors is also accomplished via dimerization of monomers, suggesting that receptor dimerization is a general mechanism for ligand-induced receptor activation.
- 23Kasai, R. S.; Kusumi, A. Single-Molecule Imaging Revealed Dynamic GPCR Dimerization. Curr. Opin. Cell Biol. 2014, 27, 78– 86, DOI: 10.1016/j.ceb.2013.11.008Google Scholar23Single-molecule imaging revealed dynamic GPCR dimerizationKasai, Rinshi S.; Kusumi, AkihiroCurrent Opinion in Cell Biology (2014), 27 (), 78-86CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)A review. Single fluorescent-mol. video imaging and tracking in living cells are revolutionizing our understanding of mol. interactions in the plasma membrane and intracellular membrane systems. They have revealed that mol. interactions occur surprisingly dynamically on much shorter time scales («1 s) than those expected from the results by conventional techniques, such as pull-down assays (minutes to hours). Single-mol. imaging has unequivocally showed that G-protein-coupled receptors (GPCRs) undergo dynamic equil. between monomers and dimers, by enabling the detn. of the 2D monomer-dimer equil. const., the dimer dissocn. rate const. (typically ∼10 s-1), and the formation rate const. Within one second, GPCRs typically undergo several cycles of monomer and homo-dimer formation with different partners.
- 24Courty, S.; Luccardini, C.; Bellaiche, Y.; Cappello, G.; Dahan, M. Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging. Nano Lett. 2006, 6, 1491– 1495, DOI: 10.1021/nl060921tGoogle Scholar24Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot ImagingCourty, Sebastien; Luccardini, Camilla; Bellaiche, Yohanns; Cappello, Giovanni; Dahan, MaximeNano Letters (2006), 6 (7), 1491-1495CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. The authors characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-mol. measurements provided important parameters of the motor, such as its velocity and processivity, as well as an est. of the force necessary to carry a QD. The authors' measurements demonstrate the importance of single-mol. expts. in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.
- 25Nishikawa, S.; Arimoto, I.; Ikezaki, K.; Sugawa, M.; Ueno, H.; Komori, T.; Iwane, A. H.; Yanagida, T. Switch between Large Hand-over-Hand and Small Inchworm-Like Steps in Myosin VI. Cell 2010, 142, 879– 888, DOI: 10.1016/j.cell.2010.08.033Google Scholar25Switch between Large Hand-Over-Hand and Small Inchworm-like Steps in Myosin VINishikawa, So; Arimoto, Ikuo; Ikezaki, Keigo; Sugawa, Mitsuhiro; Ueno, Hiroshi; Komori, Tomotaka; Iwane, Atsuko H.; Yanagida, ToshioCell (Cambridge, MA, United States) (2010), 142 (6), 879-888CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Many biol. motor mols. move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.
- 26Pierobon, P.; Achouri, S.; Courty, S.; Dunn, A. R.; Spudich, J. A.; Dahan, M.; Cappello, G. Velocity, Processivity, and Individual Steps of Single Myosin V Molecules in Live Cells. Biophys. J. 2009, 96, 4268– 4275, DOI: 10.1016/j.bpj.2009.02.045Google Scholar26Velocity, processivity, and individual steps of single myosin V molecules in live cellsPierobon, Paolo; Achouri, Sarra; Courty, Sebastien; Dunn, Alexander R.; Spudich, James A.; Dahan, Maxime; Cappello, GiovanniBiophysical Journal (2009), 96 (10), 4268-4275CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)We report the tracking of single myosin V mols. in their natural environment, the cell. Myosin V mols., labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single mol. level with high spatial and temporal resoln. We perform an intracellular measurement of key parameters of this mol. transporter: velocity, processivity, step size, and dwell time. Our expts. bridge the gap between in vitro single mol. assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.
- 27Liu, S. L.; Wang, Z. G.; Hu, Y.; Xin, Y.; Singaram, I.; Gorai, S.; Zhou, X.; Shim, Y.; Min, J. H.; Gong, L. W. Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of Akt. Mol. Cell 2018, 71, 1092– 1104, e5 DOI: 10.1016/j.molcel.2018.07.035Google Scholar27Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of AktLiu, Shu-Lin; Wang, Zhi-Gang; Hu, Yusi; Xin, Yao; Singaram, Indira; Gorai, Sukhamoy; Zhou, Xin; Shim, Yoonjung; Min, Jung-Hyun; Gong, Liang-Wei; Hay, Nissim; Zhang, Jin; Cho, WonhwaMolecular Cell (2018), 71 (6), 1092-1104.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quant. imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling mols. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.
- 28Sheng, R.; Chen, Y.; Yung Gee, H.; Stec, E.; Melowic, H. R.; Blatner, N. R.; Tun, M. P.; Kim, Y.; Kallberg, M.; Fujiwara, T. K. Cholesterol Modulates Cell Signaling and Protein Networking by Specifically Interacting with PDZ Domain-Containing Scaffold Proteins. Nat. Commun. 2012, 3, 1249, DOI: 10.1038/ncomms2221Google Scholar28Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteinsSheng Ren; Chen Yong; Yung Gee Heon; Stec Ewa; Melowic Heather R; Blatner Nichole R; Tun Moe P; Kim Yonjung; Kallberg Morten; Fujiwara Takahiro K; Hye Hong Ji; Pyo Kim Kwang; Lu Hui; Kusumi Akihiro; Goo Lee Min; Cho WonhwaNature communications (2012), 3 (), 1249 ISSN:.Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.
- 29Komura, N.; Suzuki, K. G.; Ando, H.; Konishi, M.; Koikeda, M.; Imamura, A.; Chadda, R.; Fujiwara, T. K.; Tsuboi, H.; Sheng, R. Raft-Based Interactions of Gangliosides with a GPI-Anchored Receptor. Nat. Chem. Biol. 2016, 12, 402– 410, DOI: 10.1038/nchembio.2059Google Scholar29Raft-based interactions of gangliosides with a GPI-anchored receptorKomura, Naoko; Suzuki, Kenichi G. N.; Ando, Hiromune; Konishi, Miku; Koikeda, Machi; Imamura, Akihiro; Chadda, Rahul; Fujiwara, Takahiro K.; Tsuboi, Hisae; Sheng, Ren; Cho, Wonhwa; Furukawa, Koichi; Furukawa, Keiko; Yamauchi, Yoshio; Ishida, Hideharu; Kusumi, Akihiro; Kiso, MakotoNature Chemical Biology (2016), 12 (6), 402-410CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Gangliosides, glycosphingolipids contg. one or more sialic acid(s) in the glyco-chain, are involved in various important physiol. and pathol. processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or prepns. Single-fluorescent-mol. imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside mols. were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.
- 30Liu, S. L.; Wang, Z. G.; Zhang, Z. L.; Pang, D. W. Tracking Single Viruses Infecting Their Host Cells Using Quantum Dots. Chem. Soc. Rev. 2016, 45, 1211– 1224, DOI: 10.1039/C5CS00657KGoogle Scholar30Tracking single viruses infecting their host cells using quantum dotsLiu, Shu-Lin; Wang, Zhi-Gang; Zhang, Zhi-Ling; Pang, Dai-WenChemical Society Reviews (2016), 45 (5), 1211-1224CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Single-virus tracking (SVT) technique, which uses microscopy to monitor the behaviors of viruses, is a vital tool to study the real-time and in situ infection dynamics and virus-related interactions in live cells. To make SVT a more versatile tool in biol. research, the researchers have developed a quantum dot (QD)-based SVT technique, which can be utilized for long-term and highly sensitive tracking in live cells. In this review, we describe the development of a QD-based SVT technique and its biol. applications. We first discuss the advantage of QDs as tags in the SVT field by comparing the conventional tags, and then focus on the implementation of QD-based SVT expts., including the QD labeling strategy, instrumentation, and image anal. method. Next, we elaborate the recent advances of QD-based SVT in the biol. field, and mainly emphasize the representative examples to show how to use this technique to acquire more meaningful biol. information.
- 31Brandenburg, B.; Zhuang, X. Virus Trafficking–Learning from Single-Virus Tracking. Nat. Rev. Microbiol. 2007, 5, 197– 208, DOI: 10.1038/nrmicro1615Google Scholar31Virus trafficking - learning from single-virus trackingBrandenburg, Boerries; Zhuang, XiaoweiNature Reviews Microbiology (2007), 5 (3), 197-208CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell. Single-virus tracking in living cells potentially provides the authors with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.
- 32Huang, L. L.; Xie, H. Y. Progress on the Labeling and Single-Particle Tracking Technologies of Viruses. Analyst 2014, 139, 3336– 3346, DOI: 10.1039/C4AN00038BGoogle Scholar32Progress on the labeling and single-particle tracking technologies of virusesHuang, Li-Li; Xie, Hai-YanAnalyst (Cambridge, United Kingdom) (2014), 139 (13), 3336-3346CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)A review. Understanding and unravelling the invasion mechanisms of virus infection is of high importance for preventing and treating viral diseases. Single-virion tracking is a powerful way for exploring the mechanisms of viral infection. For successful tracking, the virus and cellular structures of interest must be fluorescently labeled; the microscope imaging technol. must be sufficiently powerful for real-time single virion or viral component tracking. All fields of scientists have made great efforts and improvements. Here we will review the recent advances in virus labeling and the emerging fluorescence imaging technologies used in the imaging and tracking of viruses.
- 33Parveen, N.; Borrenberghs, D.; Rocha, S.; Hendrix, J. Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication. Viruses 2018, 10, 250, DOI: 10.3390/v10050250Google Scholar33Single viruses on the fluorescence microscope: imaging molecular mobility, interactions and structure sheds new light on viral replicationParveen, Nagma; Borrenberghs, Doortje; Rocha, Susana; Hendrix, JelleViruses (2018), 10 (5), 250/1-250/21CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virol. Indeed, viral replication is often not particularly efficient, prone to errors or contg. parallel routes. Here, we review different single-mol. sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-mol. F.ovrddot.orster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resoln. microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virol. practice.
- 34Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics. Biophys. J. 1976, 16, 1055– 1069, DOI: 10.1016/S0006-3495(76)85755-4Google Scholar34Mobility measurement by analysis of fluorescence photobleaching recovery kineticsAxelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W.Biophysical Journal (1976), 16 (9), 1055-69CODEN: BIOJAU; ISSN:0006-3495.Fluorescence photobleaching recovery (FPR) denotes a method for measuring 2-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled mols. in ∼10 μm2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. The theor. basis and some practical guidelines are presented for simple, rigorous anal. of FPR expts. Information obtainable from FPR expts. includes: identification of transport process type, i.e., the admixt. of random diffusion and uniform directed flow; detn. of the abs. mobility coeff., i.e., the diffusion const. and (or) flow velocity; and the fraction of total fluorophore that is mobile. To illustrate the exptl. method and to verify the theory for diffusion, exptl. models are described comprising aq. solns. of rhodamine 6G.
- 35Simons, K.; Gerl, M. J. Revitalizing Membrane Rafts: New Tools and Insights. Nat. Rev. Mol. Cell Biol. 2010, 11, 688– 699, DOI: 10.1038/nrm2977Google Scholar35Revitalizing membrane rafts: New tools and insightsSimons, Kai; Gerl, Mathias J.Nature Reviews Molecular Cell Biology (2010), 11 (10), 688-699CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Ten years ago, the authors wrote a review on lipid rafts and signaling. At the time, this field was suffering from ambiguous methodol. and imprecise nomenclature. Now, however, new techniques are deepening the insight into the dynamics of membrane organization. Here, the authors discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms that are important in signaling, viral infection, and membrane trafficking.
- 36Magde, D.; Elson, E.; Webb, W. W. Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy. Phys. Rev. Lett. 1972, 29, 705– 708, DOI: 10.1103/PhysRevLett.29.705Google Scholar36Thermodynamic fluctations in a reacting system. Measurement by fluorescence correlation spectroscopyMagde, Douglas; Elson, Elliot; Webb, W. W.Physical Review Letters (1972), 29 (11), 705-8CODEN: PRLTAO; ISSN:0031-9007.Correlations of thermodynamic concn. fluctuations were measured in a chem. reactive system at equil. by observing fluctuations of the fluorescence of a reaction producct. The expt. yields the chem. rate consts. and diffusion coeffs. and shows the coupling among them. Data are reported for binding of ethidium bromide to DNA.
- 37Hess, S. T.; Huang, S.; Heikal, A. A.; Webb, W. W. Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review. Biochemistry 2002, 41, 697– 705, DOI: 10.1021/bi0118512Google Scholar37Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A ReviewHess, Samuel T.; Huang, Shaohui; Heikal, Ahmed A.; Webb, Watt W.Biochemistry (2002), 41 (3), 697-705CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review on historical perspective and theor. background of fluorescence correlation spectroscopy (FCS), exptl. setup for FCS, FCS applications in soln., and cellular applications of FCS.
- 38Haustein, E.; Schwille, P. Ultrasensitive Investigations of Biological Systems by Fluorescence Correlation Spectroscopy. Methods 2003, 29, 153– 166, DOI: 10.1016/S1046-2023(02)00306-7Google Scholar38Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopyHaustein, Elke; Schwille, PetraMethods (San Diego, CA, United States) (2003), 29 (2), 153-166CODEN: MTHDE9; ISSN:1046-2023. (Elsevier Science)A review. Fluorescence correlation spectroscopy (FCS) exts. information about mol. dynamics from the tiny fluctuations that can be obsd. in the emission of small ensembles of fluorescent mols. in thermodn. equil. Employing a confocal setup in conjunction with highly dil. samples, the av. no. of fluorescent particles simultaneously within the measurement vol. (∼1 fl) is minimized. Among the multitude of chem. and phys. parameters accessible by FCS are local concns., mobility coeffs., rate consts. for assocn. and dissocn. processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resoln. of less than 0.5 μm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local mol. dynamics provides access to environmental parameters such as local oxygen concns., pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quant. assessment of interactions and dynamics of small mol. quantities in biol. relevant systems.
- 39Berg, H. C. How to Track Bacteria. Rev. Sci. Instrum. 1971, 42, 868– 871, DOI: 10.1063/1.1685246Google Scholar39How to track bacteriaBerg H CThe Review of scientific instruments (1971), 42 (6), 868-71 ISSN:0034-6748.There is no expanded citation for this reference.
- 40Berg, H. C.; Brown, D. A. Chemotaxis in Escherichia Coli Analysed by Three-Dimensional Tracking. Nature 1972, 239, 500– 504, DOI: 10.1038/239500a0Google Scholar40Chemotaxis in Escherichia coli analyzed by three-dimensional trackingBerg, Howard C.; Brown, Douglas A.Nature (London, United Kingdom) (1972), 239 (5374), 500-4CODEN: NATUAS; ISSN:0028-0836.If serine is added to suspensions (no gradients), the run-length distributions are exponential but shift toward longer runs, and twiddles are suppressed. The shift does not occur with aspartate. The shift is not a metabolic effect. The bacteria runs are longer in the up-gradient swim than down-gradient and with less frequent direction changes. For serine, the distribution of runs down the gradient is similar to the distribution in a 9 μM isotropic soln. and for aspartate it is indistinguishable from that of the control. The statistics are Poisson. When a bacterium swims down the gradient, there is no functional relation between the length of a run and the derivs. of the concn. with respect to space or time. This is true both for serine and aspartate.
- 41Barak, L. S.; Webb, W. W. Diffusion of Low Density Lipoprotein-Receptor Complex on Human Fibroblasts. J. Cell Biol. 1982, 95, 846– 852, DOI: 10.1083/jcb.95.3.846Google Scholar41Diffusion of low density lipoprotein-receptor complex on human fibroblastsBarak L S; Webb W WThe Journal of cell biology (1982), 95 (3), 846-52 ISSN:0021-9525.Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.
- 42De Brabander, M.; Geuens, G.; Nuydens, R.; Moeremans, M.; De Mey, J. Probing Microtubule-Dependent Intracellular Motility with Nanometre Particle Video Ultramicroscopy (Nanovid Ultramicroscopy). Cytobios 1985, 43, 273– 283Google Scholar42Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy)De Brabander M; Geuens G; Nuydens R; Moeremans M; De Mey JCytobios (1985), 43 (174S), 273-83 ISSN:0011-4529.Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.
- 43Lee, G. M.; Ishihara, A.; Jacobson, K. A. Direct Observation of Brownian Motion of Lipids in a Membrane. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 6274– 6278, DOI: 10.1073/pnas.88.14.6274Google Scholar43Direct observation of Brownian motion of lipids in a membraneLee, Greta M.; Ishihara, Akira; Jacobson, Ken A.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (14), 6274-8CODEN: PNASA6; ISSN:0027-8424.Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual mols. in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid mols. were followed in a supported, planar bilayer contg. fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepd. by conjugating only anti-Fl to the gold. Paucivalent probes were prepd. by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an av. lateral diffusion coeff. (D) of 0.26 × 10-8 cm2/s, and paucivalent probes had an av. D of 0.73 × 10-8 cm2/s. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 × 10-8 cm2/s, which, after allowance for stochastic variation, are consistent with the D of 1.3 × 10-8 cm2/s measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diam., resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results, nanovid microscopy is a valid method for analyzing the movements of single or small groups of mols. within membranes.
- 44Gelles, J.; Schnapp, B. J.; Sheetz, M. P. Tracking Kinesin-Driven Movements with Nanometre-Scale Precision. Nature 1988, 331, 450– 453, DOI: 10.1038/331450a0Google Scholar44Tracking kinesin-driven movements with nanometer-scale precisionGelles, Jeff; Schnapp, Bruce J.; Sheetz, Michael P.Nature (London, United Kingdom) (1988), 331 (6155), 450-3CODEN: NATUAS; ISSN:0028-0836.Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (≤10) kinesin mols. may be sufficient to drive either bead movement or organelle transport. A method is described for detg. precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Measurements reveal basic mech. features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible mol. mechanisms of movement.
- 45Geerts, H.; de Brabander, M.; Nuydens, R. Nanovid Microscopy. Nature 1991, 351, 765– 766, DOI: 10.1038/351765a0Google Scholar45Nanovid microscopyGeerts H; de Brabander M; Nuydens RNature (1991), 351 (6329), 765-6 ISSN:0028-0836.By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied.
- 46Geerts, H.; De Brabander, M.; Nuydens, R.; Geuens, S.; Moeremans, M.; De Mey, J.; Hollenbeck, P. Nanovid Tracking: A New Automatic Method for the Study of Mobility in Living Cells Based on Colloidal Gold and Video Microscopy. Biophys. J. 1987, 52, 775– 782, DOI: 10.1016/S0006-3495(87)83271-XGoogle Scholar46Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopyGeerts H; De Brabander M; Nuydens R; Geuens S; Moeremans M; De Mey J; Hollenbeck PBiophysical journal (1987), 52 (5), 775-82 ISSN:0006-3495.We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.
- 47Inoué, S. Imaging of Unresolved Objects, Superresolution, and Precision of Distance Measurement with Video Microscopy. Methods Cell Biol. 1989, 30, 85– 112, DOI: 10.1016/S0091-679X(08)60976-0Google Scholar47Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopyInoue SMethods in cell biology (1989), 30 (), 85-112 ISSN:0091-679X.There is no expanded citation for this reference.
- 48Saxton, M. J. Single-Particle Tracking: Models of Directed Transport. Biophys. J. 1994, 67, 2110– 2019, DOI: 10.1016/S0006-3495(94)80694-0Google Scholar48Single-particle tracking: models of directed transportSaxton, Michael J.Biophysical Journal (1994), 67 (5), 2110-19CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Single-particle tracking techniques make it possible to measure motion of individual particles on the cell surface. In these expts., individual trajectories are obsd., so the data anal. must take into account the randomness of individual random walks. Methods of data anal. are discussed for models combining diffusion and directed motion. In the uniform flow model, a tracer simultaneously diffuses and undergoes directed motion. In the conveyor belt model, a tracer binds and unbinds to a uniform conveyor belt moving with const. velocity. If a trcer is bound, it moves at the velocity of the conveyor belt; if it is unbound, it diffuses freely. Trajectories are analyzed using parameters that measure the extent and asymmetry of the trajectory. A method of assessing the usefulness of such parameters is presented, and pitfalls in data anal. are discussed. Joint probability distributions of pairs of extent and asymmetry parameters are obtained for a pure random walk. These distributions can be used to show that a trajectory is not likely to have resulted from a pure random walk.
- 49Kao, H. P.; Verkman, A. S. Tracking of Single Fluorescent Particles in 3 Dimensions-Use of Cylindrical Optics to Encode Particle Position. Biophys. J. 1994, 67, 1291– 1300, DOI: 10.1016/S0006-3495(94)80601-0Google Scholar49Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle positionKao H P; Verkman A SBiophysical journal (1994), 67 (3), 1291-300 ISSN:0006-3495.We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.
- 50Patwardhan, A. Subpixel Position Measurement Using 1D, 2D and 3D Centroid Algorithms with Emphasis on Applications in Confocal Microscopy. J. Microsc. 1997, 186, 246– 257, DOI: 10.1046/j.1365-2818.1997.1970761.xGoogle ScholarThere is no corresponding record for this reference.
- 51Peters, I. M.; van Kooyk, Y.; van Vliet, S. J.; de Grooth, B. G.; Figdor, C. G.; Greve, J. 3D Single-Particle Tracking and Optical Trap Measurements on Adhesion Proteins. Cytometry 1999, 36, 189– 194, DOI: 10.1002/(SICI)1097-0320(19990701)36:3<189::AID-CYTO7>3.0.CO;2-3Google Scholar513D single-particle tracking and optical trap measurements on adhesion proteinsPeters, Inge M.; Van Kooyk, Yvette; Van Vliet, Sandra J.; De Grooth, Bart G.; Figdor, Carl G.; Greve, JanCytometry (1999), 36 (3), 189-194CODEN: CYTODQ; ISSN:0196-4763. (Wiley-Liss, Inc.)A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an α and a β chain. Thus far, only the cytoplasmic tail of the β chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete β cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the β chain.
- 52Bacher, C. P.; Reichenzeller, M.; Athale, C.; Herrmann, H.; Eils, R. 4-D Single Particle Tracking of Synthetic and Proteinaceous Microspheres Reveals Preferential Movement of Nuclear Particles Along Chromatin-Poor Tracks. BMC Cell Biol. 2004, 5, 45, DOI: 10.1186/1471-2121-5-45Google Scholar524-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracksBacher Christian P; Reichenzeller Michaela; Athale Chaitanya; Herrmann Harald; Eils RolandBMC cell biology (2004), 5 (), 45 ISSN:.BACKGROUND: The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. RESULTS: We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. CONCLUSIONS: Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.
- 53Genovesio, A.; Liedl, T.; Emiliani, V.; Parak, W. J.; Coppey-Moisan, M.; Olivo-Marin, J. C. Multiple Particle Tracking in 3-D+T Microscopy: Method and Application to the Tracking of Endocytosed Quantum Dots. IEEE Trans. Image Process. 2006, 15, 1062– 1070, DOI: 10.1109/TIP.2006.872323Google Scholar53Multiple particle tracking in 3-D+t microscopy: method and application to the tracking of endocytosed quantum dotsGenovesio Auguste; Liedl Tim; Emiliani Valentina; Parak Wolfgang J; Coppey-Moisan Maite; Olivo-Marin Jean-ChristopheIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2006), 15 (5), 1062-70 ISSN:1057-7149.We propose a method to detect and track multiple moving biological spot-like particles showing different kinds of dynamics in image sequences acquired through multidimensional fluorescence microscopy. It enables the extraction and analysis of information such as number, position, speed, movement, and diffusion phases of, e.g., endosomal particles. The method consists of several stages. After a detection stage performed by a three-dimensional (3-D) undecimated wavelet transform, we compute, for each detected spot, several predictions of its future state in the next frame. This is accomplished thanks to an interacting multiple model (IMM) algorithm which includes several models corresponding to different biologically realistic movement types. Tracks are constructed, thereafter, by a data association algorithm based on the maximization of the likelihood of each IMM. The last stage consists of updating the IMM filters in order to compute final estimations for the present image and to improve predictions for the next image. The performances of the method are validated on synthetic image data and used to characterize the 3-D movement of endocytic vesicles containing quantum dots.
- 54Ram, S.; Kim, D.; Ward, E. S.; Ober, R. J. Fast 3D Single Molecule Tracking with Multifocal Plane Microscopy in Polarized Epithelia Reveals a Novel Cellular Process of Intercellular Transfer. Biophys. J. 2013, 104, 535a, DOI: 10.1016/j.bpj.2012.11.2962Google ScholarThere is no corresponding record for this reference.
- 55Ram, S.; Kim, D.; Ober, R. J.; Ward, E. S. 3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers. Biophys. J. 2012, 103, 1594– 1603, DOI: 10.1016/j.bpj.2012.08.054Google Scholar553D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell BarriersRam, Sripad; Kim, Dongyoung; Ober, Raimund J.; Ward, E. SallyBiophysical Journal (2012), 103 (7), 1594-1603CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single mol. tracking of rapid protein dynamics in a 10 μ thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf mols. between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf mols. at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.
- 56Ram, S.; Prabhat, P.; Chao, J.; Ward, E. S.; Ober, R. J. High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells. Biophys. J. 2008, 95, 6025– 6043, DOI: 10.1529/biophysj.108.140392Google Scholar56High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cellsRam, Sripad; Prabhat, Prashant; Chao, Jerry; Ward, E. Sally; Ober, Raimund J.Biophysical Journal (2008), 95 (12), 6025-6043CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biol. insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to det. the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and exptl. data and show that the 3D position of quantum dots can be detd. over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be detd. Our anal. shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody mols. undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.
- 57Levi, V.; Ruan, Q.; Kis-Petikova, K.; Gratton, E. Scanning FCS, a Novel Method for Three-Dimensional Particle Tracking. Biochem. Soc. Trans. 2003, 31, 997– 1000, DOI: 10.1042/bst0310997Google Scholar57Scanning FCS, a novel method for three-dimensional particle trackingLevi, V.; Ruan, Q.; Kis-Petikova, K.; Gratton, E.Biochemical Society Transactions (2003), 31 (5), 997-1000CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)We describe a novel method to track fluorescent particles in three dimensions with nanometer precision and millisecond time resoln. In this method, we use our two-photon excitation microscope. The galvomotor-driven x-y scanning mirrors allow the laser beam to move repetitively in a circular path with a radius of half the width of the point spread function of the laser. When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-x plane can be detd. by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective was used to change the laser focus at two planes (half width of the point spread function apart). The difference of the fluorescence intensity in the two planes is used to calc. the z-position of the fluorescent particle. The laser beam is allowed to scan multiple circular orbits before it is moved to the other plane, thus improving the signal to noise ratio. With a fast feedback mechanism, the position of the laser beam is directed to the center of the fluorescent particle, thus allowing us to track a particle in three dimensions. In this contribution we describe some calibration expts. performed to test the three-dimensional tracking capability of our system over a large range.
- 58Dupont, A.; Lamb, D. C. Nanoscale Three-Dimensional Single Particle Tracking. Nanoscale 2011, 3, 4532– 4541, DOI: 10.1039/c1nr10989hGoogle Scholar58Nanoscale three-dimensional single particle trackingDupont, Aurelie; Lamb, Don C.Nanoscale (2011), 3 (11), 4532-4541CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. Single particle tracking (SPT) in biol. systems is a quickly growing field. Many new technologies are being developed providing new tracking capabilities, which also lead to higher demands and expectations for SPT. Following a single biomol. as it performs its function provides quant. mechanistic information that cannot be obtained in classical ensemble methods. From the 3D trajectory, information is available over the diffusional behavior of the particle and precise position information can also be used to elucidate interactions of the tracked particle with its surroundings. Thus, three-dimensional (3D) SPT is a very valuable tool for investigating cellular processes. This review presents recent progress in 3D SPT, from image-based techniques toward more sophisticated feedback approaches. We focus mainly on the feedback technique known as orbital tracking. We present here a modified version of the original orbital tracking in which the intensities from two z-planes are simultaneously measured allowing a concomitant wide-field imaging. The system can track single particles with a precision down to 5 nm in the x-y plane and 7 nm in the axial direction. The capabilities of the system are demonstrated using single virus tracing to follow the infection pathway of Prototype Foamy Virus in living cells.
- 59Ruthardt, N.; Lamb, D. C.; Brauchle, C. Single-Particle Tracking as a Quantitative Microscopy-Based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical Nanoparticles. Mol. Ther. 2011, 19, 1199– 1211, DOI: 10.1038/mt.2011.102Google Scholar59Single-particle Tracking as a Quantitative Microscopy-based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical NanoparticlesRuthardt, Nadia; Lamb, Don C.; Braeuchle, ChristophMolecular Therapy (2011), 19 (7), 1199-1211CODEN: MTOHCK; ISSN:1525-0016. (Nature Publishing Group)A review. Highly sensitive fluorescence microscopy techniques allow single nanoparticles to be tracked during their uptake into living cells with high temporal and spatial resoln. From anal. of the trajectories, random motion can be discriminated from active transport and the av. transport velocity and/or diffusion coeff. detd. Such an anal. provides important information regarding the uptake pathway and location of viruses and nanoparticles. In this review, we give an introduction into single-particle tracking (SPT) and detn. of the mean-squared displacement. We also give an overview of recent advances in SPT. These include millisecond alternating-laser excitation for removal of spectral crosstalk, alternating wide-field (WF), and total internal reflection fluorescence (TIRF) microscopy for sensitive expts. at the plasma membrane and 3-dimensional tracking strategies. Throughout the review, we highlight recent advances regarding the entry (and egress) of natural and artificial viruses obtained via SPT.
- 60Hou, S.; Johnson, C.; Welsher, K. Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells. Molecules 2019, 24, 2826, DOI: 10.3390/molecules24152826Google Scholar60Real-time 3D single particle tracking: towards active feedback single molecule spectroscopy in live cellsHou, Shangguo; Johnson, Courtney; Welsher, KevinMolecules (2019), 24 (15), 2826CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)A review. Single mol. fluorescence spectroscopy has been largely implemented using methods which require tethering of mols. to a substrate in order to make high temporal resoln. measurements. However, the act of tethering a mol. requires that the mol. be removed from its environment. This is esp. perturbative when measuring biomols. such as enzymes, which may rely on the non-equil. and crowded cellular environment for normal function. A method which may be able to un-tether single mol. fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resoln. over large axial ranges. This gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single mol. tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.
- 61Montiel, D.; Yang, H. Real-Time Three-Dimensional Single-Particle Tracking Spectroscopy for Complex Systems. Laser Photonics Rev. 2010, 4, 374– 385, DOI: 10.1002/lpor.200910012Google Scholar61Real-time three-dimensional single-particle tracking spectroscopy for complex systemsMontiel, Daniel; Yang, HawLaser & Photonics Reviews (2010), 4 (3), 374-385CODEN: LPRAB8; ISSN:1863-8880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Complex systems are characterized by dynamical processes spread over multiple time and length scales. At a given instant, these systems can display spatial heterogeneities in which the local phys. and chem. properties are nonuniform, depending on the location. They can also exhibit dynamical heterogeneities in which the local dynamical characteristics vary with time. These types of systems pose unique exptl. challenges for their characterization and test of theor. ideas. Recently, real-time three-dimensional (3D) single-particle tracking spectroscopy has been developed to address these kinds of problems. With this approach, in principle, one can follow how a system evolves spatially as well as temporally. This article attempts to provide an introduction to this promising new technique by discussing the aims of studying a complex system and recent exptl. advances towards this goal.
- 62Walsh, E. E.; Hruska, J. Monoclonal Antibodies to Respiratory Syncytial Virus Proteins: Identification of the Fusion Protein. J. Virol. 1983, 47, 171– 177, DOI: 10.1128/JVI.47.1.171-177.1983Google Scholar62Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion proteinWalsh, Edward E.; Hruska, JeromeJournal of Virology (1983), 47 (1), 171-7CODEN: JOVIAM; ISSN:0022-538X.Six monoclonal antibodies directed against respiratory syncytial virus proteins were produced. Each was characterized by immunopptn. and indirect immunofluorescence. One was directed against the nucleocapsid protein, NP 44, 2 were directed against a 37,000-dalton protein, 2 were directed against the major envelope glycoprotein, GP 90, and 1 was directed against the 70,000-dalton envelope protein, VP 70. Indirect immunofluorescence stain patterns of infected HEp-2 cells defined GP 90 and VP 70 as viral proteins expressed on the cell surface, whereas NP 44 and the 37,000-dalton protein were detected as intracytoplasmic inclusions. One of the anti-GP 90 antibodies neutralized virus only in the presence of complement but did not inhibit cell-cell fusion. The anti-VP 70 antibody neutralized virus without complement and inhibited cell-cell fusion of previously infected HEp-2, thus identifying VP 70 as the fusion protein.
- 63Matlin, K. S.; Reggio, H.; Helenius, A.; Simons, K. Infectious Entry Pathway of Influenza Virus in a Canine Kidney Cell Line. J. Cell Biol. 1981, 91, 601– 613, DOI: 10.1083/jcb.91.3.601Google Scholar63Infectious entry pathway of influenza virus in a canine kidney cell lineMatlin K S; Reggio H; Helenius A; Simons KThe Journal of cell biology (1981), 91 (3 Pt 1), 601-13 ISSN:0021-9525.The entry of fowl plague virus, and avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase-resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.
- 64Bächi, T. Direct Observation of the Budding and Fusion of an Enveloped Virus by Video Microscopy of Viable Cells. J. Cell Biol. 1988, 107, 1689– 1695, DOI: 10.1083/jcb.107.5.1689Google Scholar64Direct observation of the budding and fusion of an enveloped virus by video microscopy of viable cellsBachi TThe Journal of cell biology (1988), 107 (5), 1689-95 ISSN:0021-9525.Video-enhanced microscopy and digital image processing were used to observe the assembly, budding, and fusion of Respiratory Syncytial virus. Viral filaments were seen to bud from the plasma membrane of viable infected cells to a final length of 5-10 micron with an average speed of elongation of 110-250 nm/s. The rapidity of viral assembly and its synchronous occurrence (leading to the production of several viral particles per minute from the same surface domain) suggests a directed process of recruitment of viral components to an area selected for virus maturation. Virions were also seen to adsorb to the cell surface, and to fuse with the plasma membrane. These are the first real time observations of viral morphogenesis and penetration which are crucial events in the infectious cycle of enveloped viruses.
- 65Lowy, R. J.; Sarkar, D. P.; Chen, Y.; Blumenthal, R. Observation of Single Influenza Virus-Cell Fusion and Measurement by Fluorescence Video Microscopy. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 1850– 1854, DOI: 10.1073/pnas.87.5.1850Google Scholar65Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopyLowy, R. J.; Sarkar, D. P.; Chen, Y.; Blumenthal, R.Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (5), 1850-4CODEN: PNASA6; ISSN:0027-8424.Intensified video fluorescence microscopy and digital image processing were used to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) experienced fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quant. intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calc. the fraction of R18 dye mols. transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approx. twice as fast as pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was obsd. after the pH changes, suggesting that activation processes are faster than the time resoln., 1-5 s, of the current method.
- 66Georgi, A.; Mottola-Hartshorn, C.; Warner, A.; Fields, B.; Chen, L. B. Detection of Individual Fluorescently Labeled Reovirions in Living Cells. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 6579– 6583, DOI: 10.1073/pnas.87.17.6579Google Scholar66Detection of individual fluorescently labeled reovirions in living cellsGeorgi, Ann; Mottola-Hartshorn, Cristina; Warner, Angeline; Fields, Bernard; Chen, Lan BoProceedings of the National Academy of Sciences of the United States of America (1990), 87 (17), 6579-83CODEN: PNASA6; ISSN:0027-8424.Reovirus serotype 1 (Lang) can be conjugated with rhodamine B or fluorescein isothiocyanate in a way that preserves viral infectivity. Epifluorescence microscopy was used to detect individual virions bound to the surface of cells and to follow in real time the early stages of reovirus infection in living mouse fibroblast cells. Following uptake of the virus into endocytic vesicles, the movement was inhibited by nocodazole or colchicine, which was consistent with previous findings that the movement of intracellular vesicles is often microtubule-based.
- 67Pelkmans, L.; Kartenbeck, J.; Helenius, A. Caveolar Endocytosis of Simian Virus 40 Reveals a New Two-Step Vesicular-Transport Pathway to the ER. Nat. Cell Biol. 2001, 3, 473– 483, DOI: 10.1038/35074539Google Scholar67Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ERPelkmans, Lucas; Kartenbeck, Jurgen; Helenius, AriNature Cell Biology (2001), 3 (5), 473-483CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)SV40 virus is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-color, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After assocg. with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-contg. vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-pos., smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.
- 68Lakadamyali, M.; Rust, M. J.; Babcock, H. P.; Zhuang, X. Visualizing Infection of Individual Influenza Viruses. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9280– 9285, DOI: 10.1073/pnas.0832269100Google Scholar68Visualizing infection of individual influenza virusesLakadamyali, Melike; Rust, Michael J.; Babcock, Hazen P.; Zhuang, XiaoweiProceedings of the National Academy of Sciences of the United States of America (2003), 100 (16), 9280-9285CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cell's constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.
- 69Seisenberger, G.; Ried, M. U.; Endress, T.; Buning, H.; Hallek, M.; Brauchle, C. Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus. Science 2001, 294, 1929– 1932, DOI: 10.1126/science.1064103Google Scholar69Real-time single-molecule imaging of the infection pathway of an adeno-associated virusSeisenberger, Georg; Ried, Martin U.; Endress, Thomas; Buening, Hildegard; Hallek, Michael; Brauchle, ChristophScience (Washington, DC, United States) (2001), 294 (5548), 1929-1932CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We describe a method, based on single-mol. imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye mol. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resoln. show various modes of motion of adeno-assocd. viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally obsd. so far.
- 70Suomalainen, M.; Nakano, M. Y.; Keller, S.; Boucke, K.; Stidwill, R. P.; Greber, U. F. Microtubule-Dependent Plus- and Minus End-Directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus. J. Cell Biol. 1999, 144, 657– 672, DOI: 10.1083/jcb.144.4.657Google Scholar70Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirusSuomalainen, Maarit; Nakano, Michel Y.; Keller, Stephan; Boucke, Karin; Stidwill, Robert P.; Greber, Urs F.Journal of Cell Biology (1999), 144 (4), 657-672CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was obsd. in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was obsd. in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end-directed cytoplasmic dynein and an unknown plus end-directed activity.
- 71Coller, K. E.; Berger, K. L.; Heaton, N. S.; Cooper, J. D.; Yoon, R.; Randall, G. RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis. PLoS Pathog. 2009, 5, e1000702 DOI: 10.1371/journal.ppat.1000702Google ScholarThere is no corresponding record for this reference.
- 72Rust, M. J.; Lakadamyali, M.; Zhang, F.; Zhuang, X. Assembly of Endocytic Machinery around Individual Influenza Viruses During Viral Entry. Nat. Struct. Mol. Biol. 2004, 11, 567– 573, DOI: 10.1038/nsmb769Google Scholar72Assembly of endocytic machinery around individual influenza viruses during viral entryRust, Michael J.; Lakadamyali, Melike; Zhang, Feng; Zhuang, XiaoweiNature Structural & Molecular Biology (2004), 11 (6), 567-573CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. The authors have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. The results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.
- 73van der Schaar, H. M.; Rust, M. J.; Waarts, B. L.; van der Ende-Metselaar, H.; Kuhn, R. J.; Wilschut, J.; Zhuang, X.; Smit, J. M. Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking. J. Virol. 2007, 81, 12019– 12028, DOI: 10.1128/JVI.00300-07Google Scholar73Characterization of the early events in Dengue virus cell entry by biochemical assays and single-virus trackingvan der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaar, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.Journal of Virology (2007), 81 (21), 12019-12028CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concn. of virus particles measured by biochem. assays was found to be substantially higher than the no. of infectious particles detd. by infectivity assays, leading to an infectious unit-to-particle ratio of approx. 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface d. of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was obsd. that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was obsd. in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.
- 74Chen, C.; Zhuang, X. Epsin 1 Is a Cargo-Specific Adaptor for the Clathrin-Mediated Endocytosis of the Influenza Virus. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11790– 11795, DOI: 10.1073/pnas.0803711105Google Scholar74Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virusChen, Chen; Zhuang, XiaoweiProceedings of the National Academy of Sciences of the United States of America (2008), 105 (33), 11790-11795,CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)During clathrin-mediated endocytosis, adaptor proteins recognize specific internalization signals on cargo receptors, either recruiting cargos into clathrin-coated pits (CCPs) or initiating clathrin-coat assembly around the cargo mols. Here, we identify epsin 1, a clathrin-, ubiquitin-, and phospholipid-interacting protein, as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. Using live-cell imaging to monitor the entry of individual virus particles, we obsd. recruitment of epsin 1 to the binding sites of influenza viruses in synchrony with the assembly of CCPs. Epsin 1 knockdown by siRNA significantly inhibited the clathrin-mediated endocytosis of the influenza virus and caused the majority of the virus particles to enter through a clathrin-independent pathway. The same treatment did not affect the entry of several classical ligands for clathrin-mediated endocytosis, including transferrin, LDL, and EGF. Overexpression of the dominant-neg. epsin 1 mutant lacking the ubiquitin-interaction motifs nearly completely blocked the clathrin-mediated entry of the influenza virus without affecting transferrin uptake. These results suggest that epsin 1 functions as a cargo-specific adaptor for the clathrin-mediated entry of the influenza virus.
- 75van der Schaar, H. M.; Rust, M. J.; Chen, C.; van der Ende-Metselaar, H.; Wilschut, J.; Zhuang, X.; Smit, J. M. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells. PLoS Pathog. 2008, 4, e1000244 DOI: 10.1371/journal.ppat.1000244Google Scholar75Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cellsvan der Schaar Hilde M; Rust Michael J; Chen Chen; van der Ende-Metselaar Heidi; Wilschut Jan; Zhuang Xiaowei; Smit Jolanda MPLoS pathogens (2008), 4 (12), e1000244 ISSN:.Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.
- 76Tsien, R. Y. The Green Fluorescent Protein. Annu. Rev. Biochem. 1998, 67, 509– 544, DOI: 10.1146/annurev.biochem.67.1.509Google Scholar76The green fluorescent proteinTsien, Roger Y.Annual Review of Biochemistry (1998), 67 (), 509-544CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review, with ∼114 refs. In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochem. and cell biol. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resoln. crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiol. indicators, biosensors, and photochem. memories.
- 77Baulcombe, D. C.; Chapman, S.; Santa Cruz, S. Jellyfish Green Fluorescent Protein as a Reporter for Virus Infections. Plant J. 1995, 7, 1045– 1053, DOI: 10.1046/j.1365-313X.1995.07061045.xGoogle Scholar77Jellyfish green fluorescent protein as a reporter for virus infectionsBaulcombe, David C.; Chapman, Sean; Cruz, Simon SantaPlant Journal (1995), 7 (6), 1045-53CODEN: PLJUED; ISSN:0960-7412. (Blackwell)The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Prodn. of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analyzed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of exts. from infected plants. To illustrate the potential application of this reporter gene in virol. studies a deriv. of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.
- 78Elliott, G.; O’Hare, P. Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection. J. Virol. 1999, 73, 4110– 4119, DOI: 10.1128/JVI.73.5.4110-4119.1999Google Scholar78Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infectionElliott, Gillian; O'Hare, PeterJournal of Virology (1999), 73 (5), 4110-4119CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here, the authors describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). This virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, the authors have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, the authors have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.
- 79Cruz, S. S.; Chapman, S.; Roberts, A. G.; Roberts, I. M.; Prior, D.; Oparka, K. J. Assembly and Movement of a Plant Virus Carrying a Green Fluorescent Protein Overcoat. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 6286– 6290, DOI: 10.1073/pnas.93.13.6286Google Scholar79Assembly and movement of a plant virus carrying a green fluorescent protein overcoatCruz S S; Chapman S; Roberts A G; Roberts I M; Prior D A; Oparka K JProceedings of the National Academy of Sciences of the United States of America (1996), 93 (13), 6286-90 ISSN:0027-8424.Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.
- 80Desai, P.; Person, S. Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid. J. Virol. 1998, 72, 7563– 7568, DOI: 10.1128/JVI.72.9.7563-7568.1998Google Scholar80Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsidDesai, Prashant; Person, StanleyJournal of Virology (1998), 72 (9), 7563-7568CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An anal. of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) anal. of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was obsd., confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
- 81Moradpour, D.; Evans, M. J.; Gosert, R.; Yuan, Z.; Blum, H. E.; Goff, S. P.; Lindenbach, B. D.; Rice, C. M. Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes. J. Virol. 2004, 78, 7400– 7409, DOI: 10.1128/JVI.78.14.7400-7409.2004Google Scholar81Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexesMoradpour, Darius; Evans, Matthew J.; Gosert, Rainer; Yuan, Zhenghong; Blum, Hubert E.; Goff, Stephen P.; Lindenbach, Brett D.; Rice, Charles M.Journal of Virology (2004), 78 (14), 7400-7409CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Hepatitis C virus (HCV) replicates its genome in a membrane-assocd. replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. The authors describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
- 82McDonald, D.; Vodicka, M. A.; Lucero, G.; Svitkina, T. M.; Borisy, G. G.; Emerman, M.; Hope, T. J. Visualization of the Intracellular Behavior of HIV in Living Cells. J. Cell Biol. 2002, 159, 441– 452, DOI: 10.1083/jcb.200203150Google Scholar82Visualization of the intracellular behavior of HIV in living cellsMcDonald, David; Vodicka, Marie A.; Lucero, Ginger; Svitkina, Tatyana M.; Borisy, Gary G.; Emerman, Michael; Hope, Thomas J.Journal of Cell Biology (2002), 159 (3), 441-452CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to det. the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resoln. imaging of microtubule-assocd. structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.
- 83Miyauchi, K.; Kim, Y.; Latinovic, O.; Morozov, V.; Melikyan, G. B. HIV Enters Cells via Endocytosis and Dynamin-Dependent Fusion with Endosomes. Cell 2009, 137, 433– 444, DOI: 10.1016/j.cell.2009.02.046Google Scholar83HIV enters cells via endocytosis and dynamin-dependent fusion with endosomesMiyauchi, Kosuke; Kim, Yuri; Latinovic, Olga; Morozov, Vladimir; Melikyan, Gregory B.Cell (Cambridge, MA, United States) (2009), 137 (3), 433-444CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Enveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here, the authors used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. The authors also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glycoprotein- and dynamin-dependent fusion with intracellular compartments.
- 84Koch, P.; Lampe, M.; Godinez, W. J.; Müller, B.; Rohr, K.; Kräusslich, H.-G.; Lehmann, M. J. Visualizing Fusion of Pseudotyped HIV-1 Particles in Real Time by Live Cell Microscopy. Retrovirology 2009, 6, 84, DOI: 10.1186/1742-4690-6-84Google Scholar84Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopyKoch Peter; Lampe Marko; Godinez William J; Muller Barbara; Rohr Karl; Krausslich Hans-Georg; Lehmann Maik JRetrovirology (2009), 6 (), 84 ISSN:.BACKGROUND: Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown. RESULTS: We generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env. CONCLUSION: These results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.
- 85Endreß, T.; Lampe, M.; Briggs, J. A. G.; Kräusslich, H.-G.; Bräuchle, C.; Müller, B.; Lamb, D. C. HIV-1–Cellular Interactions Analyzed by Single Virus Tracing. Eur. Biophys. J. 2008, 37, 1291– 1301, DOI: 10.1007/s00249-008-0322-zGoogle Scholar85HIV-1-cellular interactions analyzed by single virus tracingEndress, Thomas; Lampe, Marko; Briggs, John A. G.; Kraeusslich, Hans-Georg; Braeuchle, Christoph; Mueller, Barbara; Lamb, Don C.European Biophysics Journal (2008), 37 (8), 1291-1301CODEN: EBJOE8; ISSN:0175-7571. (Springer)Single virus tracing (SVT) allows the direct investigation of the entry pathway of viruses into living cells. Using fluorescently labeled virus-like particles (VLPs) and SVT, we have studied the interaction between human immunodeficiency virus type 1 (HIV-1) and the plasma membrane of living cells. From the trajectories of freely diffusing VLPs in soln., we established that the particle prepn. was homogeneous and the particles had a hydrodynamic radius of 86 ± 5 nm, consistent with the size of single HI viruses. The VLPs that come in contact with the cell surface either become immobilized or rapidly dissoc. from the cell surface. The fraction of virions that become immobilized on the plasma membrane correlates with the surface heparan sulfate linked proteoglycans (HSPG) concn. of the cell line tested. The particles that are not immobilized make an av. of 1.5 contacts with the cell surface before diffusing away. For most cell lines investigated, the contact duration follows an exponential distribution with a lifetime between 20 and 50 ms depending on the cell type.
- 86Jolly, C.; Kashefi, K.; Hollinshead, M.; Sattentau, Q. J. HIV-1 Cell to Cell Transfer Across an Env-Induced, Actin-Dependent Synapse. J. Exp. Med. 2004, 199, 283– 293, DOI: 10.1084/jem.20030648Google Scholar86HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapseJolly, Clare; Kashefi, Kirk; Hollinshead, Michael; Sattentau, Quentin J.Journal of Experimental Medicine (2004), 199 (2), 283-293CODEN: JEMEAV; ISSN:0022-1007. (Rockefeller University Press)Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4+/CXCR4+ target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-assocd. antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane mols. into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was obsd. by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV-receptors and adhesion mols. to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.
- 87Arhel, N.; Genovesio, A.; Kim, K. A.; Miko, S.; Perret, E.; Olivo-Marin, J. C.; Shorte, S.; Charneau, P. Quantitative Four-Dimensional Tracking of Cytoplasmic and Nuclear HIV-1 Complexes. Nat. Methods 2006, 3, 817– 824, DOI: 10.1038/nmeth928Google Scholar87Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexesArhel, Nathalie; Genovesio, Auguste; Kim, Kyeong-Ae; Miko, Sarah; Perret, Emmanuelle; Olivo-Marin, Jean-Christophe; Shorte, Spencer; Charneau, PierreNature Methods (2006), 3 (10), 817-824CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein deriv. FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quant. particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined vol. once assocd. with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.
- 88Hubner, W.; McNerney, G. P.; Chen, P.; Dale, B. M.; Gordon, R. E.; Chuang, F. Y.; Li, X. D.; Asmuth, D. M.; Huser, T.; Chen, B. K. Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses. Science 2009, 323, 1743– 1747, DOI: 10.1126/science.1167525Google Scholar88Quantitative 3D video microscopy of HIV transfer across T cell virological synapsesHubner Wolfgang; McNerney Gregory P; Chen Ping; Dale Benjamin M; Gordon Ronald E; Chuang Frank Y S; Li Xiao-Dong; Asmuth David M; Huser Thomas; Chen Benjamin KScience (New York, N.Y.) (2009), 323 (5922), 1743-7 ISSN:.The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized "buttons" containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.
- 89Dahan, M.; Levi, S.; Luccardini, C.; Rostaing, P.; Riveau, B.; Triller, A. Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking. Science 2003, 302, 442– 445, DOI: 10.1126/science.1088525Google Scholar89Diffusion dynamics of glycine receptors revealed by single-quantum dot trackingDahan, Maxime; Levi, Sabine; Luccardini, Camilla; Rostaing, Philippe; Riveau, Beatrice; Triller, AntoineScience (Washington, DC, United States) (2003), 302 (5644), 442-445CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biol. imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was obsd. and further confirmed by electron microscopy imaging of QD-tagged receptors.
- 90Bonneau, S.; Dahan, M.; Cohen, L. D. Single Quantum Dot Tracking Based on Perceptual Grouping Using Minimal Paths in a Spatiotemporal Volume. IEEE T. Image Process 2005, 14, 1384– 1395, DOI: 10.1109/TIP.2005.852794Google Scholar90Single quantum dot tracking based on perceptual grouping using minimal paths in a spatiotemporal volumeBonneau Stephane; Dahan Maxime; Cohen Laurent DIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2005), 14 (9), 1384-95 ISSN:1057-7149.Semiconductor quantum dots (QDs) are new fluorescent probes with great promise for ultrasensitive biological imaging. When detected at the single-molecule level, QD-tagged molecules can be observed and tracked in the membrane of live cells over unprecedented durations. The motion of these individual molecules, recorded in sequences of fluorescence images, can reveal aspects of the dynamics of cellular processes that remain hidden in conventional ensemble imaging. Due to QD complex optical properties, such as fluorescence intermittency, the quantitative analysis of these sequences is, however, challenging and requires advanced algorithms. We present here a novel approach, which, instead of a frame by frame analysis, is based on perceptual grouping in a spatiotemporal volume. By applying a detection process based on an image fluorescence model, we first obtain an unstructured set of points. Individual molecular trajectories are then considered as minimal paths in a Riemannian metric derived from the fluorescence image stack. These paths are computed with a variant of the fast marching method and few parameters are required. We demonstrate the ability of our algorithm to track intermittent objects both in sequences of synthetic data and in experimental measurements obtained with individual QD-tagged receptors in the membrane of live neurons. While developed for tracking QDs, this method can, however, be used with any fluorescent probes.
- 91Bachir, A. I.; Durisic, N.; Hebert, B.; Grütter, P.; Wiseman, P. W. Characterization of Blinking Dynamics in Quantum Dot Ensembles Using Image Correlation Spectroscopy. J. Appl. Phys. 2006, 99, 064503 DOI: 10.1063/1.2175470Google Scholar91Characterization of blinking dynamics in quantum dot ensembles using image correlation spectroscopyBachir, Alexia I.; Durisic, Nela; Hebert, Benedict; Grutter, Peter; Wiseman, Paul W.Journal of Applied Physics (2006), 99 (6), 064503/1-064503/7CODEN: JAPIAU; ISSN:0021-8979. (American Institute of Physics)Quantum dots (QDs) are being increasingly applied as luminescent labels in optical studies for biophys. and cell biol. applications due to their unique spectroscopic properties. However, their fluorescence blinking characteristics that follow power law statistics make it difficult to use QDs in some quant. biophys. applications. The authors present image correlation spectroscopy (ICS) in combination with total internal reflection fluorescence microscopy as a tool to characterize blinking dynamics in QDs. The rate of decay of the ICS measured ensemble correlation function reflects variation in blinking dynamics and can be used to distinguish different blinking distribution regimes. To test and confirm hypothesis, the authors also analyze image time series simulations of ensembles of point emitters with set blinking statistics. Optimization of the temporal sampling and the no. of QDs sampled is essential for detecting changes in blinking dynamics with ICS. Probably this exptl. characterization of the QD blinking statistics can actually serve as a sensitive reporter for certain quant. biol. applications.
- 92Durisic, N.; Bachir, A. I.; Kolin, D. L.; Hebert, B.; Lagerholm, B. C.; Grutter, P.; Wiseman, P. W. Detection and Correction of Blinking Bias in Image Correlation Transport Measurements of Quantum Dot Tagged Macromolecules. Biophys. J. 2007, 93, 1338– 1346, DOI: 10.1529/biophysj.107.106864Google Scholar92Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromoleculesDurisic, Nela; Bachir, Alexia I.; Kolin, David L.; Hebert, Benedict; Lagerholm, B. Christoffer; Grutter, Peter; Wiseman, Paul W.Biophysical Journal (2007), 93 (4), 1338-1346CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biol. applications. Here the authors demonstrate that fluorescence fluctuation anal. of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation anal. of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coeffs. can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and anal. of computer simulated image time series verified that the effect the authors observe is caused by fluorescence intermittency. The authors show that reciprocal space image correlation anal. can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. The authors also demonstrate application of the image correlation methods for measurement of the diffusion coeff. of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts.
- 93He, K.; Luo, W.; Zhang, Y.; Liu, F.; Liu, D.; Xu, L.; Qin, L.; Xiong, C.; Lu, Z.; Fang, X. Intercellular Transportation of Quantum Dots Mediated by Membrane Nanotubes. ACS Nano 2010, 4, 3015– 3022, DOI: 10.1021/nn1002198Google Scholar93Intercellular Transportation of Quantum Dots Mediated by Membrane NanotubesHe, Kangmin; Luo, Wangxi; Zhang, Yuliang; Liu, Fei; Liu, Da; Xu, Li; Qin, Lei; Xiong, Chunyang; Lu, Zhizhen; Fang, Xiaohong; Zhang, YouyiACS Nano (2010), 4 (6), 3015-3022CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)In this work, we reported that the quantum dot (QD) nanoparticles could be actively transported in the membrane nanotubes between cardiac myocytes. Single particle imaging and tracking of QDs revealed that most QDs moved in a bidirectional mode along the membrane nanotubes with a mean velocity of 1.23 μm/s. The results suggested that QDs moving in the nanotubes were coordinately motivated by mol. motors. It provides new information for the study of the intercellular transportation of nanoparticles.
- 94Chang, Y.-P.; Pinaud, F.; Antelman, J.; Weiss, S. Tracking Bio-Molecules in Live Cells Using Quantum Dots. J. Biophotonics 2008, 1, 287– 298, DOI: 10.1002/jbio.200810029Google Scholar94Tracking bio-molecules in live cells using quantum dotsChang, Yun-Pei; Pinaud, Fabien; Antelman, Joshua; Weiss, ShimonJournal of Biophotonics (2008), 1 (4), 287-298CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Single particle tracking (SPT) techniques were developed to explore bio-mols. dynamics in live cells at single mol. sensitivity and nanometer spatial resoln. Recent developments in quantum dots (Qdots) surface coating and bio-conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell expts.
- 95Pinaud, F.; Clarke, S.; Sittner, A.; Dahan, M. Probing Cellular Events, One Quantum Dot at a Time. Nat. Methods 2010, 7, 275– 285, DOI: 10.1038/nmeth.1444Google Scholar95Probing cellular events, one quantum dot at a timePinaud, Fabien; Clarke, Samuel; Sittner, Assa; Dahan, MaximeNature Methods (2010), 7 (4), 275-285CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Monitoring the behavior of single mols. in living cells is a powerful approach to investigate the details of cellular processes. Owing to their optical, chem. and biofunctional properties, semiconductor quantum dot (QD) probes promise to be tools of choice in this endeavor. Here, the authors review recent advances that allow ever more controlled expts. at the single-nanoparticle level in live cells. Several examples, related to membrane dynamics, cell signaling, or intracellular transport, illustrate how single QD tracking can be readily used to decipher complex biol. processes and address key concepts that underlie cellular organization and dynamics.
- 96Wang, Z. G.; Liu, S. L.; Tian, Z. Q.; Zhang, Z. L.; Tang, H. W.; Pang, D. W. Myosin-Driven Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer Cells. ACS Nano 2012, 6, 10033– 10041, DOI: 10.1021/nn303729rGoogle Scholar96Myosin-Driven Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer CellsWang, Zhi-Gang; Liu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Tang, Hong-Wu; Pang, Dai-WenACS Nano (2012), 6 (11), 10033-10041CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Membrane nanotubes can facilitate direct intercellular communication between cells and provide a unique channel for intercellular transfer of cellular contents. However, the transport mechanisms of membrane nanotubes remain poorly understood between cancer cells. Also largely unknown is the transport pattern mediated by membrane nanotubes. In this work, wheat germ agglutinin (WGA), a widely used drug carrier and potential antineoplastic drug, was labeled with quantum dots (QDs-WGA) as a model for exploring the intercellular transportation via membrane nanotubes. We found that membrane nanotubes allowed effective transfer of QDs-WGA. Long-term single-particle tracking indicated that the movements of QDs-WGA exhibited a slow and directed motion pattern in nanotubes. Significantly, the transport of QDs-WGA was driven by myosin mol. motors in an active and unidirectional manner. These results contribute to a better understanding of cell-to-cell communication for cancer research.
- 97Liu, S. L.; Zhang, Z. L.; Sun, E. Z.; Peng, J.; Xie, M.; Tian, Z. Q.; Lin, Y.; Pang, D. W. Visualizing the Endocytic and Exocytic Processes of Wheat Germ Agglutinin by Quantum Dot-Based Single-Particle Tracking. Biomaterials 2011, 32, 7616– 7624, DOI: 10.1016/j.biomaterials.2011.06.046Google Scholar97Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle trackingLiu, Shu-Lin; Zhang, Zhi-Ling; Sun, En-Ze; Peng, Jun; Xie, Min; Tian, Zhi-Quan; Lin, Yi; Pang, Dai-WenBiomaterials (2011), 32 (30), 7616-7624CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Wheat germ agglutinin (WGA) is a paradigm for understanding intracellular transport of lectins. As a protein exploiting the receptor-mediated endocytosis for internalization, WGA is also a valuable model system for exploring the endocytic and exocytic pathway. In this study, quantum dot-based single-particle tracking was performed to investigate the transport of WGA in live cells, revealing firstly that the endocytic and exocytic processes of WGA were both actin- and microtubule-dependent, each including five stages. The vesicle fusion event occurred near the cytomembrane, followed by two destinies with WGA: shedding to the extracellular or reversing to the cytoplasm. These findings suggest a distinct and dynamic scenario for the transport of lectins following a receptor-mediated endo/exocytic pathway in live cells. This is important for the application of lectins as drug carriers and antineoplastic drugs in medicine, and also offers insights into the pathway of endocytosis and exocytosis.
- 98Joo, K.-I.; Lei, Y.; Lee, C.-L.; Lo, J.; Xie, J.; Hamm-Alvarez, S. F.; Wang, P. Site-Specific Labeling of Enveloped Viruses with Quantum Dots for Single Virus Tracking. ACS Nano 2008, 2, 1553– 1562, DOI: 10.1021/nn8002136Google Scholar98Site-Specific Labeling of Enveloped Viruses with Quantum Dots for Single Virus TrackingJoo, Kye-Il; Lei, Yuning; Lee, Chi-Lin; Lo, Jonathon; Xie, Jiansong; Hamm-Alvarez, Sarah F.; Wang, PinACS Nano (2008), 2 (8), 1553-1562CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. It also allows for the live cell imaging of the trafficking of labeled virus to the Rab5+ endosomal compartments. This study further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway. The studies monitoring HIV particles using QD-labeling showed that the authors could detect single virions on the surface of target cells expressing either CD4/CCR5 or DC-SIGN. Further internalization studies of QD-HIV evidently showed that the clathrin pathway is the major route for DC-SIGN-mediated uptake of viruses. Taken together, the authors' data demonstrate the potential of this QD-labeling for visualizing the dynamic interactions between viruses and target cell structures.
- 99Liu, S. L.; Tian, Z. Q.; Zhang, Z. L.; Wu, Q. M.; Zhao, H. S.; Ren, B.; Pang, D. W. High-Efficiency Dual Labeling of Influenza Virus for Single-Virus Imaging. Biomaterials 2012, 33, 7828– 7833, DOI: 10.1016/j.biomaterials.2012.07.026Google Scholar99High-efficiency dual labeling of influenza virus for single-virus imagingLiu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Wu, Qiu-Mei; Zhao, Hai-Su; Ren, Bin; Pang, Dai-WenBiomaterials (2012), 33 (31), 7828-7833CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Many viruses invade host cells by entering the cells and releasing their genome for replication, which are remarkable incidents for viral infection. Therefore, the viral internal and external components should be simultaneously labeled and dynamically tracked at single-virus level for further understanding viral infection mechanisms. However, most of the previously reported methods have very low labeling efficiency and require considerable time and effort, which is laborious and inconvenient for researchers. In this work, we report a general strategy to high-efficiently label viral envelope and genome for single-virus imaging with quantum dots (QDs) and Syto 82, resp. It was found that nearly all viral envelopes could be labeled with QDs with superior stability, which makes it possible to realize global and long-term tracking of single virus in individual cells. Effectively labeling their genome with Syto 82, about 90% of QDs-labeled viruses could be used to monitor the viral genome signal, which may provide valuable information for deeply studying viral genome transport. This is very important and meaningful to investigate the viral infection mechanism. Our labeling strategy has advantage in commonality, convenience and efficiency, which is expected to be widely used in biol. research.
- 100Lv, C.; Lin, Y.; Liu, A. A.; Hong, Z. Y.; Wen, L.; Zhang, Z.; Zhang, Z. L.; Wang, H.; Pang, D. W. Labeling Viral Envelope Lipids with Quantum Dots by Harnessing the Biotinylated Lipid-Self-Inserted Cellular Membrane. Biomaterials 2016, 106, 69– 77, DOI: 10.1016/j.biomaterials.2016.08.013Google Scholar100Labeling viral envelope lipids with quantum dots by harnessing the biotinylated lipid-self-inserted cellular membraneLv, Cheng; Lin, Yi; Liu, An-An; Hong, Zheng-Yuan; Wen, Li; Zhang, Zhenfeng; Zhang, Zhi-Ling; Wang, Hanzhong; Pang, Dai-WenBiomaterials (2016), 106 (), 69-77CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Highly efficient labeling of viruses with quantum dots (QDs) is the prerequisite for the long-term tracking of virus invasion at the single virus level to reveal mechanisms of virus infection. As one of the structural components of viruses, viral envelope lipids are hard to be labeled with QDs due to the lack of efficient methods to modify viral envelope lipids. Moreover, it is still a challenge to maintain the intactness and infectivity of labeled viruses. Herein, a mild method has been developed to label viral envelope lipids with QDs by harnessing the biotinylated lipid-self-inserted cellular membrane. Biotinylated lipids can spontaneously insert in cellular membranes of host cells during culture and then be naturally assembled on progeny Pseudorabies virus (PrV) via propagation. The biotinylated PrV can be labeled with streptavidin-conjugated QDs, with a labeling efficiency of ∼90%. Such a strategy to label lipids with QDs can retain the intactness and infectivity of labeled viruses to the largest extent, facilitating the study of mechanisms of virus infection at the single virus level.
- 101Hong, Z. Y.; Lv, C.; Liu, A. A.; Liu, S. L.; Sun, E. Z.; Zhang, Z. L.; Lei, A. W.; Pang, D. W. Clicking Hydrazine and Aldehyde: The Way to Labeling of Viruses with Quantum Dots. ACS Nano 2015, 9, 11750– 11760, DOI: 10.1021/acsnano.5b03256Google Scholar101Clicking Hydrazine and Aldehyde: The Way to Labeling of Viruses with Quantum DotsHong, Zheng-Yuan; Lv, Cheng; Liu, An-An; Liu, Shu-Lin; Sun, En-Ze; Zhang, Zhi-Ling; Lei, Ai-Wen; Pang, Dai-WenACS Nano (2015), 9 (12), 11750-11760CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Real-time tracking of fluorophore-tagged viruses in living cells can help uncover virus infection mechanisms. Certainly, the indispensable prerequisite for virus-tracking is to label viruses with some bright and photostable beacons such as quantum dots (QDs) via an appropriate labeling strategy. Herein, the authors devise a convenient hydrazine-aldehyde based strategy to label viruses with QDs through the conjugation of 4-formylbenzoate (4FB) modified QDs to 6-hydrazinonicotinate acetone hydrazone (HyNic) modified viruses under mild conditions. On the basis of this strategy, viruses can be successfully labeled with QDs with high selectivity, stable conjugation, good reproducibility, high labeling efficiency of 92-93% and max. retention of both fluorescence properties of QDs and infectivity of viruses, which is very meaningful to tracking and statistical anal. of virus infection processes. By further comparing with the most widely used labeling strategy based on the Biotin-SA system, this new strategy has advantages of both high labeling efficiency and good retention of virus infectivity, thus offering a promising alternative for virus-labeling. Moreover, due to the ubiquitous presence of exposed amino groups on the surface of various viruses, this selective, efficient, reproducible and biofriendly strategy should have good universality for labeling both enveloped and nonenveloped viruses.
- 102Wen, L.; Lin, Y.; Zheng, Z. H.; Zhang, Z. L.; Zhang, L. J.; Wang, L. Y.; Wang, H. Z.; Pang, D. W. Labeling the Nucleocapsid of Enveloped Baculovirus with Quantum Dots for Single-Virus Tracking. Biomaterials 2014, 35, 2295– 2301, DOI: 10.1016/j.biomaterials.2013.11.069Google Scholar102Labeling the nucleocapsid of enveloped baculovirus with quantum dots for single-virus trackingWen, Li; Lin, Yi; Zheng, Zhen-Hua; Zhang, Zhi-Ling; Zhang, Li-Juan; Wang, Li-Ying; Wang, Han-Zhong; Pang, Dai-WenBiomaterials (2014), 35 (7), 2295-2301CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Utilization of quantum dots (QDs) for single-virus tracking is highly important for understanding virus infection mechanism. However, QD labeling site of real enveloped viruses has been confined to the external envelope so far, causing the impossibility to monitor the late infection events after the loss of envelope. Herein, a strategy to label the internal nucleocapsid of enveloped virus with QDs was proposed. The nucleocapsid of enveloped baculovirus was self-biotinylated during virus replication process in host cells and subsequently labeled with streptavidin-conjugated QDs (SA-QDs). Such host cell-assisted QD labeling was proved to be reliable, specific, efficient and capable of maintaining virus infectivity. Based on such labeling, crit. infection events before and after the envelope loss were monitored in real time, including single virus interacting with late endosomes and the subsequent nucleocapsid transporting into cell nucleus. Thus our established QD labeling of enveloped virus nucleocapsid with QDs enables the comprehensive single-virus tracking for deeply understanding virus infection mechanism.
- 103Xie, M.; Luo, K.; Huang, B.-H.; Liu, S.-L.; Hu, J.; Cui, D.; Zhang, Z.-L.; Xiao, G.-F.; Pang, D.-W. PEG-Interspersed Nitrilotriacetic Acid-Functionalized Quantum Dots for Site-Specific Labeling of Prion Proteins Expressed on Cell Surfaces. Biomaterials 2010, 31, 8362– 8370, DOI: 10.1016/j.biomaterials.2010.07.063Google Scholar103PEG-interspersed nitrilotriacetic acid-functionalized quantum dots for site-specific labeling of prion proteins expressed on cell surfacesXie, Min; Luo, Kan; Huang, Bi-Hai; Liu, Shu-Lin; Hu, Jun; Cui, Di; Zhang, Zhi-Ling; Xiao, Geng-Fu; Pang, Dai-WenBiomaterials (2010), 31 (32), 8362-8370CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)A strategy has been put forward to fabricate PEG-interspersed nitrilotriacetic acid (NTA)-functionalized QDs by one-step self-assembly using a mixt. of self-synthesized NTA-terminated amphiphilic polymer and 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(Polyethylene Glycol)2000] (DSPE-PEG-COOH). The process was highly reproducible for facile functionalization of QDs via simultaneous self-assembly of biocompatible PEG mols. onto their surface. An optimized molar ratio of NTA-terminated amphiphilic polymer to DSPE-PEG-COOH was used to obtain NTA-functionalized QDs for site-specific labeling of prion proteins (PrPC) expressed on cell surfaces. Fabricated NTA-functionalized QDs can be a good candidate used for real-time visualization of PrPC in single live cells to clarify the nosogenesis of pathogenic scrapie prion protein (PrPSc).
- 104Wen, L.; Zheng, Z. H.; Liu, A. A.; Lv, C.; Zhang, L. J.; Ao, J.; Zhang, Z. L.; Wang, H. Z.; Lin, Y.; Pang, D. W. Tracking Single Baculovirus Retrograde Transportation in Host Cell via Quantum Dot-Labeling of Virus Internal Component. J. Nanobiotechnol. 2017, 15, 37, DOI: 10.1186/s12951-017-0270-9Google Scholar104Tracking single baculovirus retrograde transportation in host cell via quantum dot-labeling of virus internal componentWen, Li; Zheng, Zhen-Hua; Liu, An-An; Lv, Cheng; Zhang, Li-Juan; Ao, Jian; Zhang, Zhi-Ling; Wang, Han-Zhong; Lin, Yi; Pang, Dai-WenJournal of Nanobiotechnology (2017), 15 (), 37/1-37/10CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)Background: Quantum dot (QD)-based single virus tracking has become a powerful tool for dissecting virus infection mechanism. However, only virus behaviors at the early stage of retrograde trafficking have been dynamically tracked so far. Monitoring of comprehensive virus retrograde transportation remains a challenge. Results: Based on the superior fluorescence properties of QDs and their labeling of virus internal component, the dynamic interactions between baculoviruses and all key transportation-related cellular structures, including vesicles, acidic endosomes, actins, nuclear pores and nuclei, were visualized at the single-virus level. Detailed scenarios and dynamic information were provided for these crit. interaction processes. Conclusions: A comprehensive model of baculovirus retrograde trafficking involving virus endocytosis, fusion with acidic endosome, translocation to nuclear periphery, internalization into nucleus, and arriving at the destination in nucleus was proposed. Thus the whole retrograde transportation of baculovirus in live host cells was elucidated at the single-virus level for the first time.
- 105Hao, J.; Huang, L. L.; Zhang, R.; Wang, H. Z.; Xie, H. Y. A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click Chemistry. Anal. Chem. 2012, 84, 8364– 8370, DOI: 10.1021/ac301918tGoogle Scholar105A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click ChemistryHao, Jian; Huang, Li-Li; Zhang, Rui; Wang, Han-Zhong; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2012), 84 (19), 8364-8370CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Real-time tracking of the dynamic process of virus invasion is crucial to understanding the infection mechanism. For successful tracking, efficient labeling methods are indispensable. The authors report a mild and reliable method for labeling viruses, esp. with regard to easily disabled enveloped viruses. The copper-free click chem. has been used to label enveloped viruses with quantum dots (QDs) by linking virions modified with azide to the QDs derived with dibenzocyclooctynes (DBCO). Both vaccinia virus (VACV) and avian influenza A virus (H9N2) can be specifically and rapidly labeled under mild conditions, with a labeling efficiency of >80%. The labeled virions were of intact infectivity, and their fluorescence was strong enough to realize single-virion tracking. Compared to previously reported methods, the authors' method is less destructive, reliable, and universal, without specific requirements for the type and structure of viruses to be labeled, which has laid the foundation for long-term dynamic visualization of virus infection process.
- 106Zhang, P.; Liu, S.; Gao, D.; Hu, D.; Gong, P.; Sheng, Z.; Deng, J.; Ma, Y.; Cai, L. Click-Functionalized Compact Quantum Dots Protected by Multidentate-Imidazole Ligands: Conjugation-Ready Nanotags for Living-Virus Labeling and Imaging. J. Am. Chem. Soc. 2012, 134, 8388– 8391, DOI: 10.1021/ja302367sGoogle Scholar106Click-Functionalized Compact Quantum Dots Protected by Multidentate-Imidazole Ligands: Conjugation-Ready Nanotags for Living-Virus Labeling and ImagingZhang, Pengfei; Liu, Shuhui; Gao, Duyang; Hu, Dehong; Gong, Ping; Sheng, Zonghai; Deng, Jizhe; Ma, Yifan; Cai, LintaoJournal of the American Chemical Society (2012), 134 (20), 8388-8391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We synthesized a new class of multifunctional multidentate-imidazole polymer ligands by one-step reaction to produce conjugation-ready QDs with great stability and compact size. Furthermore, combined with strain-promoted click chem., we developed a general strategy for efficient labeling of living-viruses with QD probes.
- 107Liu, H.; Liu, Y.; Liu, S.; Pang, D. W.; Xiao, G. Clathrin-Mediated Endocytosis in Living Host Cells Visualized through Quantum Dot Labeling of Infectious Hematopoietic Necrosis virus. J. Virol. 2011, 85, 6252– 6262, DOI: 10.1128/JVI.00109-11Google Scholar107Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virusLiu, Haibin; Liu, Yi; Liu, Shulin; Pang, Dai-Wen; Xiao, GengfuJournal of Virology (2011), 85 (13), 6252-6262CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, the authors found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked redn. in viral entry. The authors also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymn. is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compd. that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments.
- 108Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Zhao, H. S.; Liu, H.; Sun, E. Z.; Xiao, G. F.; Zhang, W.; Wang, H. Z.; Pang, D. W. Effectively and Efficiently Dissecting the Infection of Influenza Virus by Quantum-Dot-Based Single-Particle Tracking. ACS Nano 2012, 6, 141– 150, DOI: 10.1021/nn2031353Google Scholar108Effectively and Efficiently Dissecting the Infection of Influenza Virus by Quantum-Dot-Based Single-Particle TrackingLiu, Shu-Lin; Zhang, Zhi-Ling; Tian, Zhi-Quan; Zhao, Hai-Su; Liu, Haibin; Sun, En-Ze; Xiao, Geng Fu; Zhang, Wanpo; Wang, Han-Zhong; Pang, Dai-WenACS Nano (2012), 6 (1), 141-150CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Exploring the virus infection mechanisms is significant for defending against virus infection and providing a basis for studying endocytosis mechanisms. Single-particle tracking technique is a powerful tool to monitor virus infection in real time for obtaining dynamic information. In this study, the authors reported a quantum-dot-based single-particle tracking technique to efficiently and globally research the virus infection behaviors in individual cells. It was obsd. that many influenza viruses were moving rapidly, converging to the microtubule organizing center (MTOC), interacting with acidic endosomes, and finally entering the target endosomes for genome release, which provides a vivid portrayal of the five-stage virus infection process. This report settles a long-pending question of how viruses move and interact with acidic endosomes before genome release in the perinuclear region and also finds that influenza virus infection is likely to be a "MTOC rescue" model for genome release. The systemic technique developed in this report is expected to be widely used for studying the mechanisms of virus infection and uncovering the secrets of endocytosis.
- 109Luo, K.; Li, S.; Xie, M.; Wu, D.; Wang, W.; Chen, R.; Huang, L.; Huang, T.; Pang, D.; Xiao, G. Real-Time Visualization of Prion Transport in Single Live Cells Using Quantum Dots. Biochem. Biophys. Res. Commun. 2010, 394, 493– 497, DOI: 10.1016/j.bbrc.2010.02.159Google Scholar109Real-time visualization of prion transport in single live cells using quantum dotsLuo, Kan; Li, Shu; Xie, Min; Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao; Pang, Daiwen; Xiao, GengfuBiochemical and Biophysical Research Communications (2010), 394 (3), 493-497CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrPC to the infectious scrapie isoform PrPSc. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, the authors have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addn., using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, the authors show that lipid rafts play an important role in locating cellular PrPC to the cell membrane and in initiating PrPC endocytosis.
- 110Liu, S. L.; Zhang, L. J.; Wang, Z. G.; Zhang, Z. L.; Wu, Q. M.; Sun, E. Z.; Shi, Y. B.; Pang, D. W. Globally Visualizing the Microtubule-Dependent Transport Behaviors of Influenza Virus in Live Cells. Anal. Chem. 2014, 86, 3902– 3908, DOI: 10.1021/ac500640uGoogle Scholar110Globally Visualizing the Microtubule-Dependent Transport Behaviors of Influenza Virus in Live CellsLiu, Shu-Lin; Zhang, Li-Juan; Wang, Zhi-Gang; Zhang, Zhi-Ling; Wu, Qiu-Mei; Sun, En-Ze; Shi, Yun-Bo; Pang, Dai-WenAnalytical Chemistry (Washington, DC, United States) (2014), 86 (8), 3902-3908CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.
- 111Liu, S. L.; Wu, Q. M.; Zhang, L. J.; Wang, Z. G.; Sun, E. Z.; Zhang, Z. L.; Pang, D. W. Three-Dimensional Tracking of Rab5- and Rab7-Associated Infection Process of Influenza Virus. Small 2014, 10, 4746– 4753, DOI: 10.1002/smll.201400944Google Scholar111Three-Dimensional Tracking of Rab5- and Rab7- Associated Infection Process of Influenza VirusLiu, Shu-Lin; Wu, Qiu-Mei; Zhang, Li-Juan; Wang, Zhi-Gang; Sun, En-Ze; Zhang, Zhi-Ling; Pang, Dai-WenSmall (2014), 10 (22), 4746-4753CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biol. problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-assocd. infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The exptl. results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-contg. vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-pos. endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.
- 112Wang, Z. G.; Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Tang, H. W.; Pang, D. W. Exploring Sialic Acid Receptors-Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single-Particle Tracking. Small 2014, 10, 2712– 2720, DOI: 10.1002/smll.201303532Google Scholar112Exploring sialic acid receptors-related infection behavior of avian influenza virus in human bronchial epithelial cells by single-particle trackingWang, Zhi-Gang; Liu, Shu-Lin; Zhang, Zhi-Ling; Tian, Zhi-Quan; Tang, Hong-Wu; Pang, Dai-WenSmall (2014), 10 (13), 2712-2720CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomols. in live cells. In this work, the authors use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. The authors have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.
- 113Qin, C.; Li, W.; Li, Q.; Yin, W.; Zhang, X.; Zhang, Z.; Zhang, X. E.; Cui, Z. Real-Time Dissection of Dynamic Uncoating of Individual Influenza Viruses. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 2577– 2582, DOI: 10.1073/pnas.1812632116Google Scholar113Real-time dissection of dynamic uncoating of individual influenza virusesQin, Chong; Li, Wei; Li, Qin; Yin, Wen; Zhang, Xiaowei; Zhang, Zhiping; Zhang, Xian-En; Cui, ZongqiangProceedings of the National Academy of Sciences of the United States of America (2019), 116 (7), 2577-2582CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Uncoating is an obligatory step in the virus life cycle that serves as an antiviral target. Unfortunately, it is challenging to study viral uncoating due to methodol. limitations for detecting this transient and dynamic event. The uncoating of influenza A virus (IAV), which contains an unusual genome of eight segmented RNAs, is particularly poorly understood. Here, by encapsulating quantum dot (QD)-conjugated viral ribonucleoprotein complexes (vRNPs) within infectious IAV virions and applying single-particle imaging, we tracked the uncoating process of individual IAV virions. Approx. 30% of IAV particles were found to undergo uncoating through fusion with late endosomes in the "around-nucleus" region at 30 to 90 min postinfection. Inhibition of viral M2 proton channels and cellular endosome acidification prevented IAV uncoating. IAV vRNPs are released sep. into the cytosol after virus uncoating. Then, individual vRNPs undergo a three-stage movement to the cell nucleus and display two diffusion patterns when inside the nucleus. These findings reveal IAV uncoating and vRNP trafficking mechanisms, filling a crit. gap in knowledge about influenza viral infection.
- 114Sun, E. Z.; Liu, A. A.; Zhang, Z. L.; Liu, S. L.; Tian, Z. Q.; Pang, D. W. Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking. ACS Nano 2017, 11, 4395– 4406, DOI: 10.1021/acsnano.6b07853Google Scholar114Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus TrackingSun, En-Ze; Liu, An-An; Zhang, Zhi-Ling; Liu, Shu-Lin; Tian, Zhi-Quan; Pang, Dai-WenACS Nano (2017), 11 (5), 4395-4406CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Entry is the 1st crit. step for the infection of influenza A virus and of great significance for the research and development of antiflu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of a single-virus tracking technique and quantum dots, we have realized real-time and multicolor visualization of the endocytic process of individual viruses and comprehensive dissection of 2 distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of the virus, with a large population of events for short-lived recruitments of these 2 proteins being abortive. In addn., the differentiated durations of dynamin recruitment and responses to inhibitors in these 2 routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also participates in the maturation of a clathrin-coated pit in the clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.
- 115Wu, Q. M.; Liu, S. L.; Chen, G.; Zhang, W.; Sun, E. Z.; Xiao, G. F.; Zhang, Z. L.; Pang, D. W. Uncovering the Rab5-Independent Autophagic Trafficking of Influenza A Virus by Quantum-Dot-Based Single-Virus Tracking. Small 2018, 14, e1702841 DOI: 10.1002/smll.201702841Google ScholarThere is no corresponding record for this reference.
- 116Ma, Y.; Wang, M.; Li, W.; Zhang, Z.; Zhang, X.; Tan, T.; Zhang, X. E.; Cui, Z. Live Cell Imaging of Single Genomic Loci with Quantum Dot-Labeled TALEs. Nat. Commun. 2017, 8, 15318, DOI: 10.1038/ncomms15318Google Scholar116Live cell imaging of single genomic loci with quantum dot-labeled talesMa, Yingxin; Wang, Mingxiu; Li, Wei; Zhang, Zhiping; Zhang, Xiaowei; Tan, Tianwei; Zhang, Xian-En; Cui, ZongqiangNature Communications (2017), 8 (), 15318CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labeling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different color quantum dots (QDs). These QD-labeled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different colored QD-labeled TALEs, we det. and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.
- 117Li, Q.; Yin, W.; Li, W.; Zhang, Z.; Zhang, X.; Zhang, X. E.; Cui, Z. Encapsulating Quantum Dots within HIV-1 Virions through Site-Specific Decoration of the Matrix Protein Enables Single Virus Tracking in Live Primary Macrophages. Nano Lett. 2018, 18, 7457– 7468, DOI: 10.1021/acs.nanolett.8b02800Google Scholar117Encapsulating Quantum Dots within HIV-1 Virions through Site-Specific Decoration of the Matrix Protein Enables Single Virus Tracking in Live Primary MacrophagesLi, Qin; Yin, Wen; Li, Wei; Zhang, Zhiping; Zhang, Xiaowei; Zhang, Xian-En; Cui, ZongqiangNano Letters (2018), 18 (12), 7457-7468CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Labeling and imaging with quantum dots (QDs) provides powerful tools to visualize viral infection in living cells. Encapsulating QDs within virions represents a novel strategy for virus labeling. Here, the authors developed infectious HIV-1 virions encapsulating QDs through site-specific decoration of the viral matrix protein (MA) and used them to visualize early infection events in human primary macrophages by single-particle imaging. The MA protein was fused to a biotin acceptor peptide (BAP) tag, biotinylated, complexed with streptavidin-conjugated QDs in live cells, and incorporated into virions during virus assembly. The QD-encapsulated virions were tracked during infection of macrophages at a single particle level. The dynamic dissocn. of MA and Vpr was also tracked in real time, and MA has multiple dynamic behaviors and functions during virus entry. More importantly, the authors tracked the dynamic interplay of QD-encapsulated virions with cellular mitochondria in live primary macrophages. Also HIV-1 can induce fission of mitochondria during the early phases of infection. In summary, the authors have constructed a type of QD-encapsulated virus particle and used this technol. to further the authors' understanding of the early events of HIV-1 infection.
- 118Zhang, L. J.; Xia, L.; Liu, S. L.; Sun, E. Z.; Wu, Q. M.; Wen, L.; Zhang, Z. L.; Pang, D. W. A ″Driver Switchover″ Mechanism of Influenza Virus Transport from Microfilaments to Microtubules. ACS Nano 2018, 12, 474– 484, DOI: 10.1021/acsnano.7b06926Google Scholar118A "Driver Switchover" Mechanism of Influenza Virus Transport from Microfilaments to MicrotubulesZhang, Li-Juan; Xia, Li; Liu, Shu-Lin; Sun, En-Ze; Wu, Qiu-Mei; Wen, Li; Zhang, Zhi-Ling; Pang, Dai-WenACS Nano (2018), 12 (1), 474-484CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.
- 119Muller, B.; Daecke, J.; Fackler, O. T.; Dittmar, M. T.; Zentgraf, H.; Krausslich, H. G. Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative. J. Virol. 2004, 78, 10803– 10813, DOI: 10.1128/JVI.78.19.10803-10813.2004Google Scholar119Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivativeMuller Barbara; Daecke Jessica; Fackler Oliver T; Dittmar Matthias T; Zentgraf Hanswalter; Krausslich Hans-GeorgJournal of virology (2004), 78 (19), 10803-13 ISSN:0022-538X.The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.
- 120Carlson, L. A.; Briggs, J. A. G.; Glass, B.; Riches, J. D.; Simon, M. N.; Johnson, M. C.; Muller, B.; Grunewald, K.; Krausslich, H. G. Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis. Cell Host Microbe 2008, 4, 592– 599, DOI: 10.1016/j.chom.2008.10.013Google Scholar120Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesisCarlson, Lars-Anders; Briggs, John A. G.; Glass, Baerbel; Riches, James D.; Simon, Martha N.; Johnson, Marc C.; Mueller, Barbara; Gruenewald, Kay; Kraeusslich, Hans-GeorgCell Host & Microbe (2008), 4 (6), 592-599CODEN: CHMECB; ISSN:1931-3128. (Cell Press)Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomog. and scanning transmission electron microscopy, we find that virions have a morphol. and compn. distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release-akin to its role in vesicle formation-and is not restricted to severing the thin membrane tether.
- 121Sakin, V.; Paci, G.; Lemke, E. A.; Muller, B. Labeling of Virus Components for Advanced, Quantitative Imaging Analyses. FEBS Lett. 2016, 590, 1896– 1914, DOI: 10.1002/1873-3468.12131Google Scholar121Labeling of virus components for advanced, quantitative imaging analysesSakin, Volkan; Paci, Giulia; Lemke, Edward A.; Mueller, BarbaraFEBS Letters (2016), 590 (13), 1896-1914CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)In recent years, investigation of virus-cell interactions has moved from ensemble measurements to imaging analyses at the single-particle level. Advanced fluorescence microscopy techniques provide single-mol. sensitivity and subdiffraction spatial resoln., allowing observation of subviral details and individual replication events to obtain detailed quant. information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virol. questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging.
- 122Goncalves, M. S. Fluorescent Labeling of Biomolecules with Organic Probes. Chem. Rev. 2009, 109, 190– 212, DOI: 10.1021/cr0783840Google Scholar122Fluorescent Labeling of Biomolecules with Organic ProbesGoncalves, M. Sameiro T.Chemical Reviews (Washington, DC, United States) (2009), 109 (1), 190-212CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review looking at org. h labels with emissions up to 500 nm and org. labels with emissions beyond 500 nm.
- 123Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763– 775, DOI: 10.1038/nmeth.1248Google Scholar123Quantum dots versus organic dyes as fluorescent labelsResch-Genger, Ute; Grabolle, Markus; Cavaliere-Jaricot, Sara; Nitschke, Roland; Nann, ThomasNature Methods (2008), 5 (9), 763-775CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Suitable labels are at the core of luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technol. is the emerging development of quantum dots (QDs) - inorg. nanocrystals with unique optical and chem. properties but complicated surface chem. - as in vitro and in vivo fluorophores. Here the authors compare and evaluate the differences in physicochem. properties of common fluorescent labels, focusing on traditional org. dyes and QDs. The authors' aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.
- 124Miyawaki, A.; Sawano, A.; Kogure, T. Lighting Up Cells: Labelling Proteins with Fluorophores. Nat. Cell Biol. 2003, S1– S7Google Scholar124Lighting up cells: Labelling proteins with fluorophoresMiyawaki, Atsushi; Sawano, Asako; Kogure, TakakoNature Cell Biology (2003), (Suppl.), S1-S7CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)A review. During the past decade, rapid improvements have been made in the tools available for labeling proteins within cells, which has increased our ability to unravel the finer details of cellular events. One significant reason for these advances has been the development of fluorescent proteins that can be incorporated into proteins by genetic fusion to produce a fluorescent label. In addn., new techniques have made it possible to label proteins with small org. fluorophores and semiconductor nanocrystals.
- 125Sivaraman, D.; Biswas, P.; Cella, L. N.; Yates, M. V.; Chen, W. Detecting RNA Viruses in Living Mammalian Cells by Fluorescence Microscopy. Trends Biotechnol. 2011, 29, 307– 313, DOI: 10.1016/j.tibtech.2011.02.006Google Scholar125Detecting RNA viruses in living mammalian cells by fluorescence microscopySivaraman, Divya; Biswas, Payal; Cella, Lakshmi N.; Yates, Marylynn V.; Chen, WilfredTrends in Biotechnology (2011), 29 (7), 307-313CODEN: TRBIDM; ISSN:0167-7799. (Elsevier B.V.)A review. Traditional methods that rely on viral isolation and culture techniques continue to be the gold stds. used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.
- 126Wang, I. H.; Burckhardt, C. J.; Yakimovich, A.; Greber, U. F. Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton. Viruses 2018, 10, 166, DOI: 10.3390/v10040166Google Scholar126Imaging, tracking and computational analyses of virus entry and egress with the cytoskeletonWang, I-Hsuan; Burckhardt, Christoph J.; Yakimovich, Artur; Greber, Urs F.Viruses (2018), 10 (4), 166/1-166/29CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Viruses have a dual nature: particles are "passive substances" lacking chem. energy transformation, whereas infected cells are "active substances" turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and mol. biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and assocd. motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and assocd. motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in assocn. with cellular organelles. This review explores how the anal. of viral trajectories informs about mechanisms of infection. We tdiscuss the methodol. enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodol., we highlight the role of actin filaments and microtubules, and their assocd. motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolns. thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function.
- 127Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Fluorescence Imaging in Vivo: Recent Advances. Curr. Opin. Biotechnol. 2007, 18, 17– 25, DOI: 10.1016/j.copbio.2007.01.003Google Scholar127Fluorescence imaging in vivo: recent advancesRao, Jianghong; Dragulescu-Andrasi, Anca; Yao, HequanCurrent Opinion in Biotechnology (2007), 18 (1), 17-25CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-IR (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addn. of fluorescent org., inorg. and biol. nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resoln., multimodality and lifetime-based in vivo fluorescence imaging.
- 128Lakadamyali, M.; Rust, M. J.; Zhuang, X. Ligands for Clathrin-Mediated Endocytosis Are Differentially Sorted into Distinct Populations of Early Endosomes. Cell 2006, 124, 997– 1009, DOI: 10.1016/j.cell.2005.12.038Google Scholar128Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomesLakadamyali, Melike; Rust, Michael J.; Zhuang, XiaoweiCell (Cambridge, MA, United States) (2006), 124 (5), 997-1009CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Cells rely on the correct sorting of endocytic ligands and receptors for proper function. Early endosomes have been considered as the initial sorting station where cargos for degrdn. sep. from those for recycling. Using live-cell imaging to monitor individual endosomes and ligand particles in real time, we have discovered a sorting mechanism that takes place prior to early endosome entry. We show that early endosomes are in fact comprised of two distinct populations: a dynamic population that is highly mobile on microtubules and matures rapidly toward late endosomes and a static population that matures much more slowly. Several cargos destined for degrdn. are preferentially targeted to the dynamic endosomes, whereas the recycling ligand transferrin is nonselectively delivered to all early endosomes and effectively enriched in the larger, static population. This pre-early endosome sorting process begins at clathrin-coated vesicles, depends on microtubule-dependent motility, and appears to involve endocytic adaptors.
- 129Babcock, H. P.; Chen, C.; Zhuang, X. Using Single-Particle Tracking to Study Nuclear Trafficking of Viral Genes. Biophys. J. 2004, 87, 2749– 2758, DOI: 10.1529/biophysj.104.042234Google Scholar129Using single-particle tracking to study nuclear trafficking of viral genesBabcock, Hazen P.; Chen, Chen; Zhuang, XiaoweiBiophysical Journal (2004), 87 (4), 2749-2758CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)The question of how genetic materials are trafficked in and out of the cell nucleus is a problem of great importance not only for understanding viral infections but also for advancing gene-delivery technol. Here we demonstrate a phys. technique that allows gene trafficking to be studied at the single-gene level by combining sensitive fluorescence microscopy with microinjection. As a model system, we investigate the nuclear import of influenza genes, in the form of ribonucleoproteins (vRNPs), by imaging single vRNPs in living cells in real time. Our single-particle trajectories show that vRNPs are transported to the nuclear envelope by diffusion. We have obsd. heterogeneous interactions between the vRNPs and nuclear pore complexes with dissocn. rate consts. spanning two orders of magnitude. Our single-particle tracking expts. also provided new insights into the regulation mechanisms for the nuclear import of vRNPs: the influenza M1 protein, a regulatory protein for the import process, downregulates the nuclear import of vRNPs by inhibiting the interactions between vRNPs and nuclear pore complexes but has no significant effect on the transport properties of vRNPs. We expect this single-particle tracking approach to find broad application in investigations of genetic trafficking.
- 130Vaughan, J. C.; Brandenburg, B.; Hogle, J. M.; Zhuang, X. Rapid Actin-Dependent Viral Motility in Live Cells. Biophys. J. 2009, 97, 1647– 1656, DOI: 10.1016/j.bpj.2009.07.011Google Scholar130Rapid actin-dependent viral motility in live cellsVaughan, Joshua C.; Brandenburg, Boerries; Hogle, James M.; Zhuang, XiaoweiBiophysical Journal (2009), 97 (6), 1647-1656CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased anal. of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was obsd. in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymn. or by myosin motor proteins.
- 131Brandenburg, B.; Lee, L. Y.; Lakadamyali, M.; Rust, M. J.; Zhuang, X.; Hogle, J. M. Imaging Poliovirus Entry in Live Cells. PLoS Biol. 2007, 5, e183 DOI: 10.1371/journal.pbio.0050183Google ScholarThere is no corresponding record for this reference.
- 132Xu, H.; Hao, X.; Wang, S.; Wang, Z.; Cai, M.; Jiang, J.; Qin, Q.; Zhang, M.; Wang, H. Real-Time Imaging of Rabies Virus Entry into Living Vero Cells. Sci. Rep. 2015, 5, 11753, DOI: 10.1038/srep11753Google Scholar132Real-time Imaging of Rabies Virus Entry into Living Vero cellsXu Haijiao; Hao Xian; Wang Zhiyong; Cai Mingjun; Jiang Junguang; Wang Shaowen; Qin Qiwei; Zhang Maolin; Wang HongdaScientific reports (2015), 5 (), 11753 ISSN:.Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.
- 133Vonderheit, A.; Helenius, A. Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes. PLoS Biol. 2005, 3, e233 DOI: 10.1371/journal.pbio.0030233Google Scholar133Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomesVonderheit Andreas; Helenius AriPLoS biology (2005), 3 (7), e233 ISSN:.Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.
- 134Johannsdottir, H. K.; Mancini, R.; Kartenbeck, J.; Amato, L.; Helenius, A. Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry. J. Virol. 2009, 83, 440– 453, DOI: 10.1128/JVI.01864-08Google Scholar134Host cell factors and functions involved in vesicular stomatitis virus entryJohannsdottir, Hrefna Kristin; Mancini, Roberta; Kartenbeck, Jurgen; Amato, Lea; Helenius, AriJournal of Virology (2009), 83 (1), 440-453CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although assocd. with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-assocd. proteins and cellular functions.
- 135Engel, S.; Heger, T.; Mancini, R.; Herzog, F.; Kartenbeck, J.; Hayer, A.; Helenius, A. Role of Endosomes in Simian Virus 40 Entry and Infection. J. Virol. 2011, 85, 4198– 4211, DOI: 10.1128/JVI.02179-10Google Scholar135Role of endosomes in simian virus 40 entry and infectionEngel, Sabrina; Heger, Thomas; Mancini, Roberta; Herzog, Fabian; Kartenbeck, Jurgen; Hayer, Arnold; Helenius, AriJournal of Virology (2011), 85 (9), 4198-4211CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was esp. sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.
- 136Joo, K. I.; Fang, Y.; Liu, Y.; Xiao, L.; Gu, Z.; Tai, A.; Lee, C. L.; Tang, Y.; Wang, P. Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates. ACS Nano 2011, 5, 3523– 3535, DOI: 10.1021/nn102651pGoogle Scholar136Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot ConjugatesJoo, Kye-Il; Fang, Yun; Liu, Yarong; Xiao, Liang; Gu, Zhen; Tai, April; Lee, Chi-Lin; Tang, Yi; Wang, PinACS Nano (2011), 5 (5), 3523-3535CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The unique spectral properties of semiconductor quantum dots (QDs) enable long-term live-cell imaging and ultrasensitive detection of viral particles, which in turn can potentially provide a practical means for detailed anal. of the underlying mol. mechanisms of virus entry. In this study, the authors report a general method of labeling adeno-assocd. virus serotype 2 (AAV2) with QDs for enhanced visualization of the intracellular behavior of viruses in living target cells. It was found that the mild conditions required for this QD conjugation reaction allowed for the retention of viral infectivity of AAV2. Furthermore, quant. anal. of viral motility in living cells suggested that QD-labeling had no significant effect on the intracellular transport properties of AAV2 particles compared to those of conventional org. dye-labeled AAV2. The authors' imaging study demonstrated that QD-AAV2 was internalized mainly through a clathrin-dependent pathway and then trafficked through various endosomes. It was also obsd. that QD-AAV2 particles exploit the cytoskeleton network to facilitate their transport within cells, and the labeling study provided evidence that the ubiquitin-proteasome system was likely involved in the intracellular trafficking of AAV2, at least at the level of nuclear transport. Taken together, the authors' findings reveal the potential of this QD-labeling method for monitoring the intracellular dynamics of virus-host cell interactions and interrogating the mol. mechanisms of viral infection in greater detail.
- 137Schelhaas, M.; Ewers, H.; Rajamaki, M. L.; Day, P. M.; Schiller, J. T.; Helenius, A. Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions. PLoS Pathog. 2008, 4, e1000148 DOI: 10.1371/journal.ppat.1000148Google Scholar137Human papillomavirus type 16 entry: retrograde cell surface transport along actin-rich protrusionsSchelhaas Mario; Ewers Helge; Rajamaki Minna-Liisa; Day Patricia M; Schiller John T; Helenius AriPLoS pathogens (2008), 4 (9), e1000148 ISSN:.The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30-60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing.
- 138Martin-Acebes, M. A.; Vazquez-Calvo, A.; Gonzalez-Magaldi, M.; Sobrino, F. Foot-and-Mouth Disease Virus Particles Inactivated with Binary Ethylenimine Are Efficiently Internalized into Cultured Cells. Vaccine 2011, 29, 9655– 9662, DOI: 10.1016/j.vaccine.2011.10.031Google Scholar138Foot-and-mouth disease virus particles inactivated with binary ethylenimine are efficiently internalized into cultured cellsMartin-Acebes, Miguel A.; Vazquez-Calvo, Angela; Gonzalez-Magaldi, Monica; Sobrino, FranciscoVaccine (2011), 29 (52), 9655-9662CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Conventional foot-and-mouth disease (FMD) vaccines are produced from virus grown in cell culture that is chem. inactivated by using binary ethylenimide (BEI). Here, we show that BEI treatment preserves both the architecture of FMDV particles, as inactivated viral particles showed by electron microscopy characteristics similar to those of infectious virions, as well as the general features of infectious virus internalization. Binding of inactivated particles to BHK-21 cells was blocked by preincubation with either a FMDV-specific monoclonal antibody or a synthetic peptide spanning the integrin-binding viral motif Arg-Gly-Asp (RGD). In addn., these particles were internalized into cultured cells through endocytosis, being directed to early endosomes, as indicated by their colocalization with the marker protein Rab5. When purified BEI-inactivated virions were labeled and their interaction with live cultured cells analyzed by time-lapse fluorescence microscopy, a major subpopulation of virus particles, about 80%, was shown to undergo internalization into a static endosome population, insensitive to the microtubule depolymn. exerted by nocodazole, while the remaining subpopulation (about 20%) was dynamic and sensitive to this drug. Thus, BEI-inactivated particles provide an interesting tool to study early steps in FMDV-cell interactions enabling a distinction between FMDV internalization and productive infection. Possible implications for FMDV immune response elicited following vaccine administration are discussed.
- 139Ewers, H.; Smith, A. E.; Sbalzarini, I. F.; Lilie, H.; Koumoutsakos, P.; Helenius, A. Single-Particle Tracking of Murine Polyoma Virus-Like Particles on Live Cells and Artificial Membranes. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 15110– 15115, DOI: 10.1073/pnas.0504407102Google Scholar139Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranesEwers, Helge; Smith, Alicia E.; Sbalzarini, Ivo F.; Lilie, Hauke; Koumoutsakos, Petros; Helenius, AriProceedings of the National Academy of Sciences of the United States of America (2005), 102 (42), 15110-15115CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, anal. of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diam.), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside mols. by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.
- 140Cureton, D. K.; Massol, R. H.; Whelan, S. P.; Kirchhausen, T. The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly During Clathrin-Dependent Endocytosis. PLoS Pathog. 2010, 6, e1001127 DOI: 10.1371/journal.ppat.1001127Google ScholarThere is no corresponding record for this reference.
- 141Pelkmans, L.; Puntener, D.; Helenius, A. Local Actin Polymerization and Dynamin Recruitment in SV40-Induced Internalization of Caveolae. Science 2002, 296, 535– 539, DOI: 10.1126/science.1069784Google Scholar141Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolaePelkmans, Lucas; Puntener, Daniel; Helenius, AriScience (Washington, DC, United States) (2002), 296 (5567), 535-539CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. The authors found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.
- 142Cureton, D. K.; Harbison, C. E.; Cocucci, E.; Parrish, C. R.; Kirchhausen, T. Limited Transferrin Receptor Clustering Allows Rapid Diffusion of Canine Parvovirus into Clathrin Endocytic Structures. J. Virol. 2012, 86, 5330– 5340, DOI: 10.1128/JVI.07194-11Google Scholar142Limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structuresCureton, David K.; Harbison, Carole E.; Cocucci, Emanuele; Parrish, Colin R.; Kirchhausen, TomJournal of Virology (2012), 86 (9), 5330-5340CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here, the authors used fluorescence microscopy to directly visualize the assocn. of single canine parvovirus (CPV) capsids with cellular transferring receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. They found that most capsids assocd. with fewer than five TfRs and that ∼25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissocd. from the cell surface with a half-life of ∼30 s. Thus, the results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.
- 143Ehrlich, M.; Boll, W.; Van Oijen, A.; Hariharan, R.; Chandran, K.; Nibert, M. L.; Kirchhausen, T. Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits. Cell 2004, 118, 591– 605, DOI: 10.1016/j.cell.2004.08.017Google Scholar143Endocytosis by random initiation and stabilization of clathrin-coated pitsEhrlich, Marcelo; Boll, Werner; van Oijen, Antoine; Hariharan, Ramesh; Chandran, Kartik; Nibert, Max L.; Kirchhausen, TomasCell (Cambridge, MA, United States) (2004), 118 (5), 591-605CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approx. const. growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.
- 144Damm, E. M.; Pelkmans, L.; Kartenbeck, J.; Mezzacasa, A.; Kurzchalia, T.; Helenius, A. Clathrin- and Caveolin-1-Independent Endocytosis: Entry of Simian Virus 40 into Cells Devoid of Caveolae. J. Cell Biol. 2005, 168, 477– 488, DOI: 10.1083/jcb.200407113Google Scholar144Clathrin- and caveolin-1-independent endocytosis: Entry of simian virus 40 into cells devoid of caveolaeDamm, Eva-Maria; Pelkmans, Lucas; Kartenbeck, Juergen; Mezzacasa, Anna; Kurzchalia, Teymuras; Helenius, AriJournal of Cell Biology (2005), 168 (3), 477-488CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol- and tyrosine kinase- dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.
- 145Meier, R.; Franceschini, A.; Horvath, P.; Tetard, M.; Mancini, R.; Von Mering, C.; Helenius, A.; Lozach, P.-Y. Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration. J. Virol. 2014, 88, 8565– 8578, DOI: 10.1128/JVI.00388-14Google Scholar145Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetrationMeier, Roger; Franceschini, Andrea; Horvath, Peter; Tetard, Marilou; Mancini, Roberta; von Mering, Christian; Helenius, Ari; Lozach, Pierre-YvesJournal of Virology (2014), 88 (15), 8565-8578, 15 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used 2 genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence anal. of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but addnl. genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was assocd. with the VAMP3+ virus-contg. endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-neg. compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network.
- 146Kalin, S.; Amstutz, B.; Gastaldelli, M.; Wolfrum, N.; Boucke, K.; Havenga, M.; DiGennaro, F.; Liska, N.; Hemmi, S.; Greber, U. F. Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35. J. Virol. 2010, 84, 5336– 5350, DOI: 10.1128/JVI.02494-09Google Scholar146Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35Kalin, Stefan; Amstutz, Beat; Gastaldelli, Michele; Wolfrum, Nina; Boucke, Karin; Havenga, Menzo; Di Gennaro, Fabienne; Liska, Nicole; Hemmi, Silvio; Greber, Urs F.Journal of Virology (2010), 84 (10), 5336-5350CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here, the authors show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chems. against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-neg. proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were pos. for CD46, αν integrins, and also CtBP1. The results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.
- 147Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B. Cyanines During the 1990s: A Review. Chem. Rev. 2000, 100, 1973– 2011, DOI: 10.1021/cr990402tGoogle Scholar147Cyanines during the 1990s: A ReviewMishra, Amaresh; Behera, Rajani K.; Behera, Pradipta K.; Mishra, Bijaya K.; Behera, Gopa B.Chemical Reviews (Washington, D. C.) (2000), 100 (6), 1973-2011CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 401 refs. on synthesis, self-aggregation, nonlinear optical properties, adsorption, photodimerization and -isomerization, photodynamic therapy, dyes in organized systems, and as fluorescent ion sensors.
- 148Kvach, M. V.; Ustinov, A. V.; Stepanova, I. A.; Malakhov, A. D.; Skorobogatyi, M. V.; Shmanai, V. V.; Korshun, V. A. A Convenient Synthesis of Cyanine Dyes: Reagents for the Labeling of Biomolecules. Eur. J. Org. Chem. 2008, 2008, 2107– 2117, DOI: 10.1002/ejoc.200701190Google ScholarThere is no corresponding record for this reference.
- 149Yang, X.; Shi, C.; Tong, R.; Qian, W.; Zhau, H. E.; Wang, R.; Zhu, G.; Cheng, J.; Yang, V. W.; Cheng, T. Near IR Heptamethine Cyanine Dye-Mediated Cancer Imaging. Clin. Cancer Res. 2010, 16, 2833– 2844, DOI: 10.1158/1078-0432.CCR-10-0059Google Scholar149Near IR Heptamethine Cyanine Dye-Mediated Cancer ImagingYang, Xiaojian; Shi, Chunmeng; Tong, Rong; Qian, Weiping; Zhau, Haiyen E.; Wang, Ruoxiang; Zhu, Guodong; Cheng, Jianjun; Yang, Vincent W.; Cheng, Tianmin; Henary, Maged; Strekowski, Lucjan; Chung, Leland W. K.Clinical Cancer Research (2010), 16 (10), 2833-2844CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Near-IR fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we show the preferential uptake and retention of two heptamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues. IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts, and spontaneous mouse tumors in transgenic animals. Time- and concn.-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein, a competitive inhibitor of org. anion transporting peptides. These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood. These near-IR heptamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to conc. in the mitochondria and lysosomes of cancer cells, probably through org. anion transporting peptides, because the dye uptake and retention in cancer cells can be blocked completely by bromosulfophthalein. These dyes, when injected to mice, did not cause systemic toxicity. These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis, and treatment.
- 150Brauchle, C.; Seisenberger, G.; Endress, T.; Ried, M. U.; Buning, H.; Hallek, M. Single Virus Tracing: Visualization of the Infection Pathway of a Virus into a Living Cell. ChemPhysChem 2002, 3, 299– 303, DOI: 10.1002/1439-7641(20020315)3:3<299::AID-CPHC299>3.0.CO;2-RGoogle Scholar150Single virus tracing: visualization of the infection pathway of a virus into a living cellBrauchle, Christoph; Seisenberger, Georg; Endress, Thomas; Ried, Martin U.; Buning, Hildegard; Hallek, MichaelChemPhysChem (2002), 3 (3), 299-303CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH)The novel technique, known as single virus tracing (SVT), allows the visualization of the infection pathway of an individual virus labeled with only one fluorescent dye mol. into a living cell. The fluorescence of the marker mol. is imaged with single-mol. techniques and used to follow the pathway of the virus with high spatial (>40 nm) and time (>10 ms) resoln. An overview of the different steps, which can be visualized and kinetically characterized by SVT, is presented for the model system, the adeno-assocd. virus (AAV). Since the capsid of the AAV becomes internalized into the cell, a fluorescent label can be attached either to the protein capsid, the DNA genome, or both. When this labeled virus is given to a living cell, a sequence of events can be monitored by SVT starting with the virus approaching the cell surface.
- 151Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.; Filanoski, B. J.; Telford, W. G.; Yue, S.; Liu, J. X.; Cheung, C. Y. Quantitative Comparison of Long-Wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates. J. Histochem. Cytochem. 2003, 51, 1699– 1712, DOI: 10.1177/002215540305101214Google Scholar151Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: Fluorescence of the dyes and their bioconjugatesBerlier, Judith E.; Rothe, Anca; Buller, Gayle; Bradford, Jolene; Gray, Diane R.; Filanoski, Brian J.; Telford, William G.; Yue, Stephen; Liu, Jixiang; Cheung, Ching-Ying; Chang, Wesley; Hirsch, James D.; Beechem, Joseph M.; Haugland, Rosaria P.; Haugland, Richard P.Journal of Histochemistry and Cytochemistry (2003), 51 (12), 1699-1712CODEN: JHCYAS; ISSN:0022-1554. (Histochemical Society, Inc.)Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coeffs. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepd. from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously obsd. for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, esp. at high degrees of labeling. The results from our flow cytometry, immunocytochem., and immunohistochem. expts. demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.
- 152Mahmoudian, J.; Hadavi, R.; Jeddi-Tehrani, M.; Mahmoudi, A. R.; Bayat, A. A.; Shaban, E.; Vafakhah, M.; Darzi, M.; Tarahomi, M.; Ghods, R. Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568-and Fluorescein Isothiocyanate- Conjugated Antibody. Cell J. 2011, 13, 169– 172Google Scholar152Comparison of the photobleaching and photostability traits of Alexa fluor 568- and fluorescein isothiocyanate- conjugated antibodyMahmoudian, Jafar; Hadavi, Reza; Jeddi-Tehrani, Mahmood; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Shaban, Elham; Vafakhah, Mohtaram; Darzi, Maryam; Tarahomi, Majid; Ghods, RoyaCell Journal (2011), 13 (3), 169-172,, 1 plateCODEN: CJEOAD; ISSN:2228-5806. (Royan Institute)Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe mols. Based on dye chem. structures, their photobleaching and photostability indexes are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleaching profiles than FITC. Materials and Methods: In this exptl. study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indexes. Then, the fluorophore/antibody ratios were calcd. using a spectrophotometer. The photobleaching profiles and photostability indexes of conjugated antibodies were subsequently studied by immunocytochem. (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.
- 153Hoekstra, D.; de Boer, T.; Klappe, K.; Wilschut, J. Fluorescence Method for Measuring the Kinetics of Fusion between Biological Membranes. Biochemistry 1984, 23, 5675– 5681, DOI: 10.1021/bi00319a002Google Scholar153Fluorescence method for measuring the kinetics of fusion between biological membranesHoekstra, Dick; De Boer, Tiny; Klappe, Karin; Wilschut, JanBiochemistry (1984), 23 (24), 5675-81CODEN: BICHAW; ISSN:0006-2960.An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biol. membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concns. up to 9 mol % with respect to total lipid, the efficiency of self-quenching is proportional to its surface d. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface d. of the fluorophores results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quant. measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were the same as those detd. with a fusion assay based on resonance energy transfer (Struck, D. K. et al., 1981). Octadecyl Rhodamine B chloride can be readily inserted into native biol. membranes by addn. of an ethanolic soln. of the probe. Evidence is presented showing that the diln. of the fluorophore, occurring when octadecyl Rhodamine contg. influenza viruses are mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Diln. of the probe was not obsd. upon prior treatment of fluorescently labeled Sendai virus with trypsin. Virus-induced fusion between fluorescently tagged ghosts and ghosts devoid of the probe was only obsd. (at 37°) after a low-temp. preincubation; no fluorescence developed was seen during virus-induced aggregation at low temp. nor when ghosts and the virus were directly mixed at 37°. Apparently, spontaneous intermembrane transfer of the fluorophore did not occur. This technique may be of considerable value for investigating fusion between biol. membranes and, hence, provides an important tool in elucidating the mechanism of fusion in such systems.
- 154Floyd, D. L.; Ragains, J. R.; Skehel, J. J.; Harrison, S. C.; van Oijen, A. M. Single-Particle Kinetics of Influenza Virus Membrane Fusion. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15382– 15387, DOI: 10.1073/pnas.0807771105Google Scholar154Single-particle kinetics of influenza virus membrane fusionFloyd, Daniel L.; Ragains, Justin R.; Skehel, John J.; Harrison, Stephen C.; van Oijen, Antoine M.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (40), 15382-15387CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed anal. of the sequence of the mol. steps involved. The authors have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Anal. of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. The authors interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.
- 155Jha, N. K.; Latinovic, O.; Martin, E.; Novitskiy, G.; Marin, M.; Miyauchi, K.; Naughton, J.; Young, J. A.; Melikyan, G. B. Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion. PLoS Pathog. 2011, 7, e1001260 DOI: 10.1371/journal.ppat.1001260Google Scholar155Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusionJha, Naveen K.; Latinovic, Olga; Martin, Erik; Novitskiy, Gennadiy; Marin, Mariana; Miyauchi, Kosuke; Naughton, John; Young, John A. T.; Melikyan, Gregory B.PLoS Pathogens (2011), 7 (1), e1001260CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A crit. barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor d. and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a crit. role in establishing low pH-dependent virus entry from within acidic endosomes.
- 156Hoornweg, T. E.; van Duijl-Richter, M. K. S.; Ayala Nunez, N. V.; Albulescu, I. C.; van Hemert, M. J.; Smit, J. M. Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells. J. Virol. 2016, 90, 4745– 4756, DOI: 10.1128/JVI.03184-15Google Scholar156Dynamics of chikungunya virus cell entry unraveled by single-virus tracking in living cellsHoornweg, Tabitha E.; van Duijl-Richter, Mareike K. S.; Nunez, Nilda V. Ayala; Albulescu, Irina C.; van Hemert, Martijn J.; Smit, Jolanda M.Journal of Virology (2016), 90 (9), 4745-4756CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We obsd. that almost all particles fused within 20 min after addn. to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The av. time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was obsd. predominantly from within Rab5-pos. endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes.
- 157Li, Q.; Li, W.; Yin, W.; Guo, J.; Zhang, Z. P.; Zeng, D.; Zhang, X.; Wu, Y.; Zhang, X. E.; Cui, Z. Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages. ACS Nano 2017, 11, 3890– 3903, DOI: 10.1021/acsnano.7b00275Google Scholar157Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary MacrophagesLi, Qin; Li, Wei; Yin, Wen; Guo, Jia; Zhang, Zhi-Ping; Zeng, Dejun; Zhang, Xiaowei; Wu, Yuntao; Zhang, Xian-En; Cui, ZongqiangACS Nano (2017), 11 (4), 3890-3903CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and obsd. that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-pos. endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Addnl., we obsd. that a dynamic actin cytoskeleton is crit. for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.
- 158Nanbo, A.; Imai, M.; Watanabe, S.; Noda, T.; Takahashi, K.; Neumann, G.; Halfmann, P.; Kawaoka, Y. Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner. PLoS Pathog. 2010, 6, e1001121 DOI: 10.1371/journal.ppat.1001121Google ScholarThere is no corresponding record for this reference.
- 159Hao, X.; Shang, X.; Wu, J. Z.; Shan, Y. P.; Cai, M. J.; Jiang, J. G.; Huang, Z.; Tang, Z. Y.; Wang, H. D. Single-Particle Tracking of Hepatitis B Virus-Like Vesicle Entry into Cells. Small 2011, 7, 1212– 1218, DOI: 10.1002/smll.201002020Google Scholar159Single-Particle Tracking of Hepatitis B Virus-like Vesicle Entry into CellsHao, Xian; Shang, Xin; Wu, Jiazhen; Shan, Yuping; Cai, Mingjun; Jiang, Junguang; Huang, Zhong; Tang, Zhiyong; Wang, HongdaSmall (2011), 7 (9), 1212-1218CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)HBsAg, the surface antigen of the hepatitis B virus (HBV), is used as a model to study the mechanisms and dynamics of a single-enveloped virus infecting living cells by imaging and tracking at the single-particle level. By monitoring the fluorescent indicator of HBsAg particles, it is found that HBsAg enters cells via a caveolin-mediated endocytic pathway. Tracking of individual HBsAg particles in living cells reveals the anomalously actin-dependent but not microtubule-dependent motility of the internalized HBsAg particle. The motility of HBsAg particles in living cells is also analyzed quant. These results may settle the long-lasting debate of whether HBV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell, and how the virus moves in the cell.
- 160Le Blanc, I.; Luyet, P. P.; Pons, V.; Ferguson, C.; Emans, N.; Petiot, A.; Mayran, N.; Demaurex, N.; Faure, J.; Sadoul, R. Endosome-to-Cytosol Transport of Viral Nucleocapsids. Nat. Cell Biol. 2005, 7, 653– 664, DOI: 10.1038/ncb1269Google Scholar160Endosome-to-cytosol transport of viral nucleocapsidsLe Blanc, Isabelle; Luyet, Pierre-Philippe; Pons, Veronique; Ferguson, Charles; Emans, Neil; Petiot, Anne; Mayran, Nathalie; Demaurex, Nicolas; Faure, Julien; Sadoul, Remy; Parton, Robert G.; Gruenberg, J.Nature Cell Biology (2005), 7 (7), 653-664CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns(3)P) signaling via the PtdIns(3)P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns(3)P and their effectors.
- 161Lozach, P.-Y.; Kühbacher, A.; Meier, R.; Mancini, R.; Bitto, D.; Bouloy, M.; Helenius, A. DC-SIGN as a Receptor for Phleboviruses. Cell Host Microbe 2011, 10, 75– 88, DOI: 10.1016/j.chom.2011.06.007Google Scholar161DC-SIGN as a Receptor for PhlebovirusesLozach, Pierre-Yves; Kuehbacher, Andreas; Meier, Roger; Mancini, Roberta; Bitto, David; Bouloy, Michele; Helenius, AriCell Host & Microbe (2011), 10 (1), 75-88CODEN: CHMECB; ISSN:1931-3128. (Cell Press)Summary: During natural transmission, bunyaviruses are introduced into the skin through arthropod bites, and dermal dendritic cells (DCs) are the first to encounter incoming viruses. DC-SIGN is a C-type lectin highly expressed on the surface of dermal DCs. We found that several arthropod-borne phleboviruses (Bunyaviridae), including Rift Valley fever and Uukuniemi viruses, exploit DC-SIGN to infect DCs and other DC-SIGN-expressing cells. DC-SIGN binds the virus directly via interactions with high-mannose N-glycans on the viral glycoproteins and is required for virus internalization and infection. In live cells, virus-induced clustering of cell surface DC-SIGN could be visualized. An endocytosis-defective mutant of DC-SIGN was unable to mediate virus uptake, indicating that DC-SIGN is an authentic receptor required for both attachment and endocytosis. After internalization, viruses sepd. from DC-SIGN and underwent trafficking to late endosomes. Our study provides real-time visualization of virus-receptor interactions on the cell surface and establishes DC-SIGN as a phlebovirus entry receptor.
- 162Ayala-Nuñez, N. V.; Wilschut, J.; Smit, J. M. Monitoring Virus Entry into Living Cells Using DiD-Labeled Dengue Virus Particles. Methods 2011, 55, 137– 143, DOI: 10.1016/j.ymeth.2011.07.009Google Scholar162Monitoring virus entry into living cells using DiD-labeled dengue virus particlesAyala-Nunez, Nilda V.; Wilschut, Jan; Smit, Jolanda M.Methods (Amsterdam, Netherlands) (2011), 55 (2), 137-143CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)A review. A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addn. - and using the same exptl. setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data anal., and hints for troubleshooting are described throughout the paper.
- 163Crowther, D.; Melnick, J. L. The Incorporation of Neutral Red and Acridine Orange into Developing Poliovirus Particles Making Them Photosensitive. Virology 1961, 14, 11– 21, DOI: 10.1016/0042-6822(61)90127-1Google Scholar163Incorporation of neutral red and Acridine Orange into developing poliovirus particles making them photosensitiveCrowther, Derek; Melnick, Joseph L.Virology (1961), 14 (1), 11-21CODEN: VIRLAX; ISSN:0042-6822.Mature, infective virus incubated with neutral red and Acridine orange for one hour in the absence of cells and then exposed to white light showed no redn. in titer. Virus grown in cells contg. neutral red or Acridine Orange may be subsequently inactivated by light, thus indicating an incorporation of the dye into the developing virus. The effect of neutral red and light was the same for the virulent and the attenuated strains. The biol. and chem. similarities between the quinonimide dyes (neutral red, toluidine blue) and the acridine (Acridine Orange, proflavine) are discussed.
- 164Wilson, J. N.; Cooper, P. D. Aspects of the Growth of Poliovirus as Revealed by the Photodynamic Effects of Neutral Red and Acridine Orange. Virology 1963, 21, 135– 145, DOI: 10.1016/0042-6822(63)90249-6Google Scholar164ASPECTS OF THE GROWTH OF POLIOVIRUS AS REVEALED BY THE PHOTODYNAMIC EFFECTS OF NEUTRAL RED AND ACRIDINE ORANGEWILSON J N; COOPER P DVirology (1963), 21 (), 135-45 ISSN:0042-6822.There is no expanded citation for this reference.
- 165Glynn, T. J.; Power, S.; Ryder, A. G.; Morrison, J. J. Time-Domain Measurement of Fluorescence Lifetime Variation with pH. Proc. SPIE-Int. Soc. Opt. Eng.; Society of Photo-optical Instrumentation Engineers: 2001.Google ScholarThere is no corresponding record for this reference.
- 166Kremser, L.; Okun, V. M.; Nicodemou, A.; Blaas, D.; Kenndler, E. Binding of Fluorescent Dye to Genomic RNA inside Intact Human Rhinovirus after Viral Capsid Penetration Investigated by Capillary Electrophoresis. Anal. Chem. 2004, 76, 882– 887, DOI: 10.1021/ac034898xGoogle Scholar166Binding of Fluorescent Dye to Genomic RNA Inside Intact Human Rhinovirus after Viral Capsid Penetration Investigated by Capillary ElectrophoresisKremser, Leopold; Okun, Vadim M.; Nicodemou, Andreas; Blaas, Dieter; Kenndler, ErnstAnalytical Chemistry (2004), 76 (4), 882-887CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)RiboGreen is used for concn. measurements of RNA. Upon binding to the RNA, an ∼1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is obsd. In the present work, we demonstrate that this dye can penetrate in a time- and temp.-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant sol. receptor fragment derived from the very low d. lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degrdn. was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-mol.-mass compd. to the otherwise protected RNA. It might be of great value for expts. requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single mol. level.
- 167Kremser, L.; Petsch, M.; Blaas, D.; Kenndler, E. Labeling of Capsid Proteins and Genomic RNA of Human Rhinovirus with Two Different Fluorescent Dyes for Selective Detection by Capillary Electrophoresis. Anal. Chem. 2004, 76, 7360– 7365, DOI: 10.1021/ac048999mGoogle Scholar167Labeling of Capsid Proteins and Genomic RNA of Human Rhinovirus with Two Different Fluorescent Dyes for Selective Detection by Capillary ElectrophoresisKremser, Leopold; Petsch, Martina; Blaas, Dieter; Kenndler, ErnstAnalytical Chemistry (2004), 76 (24), 7360-7365CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)During uncoating of human rhinoviruses, the innermost capsid protein VP4 and the genomic RNA are released from the viral protein shell. This process gives rise to subviral particles that are composed of the remaining three capsid proteins VP1, VP2, and VP3. The process is believed to take place in a sequential manner in that first VP4 is expelled resulting in A-particles sedimenting at 135S followed by the RNA resulting in B-particles sedimenting at 80S. Aiming at ultimately analyzing this process in vivo, we introduced two different fluorophores into the RNA and the viral capsid proteins, resp. Incubation of the virus with RiboGreen resulted in formation of a RNA-dye complex with λex/λem = 500/525 nm, whereas subsequent derivatization of the viral protein shell in the same sample with AMCA-S introduced a label with λex/λem = 345-350/440-460 nm. In this way, both viral components could be selectively detected via fluorescence in a capillary electrophoresis system. The intact virus delivers two superimposed signals in the electropherogram. Derivatization of the free amino groups of the capsid proteins partially preserved the bioaffinity of the virus toward a synthetic receptor fragment, an artificial recombinant concatemer of repeat no. 3 of the very low d. lipoprotein receptor. Between 10 and 20% of the infectivity were recovered after labeling when compared to native virus. In addn. to anal. of factors influencing the stability of the virus by CE, double-labeled virions might be useful for the investigation of the uncoating process by real-time confocal fluorescence microscopy.
- 168Jones, L. J.; Yue, S. T.; Cheung, C. Y.; Singer, V. L. RNA Quantitation by Fluorescence-Based Solution Assay: RiboGreen Reagent Characterization. Anal. Biochem. 1998, 265, 368– 374, DOI: 10.1006/abio.1998.2914Google Scholar168RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterizationJones, Laurie J.; Yue, Stephen T.; Cheung, Ching-Ying; Singer, Victoria L.Analytical Biochemistry (1998), 265 (2), 368-374CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)We described the development of a sensitive fluorescence-based soln. assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits > 1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coeff. (67,000 cm-1 M-1). The RiboGreen assay allows detection of as little as 1.0 ng/mL RNA in a std. fluorometer, filter fluorometer, or fluorescence microplate reader-surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range for RiboGreen reagent extends over three orders of magnitude in RNA concn. Using 750 nM RiboGreen reagent, we quantitated 20 ng/mL to 1.0 μg/mL RNA. By dilg. the reagent to 75 nM, we could quantitate 1.0 to 50 ng/mL RNA. Both assay ranges exhibited linear fluorescence increases vs. RNA concn. (r2 = 0.999). Assay linearity was maintained in the presence of salts, protein, urea, ethanol, chloroform, agarose, and some detergents. Several different RNA types yielded similar signal intensities and detection sensitivities. The assay is easy to use, rapid, and readily adaptable for automation. (c) 1998 Academic Press.
- 169Lin, Y.-W.; Chiu, T.-C.; Chang, H.-T. Laser-Induced Fluorescence Technique for DNA and Proteins Separated by Capillary Electrophoresis. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2003, 793, 37– 48, DOI: 10.1016/S1570-0232(03)00363-5Google Scholar169Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresisLin, Yang-Wei; Chiu, Tai-Chia; Chang, Huan-TsungJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2003), 793 (1), 37-48CODEN: JCBAAI; ISSN:1570-0232. (Elsevier Science B.V.)A review. Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (max. excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chems. can be carried out prior to, during, or after sepns. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the anal. of polymerase chain reaction products, DNA sequencing, trace anal. of proteins, and single cell anal. were presented.
- 170Wen, L.; Lin, Y.; Zhang, Z. L.; Lu, W.; Lv, C.; Chen, Z. L.; Wang, H. Z.; Pang, D. W. Intracellular Self-Assembly Based Multi-Labeling of Key Viral Components: Envelope, Capsid and Nucleic Acids. Biomaterials 2016, 99, 24– 33, DOI: 10.1016/j.biomaterials.2016.04.038Google Scholar170Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acidsWen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-WenBiomaterials (2016), 99 (), 24-33CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissocn. and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment.
- 171Zhou, P.; Zheng, Z.; Lu, W.; Zhang, F.; Zhang, Z.; Pang, D.; Hu, B.; He, Z.; Wang, H. Multicolor Labeling of Living-Virus Particles in Live Cells. Angew. Chem., Int. Ed. 2012, 51, 670– 674, DOI: 10.1002/anie.201105701Google Scholar171Multicolor Labeling of Living-Virus Particles in Live CellsZhou, Peng; Zheng, Zhenhua; Lu, Wen; Zhang, Fuxian; Zhang, Zhenfeng; Pang, Daiwen; Hu, Bin; He, Zhike; Wang, HanzhongAngewandte Chemie, International Edition (2012), 51 (3), 670-674, S670/1-S670/3CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Herein, the authors describe a new strategy for synchronous multicolor labeling of a living virus in live cells. The viral genomes were labeled by using an in vivo virus self-assembly system in combination with a novel nucleic acid probe based on a ruthenium complex, and the viral envelope was labeled by fusing a characteristic external viral protein with green fluorescent protein (GFP). Multicolor labeling of distinct viral components can be done during the viral replication in host cells. More importantly, labeling a virus in this manner can overcome the photobleaching and self-quenching effects between dye mols. and does not affect the viral infectivity.
- 172Huang, L. L.; Zhou, P.; Wang, H. Z.; Zhang, R.; Hao, J.; Xie, H. Y.; He, Z. K. A New Stable and Reliable Method for Labeling Nucleic Acids of Fully Replicative Viruses. Chem. Commun. 2012, 48, 2424– 2426, DOI: 10.1039/c2cc17069hGoogle Scholar172A new stable and reliable method for labeling nucleic acids of fully replicative virusesHuang, Li-Li; Zhou, Peng; Wang, Han-Zhong; Zhang, Rui; Hao, Jian; Xie, Hai-Yan; He, Zhi-KeChemical Communications (Cambridge, United Kingdom) (2012), 48 (18), 2424-2426CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Efficiently labeling nucleic acids of fully replicative viruses is a challenge. In this work, a mol. light switch complex [Ru(phen)2(dppz)]2+, where phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2',3'-c]phenazine, has been exploringly used to label vaccinia virus nucleic acid. The labeled virions exhibited strong and stable fluorescence and could be imaged at the single-virion level. Moreover, they were fully infectious and can be used to study the behaviors of invasion into their host cells. The method is general and suitable for labeling various DNA viruses.
- 173Shimomura, O.; Johnson, F. H.; Saiga, Y. Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. J. Cell. Comp. Physiol. 1962, 59, 223– 239, DOI: 10.1002/jcp.1030590302Google Scholar173Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, AequoreaShimomura, Osamu; Johnson, Frank H.; Saiga, YoJournal of Cellular and Comparative Physiology (1962), 59 (), 223-39CODEN: JCCPAY; ISSN:0095-9898.cf. CA 57, 1392b. The material obtained by squeezing marginal strips of Aequorea through cloth was filtered with filtercel and the cake extd. with di-Na ethylenediaminetetraacetate (EDTA). Pptn. with (NH4)2SO4 and chromatography on diethylaminoethyl cellulose gave aequorin (I) with the properties of a protein of mol. wt. approx. 35,000. Addn. of Ca++ in an amt. at least the molar equiv. to that of EDTA present produced a flash of light. No other ion could replace Ca++. The rate of light emission followed 1st order reaction kinetics; O was not necessary and only a single component (in addn. to Ca++) could be identified. Total light produced decreased with rise in temp. from 0° to 40° and was independent of pH between 5.1 and 8.3. The rate of emission was nearly const. between pH 6.5 and 7.5, decreasing at lower and increasing at higher values. Malonate, EDTA, and a series of aliphatic aldehydes (C3-C7) reversibly inhibited the rate but not the total of light emission. Aromatic aldehydes, inorg. reducing or strong oxidizing agents, p-ClHgC6H4CO2H, HgCl2, N1-methylnicotinamide chloride, hydroquinone, benzoquinone, histidine-HCl, dinitrofluorobenzene-NaHCO3, and NCCH2CO2H reduced the total light without affecting the rate of emission; H2O2 had no effect. Aliphatic alcs. increased light emission; C6H5CH2OH caused a slight decrease. The absorption spectrum of I shows a max. at 280 mμ with a bulge at 310 mμ. In the luminescent reaction the bulge is replaced by a new max. at 333 mμ. These changes suggest the presence of a reduced pyridinium deriv. combined with the protein.
- 174Chalfie, M. Green Fluorescent Protein as a Marker for Gene Expression. Trends Genet. 1994, 10, 151, DOI: 10.1016/0168-9525(94)90088-4Google ScholarThere is no corresponding record for this reference.
- 175Lippincott-Schwartz, J.; Patterson, G. H. Development and Use of Fluorescent Protein Markers in Living Cells. Science 2003, 300, 87– 91, DOI: 10.1126/science.1082520Google Scholar175Development and use of fluorescent protein markers in living cellsLippincott-Schwartz, Jennifer; Patterson, George H.Science (Washington, DC, United States) (2003), 300 (5616), 87-91CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. The ability to visualize, track, and quantify mols. and events in living cells with high spatial and temporal resoln. is essential for understanding biol. systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technol. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.
- 176Day, R. N.; Davidson, M. W. The Fluorescent Protein Palette: Tools for Cellular Imaging. Chem. Soc. Rev. 2009, 38, 2887– 2921, DOI: 10.1039/b901966aGoogle Scholar176The fluorescent protein palette: Tools for cellular imagingDay, Richard N.; Davidson, Michael W.Chemical Society Reviews (2009), 38 (10), 2887-2921CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)This crit. review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biol. and physiol. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quant. measurements in living systems (227 refs.).
- 177Wiedenmann, J.; Oswald, F.; Nienhaus, G. U. Fluorescent Proteins for Live Cell Imaging: Opportunities, Limitations, and Challenges. IUBMB Life 2009, 61, 1029– 1042, DOI: 10.1002/iub.256Google Scholar177Fluorescent proteins for live cell imaging: opportunities, limitations, and challengesWiedenmann, Jorg; Oswald, Franz; Nienhaus, Gerd UlrichIUBMB Life (2009), 61 (11), 1029-1042CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons Inc.)A review. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse mol. properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.
- 178Shaner, N. C.; Campbell, R. E.; Steinbach, P. A.; Giepmans, B. N.; Palmer, A. E.; Tsien, R. Y. Improved Monomeric Red, Orange and Yellow Fluorescent Proteins Derived from Discosoma sp. Red Fluorescent Protein. Nat. Biotechnol. 2004, 22, 1567– 1572, DOI: 10.1038/nbt1037Google Scholar178Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteinShaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y.Nature Biotechnology (2004), 22 (12), 1567-1572CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biol. and biotechnol. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
- 179Shkrob, M. A.; Yanushevich, Y. G.; Chudakov, D. M.; Gurskaya, N. G.; Labas, Y. A.; Poponov, S. Y.; Mudrik, N. N.; Lukyanov, S.; Lukyanov, K. A. Far-Red Fluorescent Proteins Evolved From a Blue Chromoprotein from Actinia Equina. Biochem. J. 2005, 392, 649– 654, DOI: 10.1042/BJ20051314Google Scholar179Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equinaShkrob, Maria A.; Yanushevich, Yurii G.; Chudakov, Dmitriy M.; Gurskaya, Nadya G.; Labas, Yulii A.; Poponov, Sergey Y.; Mudrik, Nikolay N.; Lukyanov, Sergey; Lukyanov, Konstantin A.Biochemical Journal (2005), 392 (3), 649-654CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-colored Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disk. A novel blue chromoprotein, aeCP597, with an absorption max. at 597 nm detg. the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chem. identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is detd. by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm resp. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.
- 180Nienhaus, K.; Nienhaus, G. U. Fluorescent Proteins for Live-Cell Imaging with Super-Resolution. Chem. Soc. Rev. 2014, 43, 1088– 1106, DOI: 10.1039/C3CS60171DGoogle Scholar180Fluorescent proteins for live-cell imaging with super-resolutionNienhaus, Karin; Ulrich Nienhaus, G.Chemical Society Reviews (2014), 43 (4), 1088-1106CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradn. at specific wavelengths. They enable regional optical marking in pulse-chase expts. on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resoln. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chem. reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resoln. microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many labs., there is still room for further improvement of these important markers for live cell imaging.
- 181Shcherbakova, D. M.; Verkhusha, V. V. Near-Infrared Fluorescent Proteins for Multicolor in Vivo Imaging. Nat. Methods 2013, 10, 751– 754, DOI: 10.1038/nmeth.2521Google Scholar181Near-infrared fluorescent proteins for multicolor in vivo imagingShcherbakova, Daria M.; Verkhusha, Vladislav V.Nature Methods (2013), 10 (8), 751-754CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Near-IR fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-IR FPs-iRFP670, iRFP682, iRFP702 and iRFP720-from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with std. approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.
- 182Shcherbakova, D. M.; Subach, O. M.; Verkhusha, V. V. Red Fluorescent Proteins: Advanced Imaging Applications and Future Design. Angew. Chem., Int. Ed. 2012, 51, 10724– 10738, DOI: 10.1002/anie.201200408Google Scholar182Red Fluorescent Proteins: Advanced Imaging Applications and Future DesignShcherbakova, Daria M.; Subach, Oksana M.; Verkhusha, Vladislav V.Angewandte Chemie, International Edition (2012), 51 (43), 10724-10738CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. In the past few years a large series of the advanced red shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biol. processes at the levels ranging from single mols. to whole organisms. Herein the relation between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chem. transformations of red chromophores allows the future RFP phenotypes and their resp. novel imaging applications to be foreseen.
- 183Tomosugi, W.; Matsuda, T.; Tani, T.; Nemoto, T.; Kotera, I.; Saito, K.; Horikawa, K.; Nagai, T. An Ultramarine Fluorescent Protein with Increased Photostability and pH Insensitivity. Nat. Methods 2009, 6, 351– 353, DOI: 10.1038/nmeth.1317Google Scholar183An ultramarine fluorescent protein with increased photostability and pH insensitivityTomosugi, Wataru; Matsuda, Tomoki; Tani, Tomomi; Nemoto, Tomomi; Kotera, Ippei; Saito, Kenta; Horikawa, Kazuki; Nagai, TakeharuNature Methods (2009), 6 (5), 351-353CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biol. events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concn. and caspase-3 activation in the same apoptotic cells.
- 184Ai, H. W.; Shaner, N. C.; Cheng, Z.; Tsien, R. Y.; Campbell, R. E. Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins. Biochemistry 2007, 46, 5904– 5910, DOI: 10.1021/bi700199gGoogle Scholar184Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent ProteinsAi, Hui-Wang; Shaner, Nathan C.; Cheng, Zihao; Tsien, Roger Y.; Campbell, Robert E.Biochemistry (2007), 46 (20), 5904-5910CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein the authors report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".
- 185Subach, O. M.; Gundorov, I. S.; Yoshimura, M.; Subach, F. V.; Zhang, J.; Grüenwald, D.; Souslova, E. A.; Chudakov, D. M.; Verkhusha, V. V. Conversion of Red Fluorescent Protein into a Bright Blue Probe. Chem. Biol. 2008, 15, 1116– 1124, DOI: 10.1016/j.chembiol.2008.08.006Google Scholar185Conversion of Red Fluorescent Protein into a Bright Blue ProbeSubach, Oksana M.; Gundorov, Illia S.; Yoshimura, Masami; Subach, Fedor V.; Zhang, Jinghang; Gruenwald, David; Souslova, Ekaterina A.; Chudakov, Dmitriy M.; Verkhusha, Vladislav V.Chemistry & Biology (Cambridge, MA, United States) (2008), 15 (10), 1116-1124CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochem. and photochem. anal. indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Foerster resonance energy transfer applications.
- 186Goedhart, J.; van Weeren, L.; Hink, M. A.; Vischer, N. O.; Jalink, K.; Gadella, T. W., Jr. Bright Cyan Fluorescent Protein Variants Identified by Fluorescence Lifetime Screening. Nat. Methods 2010, 7, 137– 139, DOI: 10.1038/nmeth.1415Google Scholar186Bright cyan fluorescent protein variants identified by fluorescence lifetime screeningGoedhart, Joachim; van Weeren, Laura; Hink, Mark A.; Vischer, Norbert O. E.; Jalink, Kees; Gadella, Theodorus W. J., Jr.Nature Methods (2010), 7 (2), 137-139CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.
- 187Ai, H. W.; Henderson, J. N.; Remington, S. J.; Campbell, R. E. Directed Evolution of a Monomeric, Bright and Photostable Version of Clavularia Cyan Fluorescent Protein: Structural Characterization and Applications in Fluorescence Imaging. Biochem. J. 2006, 400, 531– 540, DOI: 10.1042/BJ20060874Google Scholar187Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imagingAi, Hui-wang; Henderson, J. Nathan; Remington, S. James; Campbell, Robert E.Biochemical Journal (2006), 400 (3), 531-540CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochem. and cell biol. research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, the authors report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, the authors identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, the authors arrived at an optimized variant that the authors have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addn., the fluorescence is insensitive to physiol. relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 Å crystal structure (1 Å = 0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As the authors exptl. demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
- 188Samarkina, O. N.; Popova, A. G.; Gvozdik, E. Y.; Chkalina, A. V.; Zvyagin, I. V.; Rylova, Y. V.; Rudenko, N. V.; Lusta, K. A.; Kelmanson, I. V.; Gorokhovatsky, A. Y. Universal and Rapid Method for Purification of GFP-Like Proteins by the Ethanol Extraction. Protein Expression Purif. 2009, 65, 108– 113, DOI: 10.1016/j.pep.2008.11.008Google Scholar188Universal and rapid method for purification of GFP-like proteins by the ethanol extractionSamarkina, Olga N.; Popova, Anastasia G.; Gvozdik, Elena Yu.; Chkalina, Anna V.; Zvyagin, Ivan V.; Rylova, Yulia V.; Rudenko, Natalia V.; Lusta, Konstantin A.; Kelmanson, Ilya V.; Gorokhovatsky, Andrey Yu.; Vinokurov, Leonid M.Protein Expression & Purification (2009), 65 (1), 108-113CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)GFP-like fluorescent proteins (FPs) are crucial in biol. and biomedical studies. The majority of FP purifn. techniques either include multiple time-consuming chromatog. steps with a low yield of the desired product or require prior protein modification (addn. of special tags). In the present work, the authors propose an alternative ethanol extn.-based technique previously used for GFP purifn. and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purifn. of any amt. of protein and requires no expensive reagents and equipment.
- 189Ai, H. W.; Olenych, S. G.; Wong, P.; Davidson, M. W.; Campbell, R. E. Hue-Shifted Monomeric Variants of Clavularia Cyan Fluorescent Protein: Identification of the Molecular Determinants of Color and Applications in Fluorescence Imaging. BMC Biol. 2008, 6, 13, DOI: 10.1186/1741-7007-6-13Google Scholar189Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imagingAi Hui-wang; Olenych Scott G; Wong Peter; Davidson Michael W; Campbell Robert EBMC biology (2008), 6 (), 13 ISSN:.BACKGROUND: In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. RESULTS: Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. CONCLUSION: Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.
- 190Shaner, N. C.; Lambert, G. G.; Chammas, A.; Ni, Y.; Cranfill, P. J.; Baird, M. A.; Sell, B. R.; Allen, J. R.; Day, R. N.; Israelsson, M. A Bright Monomeric Green Fluorescent Protein Derived from Branchiostoma Lanceolatum. Nat. Methods 2013, 10, 407– 409, DOI: 10.1038/nmeth.2413Google Scholar190A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatumShaner, Nathan C.; Lambert, Gerard G.; Chammas, Andrew; Ni, Yuhui; Cranfill, Paula J.; Baird, Michelle A.; Sell, Brittney R.; Allen, John R.; Day, Richard N.; Israelsson, Maria; Davidson, Michael W.; Wang, JiwuNature Methods (2013), 10 (5), 407-409CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. MNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to the authors' knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-mol. superresoln. imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
- 191Miyawaki, A.; Griesbeck, O.; Heim, R.; Tsien, R. Y. Dynamic and Quantitative Ca2+ Measurements Using Improved Cameleons. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2135– 2140, DOI: 10.1073/pnas.96.5.2135Google Scholar191Dynamic and quantitative Ca2+ measurements using improved cameleonsMiyawaki, Atsushi; Griesbeck, Oliver; Heim, Roger; Tsien, Roger Y.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 2135-2140CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+ -buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.
- 192Griesbeck, O.; Baird, G. S.; Campbell, R. E.; Zacharias, D. A.; Tsien, R. Y. Reducing the Environmental Sensitivity of Yellow Fluorescent Protein Mechanism and Applications. J. Biol. Chem. 2001, 276, 29188– 29194, DOI: 10.1074/jbc.M102815200Google Scholar192Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applicationsGriesbeck, Oliver; Baird, Geoffrey S.; Campbell, Robert E.; Zacharias, David A.; Tsien, Roger Y.Journal of Biological Chemistry (2001), 276 (31), 29188-29194CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37°. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pKα (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37° and in organelles. The halide resistance is explained by a 2.2-Å x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca2+] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiol. signals.
- 193Sakaue-Sawano, A.; Kurokawa, H.; Morimura, T.; Hanyu, A.; Hama, H.; Osawa, H.; Kashiwagi, S.; Fukami, K.; Miyata, T.; Miyoshi, H. Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell 2008, 132, 487– 498, DOI: 10.1016/j.cell.2007.12.033Google Scholar193Visualizing spatiotemporal dynamics of multicellular cell-cycle progressionSakaue-Sawano, Asako; Kurokawa, Hiroshi; Morimura, Toshifumi; Hanyu, Aki; Hama, Hiroshi; Osawa, Hatsuki; Kashiwagi, Saori; Fukami, Kiyoko; Miyata, Takaki; Miyoshi, Hiroyuki; Imamura, Takeshi; Ogawa, Masaharu; Masai, Hisao; Miyawaki, AtsushiCell (Cambridge, MA, United States) (2008), 132 (3), 487-498CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resoln. to help us better understand how the cell cycle is coordinated with various biol. events.
- 194Merzlyak, E. M.; Goedhart, J.; Shcherbo, D.; Bulina, M. E.; Shcheglov, A. S.; Fradkov, A. F.; Gaintzeva, A.; Lukyanov, K. A.; Lukyanov, S.; Gadella, T. W. Bright Monomeric Red Fluorescent Protein with an Extended Fluorescence Lifetime. Nat. Methods 2007, 4, 555– 557, DOI: 10.1038/nmeth1062Google Scholar194Bright monomeric red fluorescent protein with an extended fluorescence lifetimeMerzlyak, Ekaterina M.; Goedhart, Joachim; Shcherbo, Dmitry; Bulina, Mariya E.; Shcheglov, Aleksandr S.; Fradkov, Arkady F.; Gaintzeva, Anna; Lukyanov, Konstantin A.; Lukyanov, Sergey; Gadella, Theodorus W. J.; Chudakov, Dmitriy M.Nature Methods (2007), 4 (7), 555-557CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Fluorescent proteins have become extremely popular tools for in vivo imaging and esp. for the study of localization, motility and interaction of proteins in living cells. Here the authors report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
- 195Lam, A. J.; St-Pierre, F.; Gong, Y.; Marshall, J. D.; Cranfill, P. J.; Baird, M. A.; McKeown, M. R.; Wiedenmann, J.; Davidson, M. W.; Schnitzer, M. J. Improving FRET Dynamic Range with Bright Green and Red Fluorescent Proteins. Nat. Methods 2012, 9, 1005– 1012, DOI: 10.1038/nmeth.2171Google Scholar195Improving FRET dynamic range with bright green and red fluorescent proteinsLam, Amy J.; St-Pierre, Francois; Gong, Yiyang; Marshall, Jesse D.; Cranfill, Paula J.; Baird, Michelle A.; McKeown, Michael R.; Wiedenmann, Joerg; Davidson, Michael W.; Schnitzer, Mark J.; Tsien, Roger Y.; Lin, Michael Z.Nature Methods (2012), 9 (10), 1005-1012CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A variety of genetically encoded reporters use changes in fluorescence (or Foerster) resonance energy transfer (FRET) to report on biochem. processes in living cells. The std. genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Foerster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochem. events such as neuronal action-potential firing and RhoA activation in growth cones.
- 196Shcherbo, D.; Murphy, C. S.; Ermakova, G. V.; Solovieva, E. A.; Chepurnykh, T. V.; Shcheglov, A. S.; Verkhusha, V. V.; Pletnev, V. Z.; Hazelwood, K. L.; Roche, P. M. Far-Red Fluorescent Tags for Protein Imaging in Living Tissues. Biochem. J. 2009, 418, 567– 574, DOI: 10.1042/BJ20081949Google Scholar196Far-red fluorescent tags for protein imaging in living tissuesShcherbo, Dmitry; Murphy, Christopher S.; Ermakova, Galina V.; Solovieva, Elena A.; Chepurnykh, Tatiana V.; Shcheglov, Aleksandr S.; Verkhusha, Vladislav V.; Pletnev, Vladimir Z.; Hazelwood, Kristin L.; Roche, Patrick M.; Lukyanov, Sergey; Zaraisky, Andrey G.; Davidson, Michael W.; Chudakov, Dmitriy M.Biochemical Journal (2009), 418 (3), 567-574CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A vast color palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolor labeling and whole-body imaging techniques. In the present paper, the authors report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. The authors also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.
- 197Lin, M. Z.; McKeown, M. R.; Ng, H. L.; Aguilera, T. A.; Shaner, N. C.; Campbell, R. E.; Adams, S. R.; Gross, L. A.; Ma, W.; Alber, T. Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in Mammals. Chem. Biol. 2009, 16, 1169– 1179, DOI: 10.1016/j.chembiol.2009.10.009Google Scholar197Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in MammalsLin, Michael Z.; McKeown, Michael R.; Ng, Ho-Leung; Aguilera, Todd A.; Shaner, Nathan C.; Campbell, Robert E.; Adams, Stephen R.; Gross, Larry A.; Ma, Wendy; Alber, Tom; Tsien, Roger Y.Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (11), 1169-1179CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by Hb. Fluorescent proteins that excite efficiently in the "optical window" above 600 nm are therefore highly desirable. The authors report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.
- 198Piatkevich, K. D.; Malashkevich, V. N.; Morozova, K. S.; Nemkovich, N. A.; Almo, S. C.; Verkhusha, V. V. Extended Stokes Shift in Fluorescent Proteins: Chromophore-Protein Interactions in a Near-Infrared TagRFP675 Variant. Sci. Rep. 2013, 3, 1847, DOI: 10.1038/srep01847Google Scholar198Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variantPiatkevich, Kiryl D.; Malashkevich, Vladimir N.; Morozova, Kateryna S.; Nemkovich, Nicolai A.; Almo, Steven C.; Verkhusha, Vladislav V.Scientific Reports (2013), 3 (), 1847, 11 pp.CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Most green fluorescent protein (GFP)-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to the rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-IR deriv. of red fluorescent protein mKate, named TagRFP675, exhibits a Stokes shift, which is 30-nm extended in comparison to that of the parental protein. In physiol. conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addn., its emission max. strongly depends on the excitation wavelength. Here, crystal structure studies of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacted with the protein matrix via an extensive network of H-bonds capable of large flexibility. Based on spectroscopic, biochem., and structural analyses, the authors suggest that the rearrangement of the H-bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.
- 199Sankaranarayanan, S.; De Angelis, D.; Rothman, J. E.; Ryan, T. A. The Use of pHluorins for Optical Measurements of Presynaptic Activity. Biophys. J. 2000, 79, 2199– 2208, DOI: 10.1016/S0006-3495(00)76468-XGoogle Scholar199The use of pHluorins for optical measurements of presynaptic activitySankaranarayanan, Sethuraman; De Angelis, Dino; Rothman, James E.; Ryan, Timothy A.Biophysical Journal (2000), 79 (4), 2199-2208CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. PHluorins have a pK ∼ 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theor. anal. of the expected signals using this approach and guidelines for future reporter development are provided.
- 200Shen, Y.; Rosendale, M.; Campbell, R. E.; Perrais, D. pHuji, A pH-Sensitive Red Fluorescent Protein for Imaging of Exo- and Endocytosis. J. Cell Biol. 2014, 207, 419– 432, DOI: 10.1083/jcb.201404107Google Scholar200pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosisShen, Yi; Rosendale, Morgane; Campbell, Robert E.; Perrais, DavidJournal of Cell Biology (2014), 207 (3), 419-432CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These expts. reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.
- 201Rodriguez, E. A.; Campbell, R. E.; Lin, J. Y.; Lin, M. Z.; Miyawaki, A.; Palmer, A. E.; Shu, X.; Zhang, J.; Tsien, R. Y. The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem. Sci. 2017, 42, 111– 129, DOI: 10.1016/j.tibs.2016.09.010Google Scholar201The Growing and Glowing Toolbox of Fluorescent and Photoactive ProteinsRodriguez, Erik A.; Campbell, Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, Amy E.; Shu, Xiaokun; Zhang, Jin; Tsien, Roger Y.Trends in Biochemical Sciences (2017), 42 (2), 111-129CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophys. and biochem. extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
- 202Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y. A Guide to Choosing Fluorescent Proteins. Nat. Methods 2005, 2, 905– 909, DOI: 10.1038/nmeth819Google Scholar202A guide to choosing fluorescent proteinsShaner, Nathan C.; Steinbach, Paul A.; Tsien, Roger Y.Nature Methods (2005), 2 (12), 905-909CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biol. imaging. With no unified std. for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use. Which are the brightest. What addnl. factors det. which are best for a given expt.. Although in many cases, a trial-and-error approach may still be necessary in detg. the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
- 203Chudakov, D. M.; Matz, M. V.; Lukyanov, S.; Lukyanov, K. A. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol. Rev. 2010, 90, 1103– 1163, DOI: 10.1152/physrev.00038.2009Google Scholar203Fluorescent proteins and their applications in imaging living cells and tissuesChudakov, Dmitriy M.; Matz, Mikhail V.; Lukyanov, Sergey; Lukyanov, Konstantin A.Physiological Reviews (2010), 90 (3), 1103-1163CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)A review. Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many labs. have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled mols. and cells in space and time and can also be used for super-resoln. imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concns. of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.
- 204Foo, Y. H.; Naredi-Rainer, N.; Lamb, D. C.; Ahmed, S.; Wohland, T. Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy. Biophys. J. 2012, 102, 1174– 1183, DOI: 10.1016/j.bpj.2012.01.040Google Scholar204Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation SpectroscopyFoo, Yong Hwee; Naredi-Rainer, Nikolaus; Lamb, Don C.; Ahmed, Sohail; Wohland, ThorstenBiophysical Journal (2012), 102 (5), 1174-1183CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence cross-correlation spectroscopy (FCCS) is used to det. interactions and dissocn. consts. (Kds) of biomols. The detn. of a Kd depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ∼0.5 or less for different FCCS schemes. The authors use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and det. the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation vols. for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Forster (or fluorescence) resonance energy transfer between the labels. The authors deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation vols. The authors use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the Kd.
- 205McDonald, D.; Wu, L.; Bohks, S. M.; KewalRamani, V. N.; Unutmaz, D.; Hope, T. J. Recruitment of HIV and Its Receptors to Dendritic Cell-T Cell Junctions. Science 2003, 300, 1295– 1297, DOI: 10.1126/science.1084238Google Scholar205Recruitment of HIV and Its Receptors to Dendritic Cell-T Cell JunctionsMcDonald, David; Wu, Li; Bohks, Stacy M.; KewalRamani, Vineet N.; Unutmaz, Derya; Hope, Thomas J.Science (Washington, DC, United States) (2003), 300 (5623), 1295-1297CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Monocyte-derived dendritic cells (MDDCs) can efficiently bind and transfer HIV infectivity without themselves becoming infected. Using live-cell microscopy, we found that HIV was recruited to sites of cell contact in MDDCs. Anal. of conjugates between MDDCs and T cells revealed that, in the absence of antigen-specific signaling, the HIV receptors CD4, CCR5, and CXCR4 on the T cell were recruited to the interface while the MDDCs concd. HIV to the same region. We propose that contact between dendritic cells and T cells facilitates transmission of HIV by locally concg. virus, receptor, and coreceptor during the formation of an infectious synapse.
- 206Zacharias, D. A.; Tsien, R. Y. Molecular Biology and Mutation of Green Fluorescent Protein. Methods Biochem. Anal. 2005, 47, 83– 120, DOI: 10.1002/0471739499.ch5Google ScholarThere is no corresponding record for this reference.
- 207Klingen, Y.; Conzelmann, K. K.; Finke, S. Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport. J. Virol. 2008, 82, 237– 245, DOI: 10.1128/JVI.01342-07Google Scholar207Double-labeled rabies virus: live tracking of enveloped virus transportKlingen, Yvonne; Conzelmann, Karl-Klaus; Finke, StefanJournal of Virology (2008), 82 (1), 237-245CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addn. to G yielded infectious viruses with mosaic envelopes contg. both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addn. an enhanced green fluorescent protein-phosphoprotein fusion construct. Individual enveloped virus particles were obsd. under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.
- 208Lehmann, M. J.; Sherer, N. M.; Marks, C. B.; Pypaert, M.; Mothes, W. Actin- and Myosin-Driven Movement of Viruses along Filopodia Precedes Their Entry into Cells. J. Cell Biol. 2005, 170, 317– 325, DOI: 10.1083/jcb.200503059Google Scholar208Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cellsLehmann, Maik J.; Sherer, Nathan M.; Marks, Carolyn B.; Pypaert, Marc; Mothes, WaltherJournal of Cell Biology (2005), 170 (2), 317-325CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Viruses have often been obsd. in assocn. with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.
- 209Sugimoto, K.; Uema, M.; Sagara, H.; Tanaka, M.; Sata, T.; Hashimoto, Y.; Kawaguchi, Y. Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1. J. Virol. 2008, 82, 5198– 5211, DOI: 10.1128/JVI.02681-07Google Scholar209Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1Sugimoto, Ken; Uema, Masashi; Sagara, Hiroshi; Tanaka, Michiko; Sata, Tetsutaro; Hashimoto, Yasuhiro; Kawaguchi, YasushiJournal of Virology (2008), 82 (11), 5198-5211CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, resp. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.
- 210Liesche, J.; Ziomkiewicz, I.; Schulz, A. Super-Resolution Imaging with Pontamine Fast Scarlet 4BS Enables Direct Visualization of Cellulose Orientation and Cell Connection Architecture in Onion Epidermis Cells. BMC Plant Biol. 2013, 13, 226, DOI: 10.1186/1471-2229-13-226Google Scholar210Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cellsLiesche Johannes; Ziomkiewicz Iwona; Schulz AlexanderBMC plant biology (2013), 13 (), 226 ISSN:.BACKGROUND: In plants, a complex cell wall protects cells and defines their shape. Cellulose fibrils form a multilayered network inside the cell-wall matrix that plays a direct role in controlling cell expansion. Resolving the structure of this network will allow us to comprehend the relationship of cellulose fibril orientation and growth.The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal microscopy of some 200 nm in xy and 550 nm in z for green light, restricts the direct visualization of cellulose to relatively large bundles, whereas the structure of cellulose microfibrils with their diameter below 10 nm remains unresolved. Over the last decade, several so-called super-resolution microscopy approaches have been developed; in this paper we explore the potential of such approaches for the direct visualization of cellulose. RESULTS: To ensure optimal imaging we determined the spectral properties of PFS-stained tissue. PFS was found not to affect cell viability in the onion bulb scale epidermis. We present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. CONCLUSIONS: Super-resolution light microscopy of PFS-stained cellulose fibrils is possible and the increased resolution over conventional approaches makes it a valuable tool for the investigation of the cell-wall structure. This is one step in method developments that will close the gap to more invasive techniques, such as atomic force and electron microscopy.
- 211Melikyan, G. B.; Barnard, R. J.; Abrahamyan, L. G.; Mothes, W.; Young, J. A. Imaging Individual Retroviral Fusion Events: From Hemifusion to Pore Formation and Growth. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8728– 8733, DOI: 10.1073/pnas.0501864102Google Scholar211Imaging individual retroviral fusion events: From hemifusion to pore formation and growthMelikyan, Gregory B.; Barnard, Richard J. O.; Abrahamyan, Levon G.; Mothes, Walther; Young, John A. T.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (24), 8728-8733CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming following by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.
- 212Dixit, R.; Tiwari, V.; Shukla, D. Herpes Simplex Virus Type 1 Induces Filopodia in Differentiated P19 Neural Cells to Facilitate Viral Spread. Neurosci. Lett. 2008, 440, 113– 118, DOI: 10.1016/j.neulet.2008.05.031Google Scholar212Herpes simplex virus type 1 induces filopodia in differentiated P19 neural cells to facilitate viral spreadDixit, Rohan; Tiwari, Vaibhav; Shukla, DeepakNeuroscience Letters (2008), 440 (2), 113-118CODEN: NELED5; ISSN:0304-3940. (Elsevier Ireland Ltd.)Herpes simplex virus type-1 (HSV-1) is a neurotropic virus with significant potential as a viral vector for central nervous system (CNS) gene therapy. This study provides visual evidence that recombinant green fluorescent protein (GFP)-expressing HSV-1 travel down dendrites in differentiated P19 neuronal-like cells to efficiently reach the soma. The virus also promotes cytoskeletal rearrangements which facilitate viral spread in vitro, including often dramatic increases in dendritic filopodia. Viral movements, cell infection and filopodia induction were each reduced with the actin polymn. inhibitor cytochalasin D, suggesting the involvement of the actin cortex in these processes. The observation of neural cytoskeletal reorganization in response to HSV-1 may shed light on the mechanisms by which acute viral infection assocd. with herpes encephalitis produces cognitive deficits in patients.
- 213Adu-Gyamfi, E.; Digman, M. A.; Gratton, E.; Stahelin, R. V. Single-Particle Tracking Demonstrates That Actin Coordinates the Movement of the Ebola Virus Matrix Protein. Biophys. J. 2012, 103, L41– 143, DOI: 10.1016/j.bpj.2012.09.026Google Scholar213Single-Particle Tracking Demonstrates that Actin Coordinates the Movement of the Ebola Virus Matrix ProteinAdu-Gyamfi, Emmanuel; Digman, Michelle A.; Gratton, Enrico; Stahelin, Robert V.Biophysical Journal (2012), 103 (9), L41-L43CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to the authors' knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. The assembly and egress of VP40 at the plasma membrane of human cells were investigated using single-particle tracking. The results demonstrate that actin coordinates the movement and assembly of VP40, a crit. step in viral egress. These findings underscore the ability of single-mol. techniques to investigate the interplay of VP40 and host proteins in viral replication.
- 214Miyauchi, K.; Marin, M.; Melikyan, G. B. Visualization of Retrovirus Uptake and Delivery into Acidic Endosomes. Biochem. J. 2011, 434, 559– 569, DOI: 10.1042/BJ20101588Google Scholar214Visualization of retrovirus uptake and delivery into acidic endosomesMiyauchi, Kosuke; Marin, Mariana; Melikyan, Gregory B.Biochemical Journal (2011), 434 (3), 559-569CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solns. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solns. of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alk. buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alk. buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.
- 215Padilla-Parra, S.; Marin, M.; Kondo, N.; Melikyan, G. B. Pinpointing Retrovirus Entry Sites in Cells Expressing Alternatively Spliced Receptor Isoforms by Single Virus Imaging. Retrovirology 2014, 11, 47, DOI: 10.1186/1742-4690-11-47Google Scholar215Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imagingPadilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki; Melikyan, Gregory B.Retrovirology (2014), 11 (), 47/1-47/14CODEN: RETRBO; ISSN:1742-4690. (BioMed Central Ltd.)Background: The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacol. interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiol. conditions. Results: Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-pos. early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-pos.) or intermediate (Rab5- and Rab7-pos.) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-pos.) and intermediate (Rab5- and Rab7-pos.) compartments. Conclusions: Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are detd. by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.
- 216Mercer, J.; Helenius, A. Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 2008, 320, 531– 535, DOI: 10.1126/science.1155164Google Scholar216Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host CellsMercer, Jason; Helenius, AriScience (Washington, DC, United States) (2008), 320 (5875), 531-535CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles assocd. with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.
- 217Manicassamy, B.; Manicassamy, S.; Belicha-Villanueva, A.; Pisanelli, G.; Pulendran, B.; Garcia-Sastre, A. Analysis of in Vivo Dynamics of Influenza Virus Infection in Mice Using a GFP Reporter Virus. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11531– 11536, DOI: 10.1073/pnas.0914994107Google Scholar217Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virusManicassamy, Balaji; Manicassamy, Santhakumar; Belicha-Villanueva, Alan; Pisanelli, Giuseppe; Pulendran, Bali; Garcia-Sastre, AdolfoProceedings of the National Academy of Sciences of the United States of America (2010), 107 (25), 11531-11536, S11531/1-S11531/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to det., partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, the authors report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, the authors have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addn. to epithelial cells, the authors found GFP-pos. antigen-presenting cells, such as CD11b+CD11c-, CD11b-CD11c+, and CD11b+CD11c+, as early as 24 h after intranasal infection. In addn., a significant proportion of NK and B cells were GFP pos., suggesting active infection of these cells. The authors next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. The authors' results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening addnl. avenues of research in the influenza virus field.
- 218Bencina, M. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors. Sensors 2013, 13, 16736– 16758, DOI: 10.3390/s131216736Google Scholar218Illumination of the spatial order of intracellular pH by genetically encoded pH-sensitive sensorsBencina, MojcaSensors (2013), 13 (12), 16736-16758, 23 pp.CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)A review. Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiol. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.
- 219Hogue, I. B.; Bosse, J. B.; Engel, E. A.; Scherer, J.; Hu, J. R.; Del Rio, T.; Enquist, L. W. Fluorescent Protein Approaches in Alpha Herpesvirus Research. Viruses 2015, 7, 5933– 5961, DOI: 10.3390/v7112915Google Scholar219Fluorescent protein approaches in alpha herpesvirus researchHogue, Ian B.; Bosse, Jens B.; Engel, Esteban A.; Scherer, Julian; Hu, Jiun-Ruey; del Rio, Tony; Enquist, Lynn W.Viruses (2015), 7 (11), 5933-5961CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized mol. and cell biol., allowing us to literally see biol. processes as never before. Naturally, this revolution has extended to virol. in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats assocd. with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful expts. these tools now offer.
- 220Jouvenet, N.; Bieniasz, P. D.; Simon, S. M. Imaging the Biogenesis of Individual HIV-1 Virions in Live Cells. Nature 2008, 454, 236– 240, DOI: 10.1038/nature06998Google Scholar220Imaging the biogenesis of individual HIV-1 virions in live cellsJouvenet, Nolwenn; Bieniasz, Paul D.; Simon, Sanford M.Nature (London, United Kingdom) (2008), 454 (7201), 236-240CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport. However, the assembly of individual virions has never been obsd. in real time. Insights into this process have come primarily from biochem. analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial. Here we describe quant. the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivs. of Gag, the major structural component of HIV-1-which is sufficient to drive the assembly of virus-like particles-with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5-6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the detn. of parameters of viral assembly that are inaccessible with conventional techniques.
- 221Hogue, I. B.; Bosse, J. B.; Hu, J. R.; Thiberge, S. Y.; Enquist, L. W. Cellular Mechanisms of Alpha Herpesvirus Eegress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis. PLoS Pathog. 2014, 10, e1004535 DOI: 10.1371/journal.ppat.1004535Google Scholar221Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosisHogue, Ian B.; Bosse, Jens B.; Hu, Jiun-Ruey; Thiberge, Stephan Y.; Enquist, Lynn W.PLoS Pathogens (2014), 10 (12), e1004535/1-e1004535/12, 12 pp.CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and mol. mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement anal. to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically assocd. with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biol. and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
- 222Adam, V.; Berardozzi, R.; Byrdin, M.; Bourgeois, D. Phototransformable Fluorescent Proteins: Future Challenges. Curr. Opin. Chem. Biol. 2014, 20, 92– 102, DOI: 10.1016/j.cbpa.2014.05.016Google Scholar222Phototransformable fluorescent proteins: Future challengesAdam, Virgile; Berardozzi, Romain; Byrdin, Martin; Bourgeois, DominiqueCurrent Opinion in Chemical Biology (2014), 20 (), 92-102CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. In fluorescence microscopy, the photophys. properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biol. cells or organisms dynamically, quant. and with unprecedented resoln. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.
- 223Chudakov, D. M.; Verkhusha, V. V.; Staroverov, D. B.; Souslova, E. A.; Lukyanov, S.; Lukyanov, K. A. Photoswitchable Cyan Fluorescent Protein for Protein Tracking. Nat. Biotechnol. 2004, 22, 1435– 1439, DOI: 10.1038/nbt1025Google Scholar223Photoswitchable cyan fluorescent protein for protein trackingChudakov, Dmitriy M.; Verkhusha, Vladislav V.; Staroverov, Dmitry B.; Souslova, Ekaterina A.; Lukyanov, Sergey; Lukyanov, Konstantin A.Nature Biotechnology (2004), 22 (11), 1435-1439CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradn. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.
- 224Nemet, I.; Ropelewski, P.; Imanishi, Y. Applications of Phototransformable Fluorescent Proteins for Tracking the Dynamics of Cellular Components. Photochem. Photobiol. Sci. 2015, 14, 1787– 1806, DOI: 10.1039/C5PP00174AGoogle Scholar224Applications of phototransformable fluorescent proteins for tracking the dynamics of cellular componentsNemet, Ina; Ropelewski, Philip; Imanishi, YoshikazuPhotochemical & Photobiological Sciences (2015), 14 (10), 1787-1806CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)A review. In the past few decades, fluorescent proteins have revolutionized the field of cell biol. Phototransformable fluorescent proteins are capable of changing their excitation and emission spectra after being exposed to specific wavelength(s) of light. The majority of phototransformable fluorescent proteins have originated from marine organisms. Genetic engineering of these proteins has made available many choices for different colors, modes of conversion, and other biophys. properties. Their phototransformative property has allowed the highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. Furthermore, phototransformable fluorescent proteins have offered new methods for superresoln. fluorescence microscopy and optogenetics manipulation of proteins. One of the major advantages of phototransformable fluorescent proteins is their applicability for visualizing newly synthesized proteins that are en route to their final destinations. In this paper, we will discuss the biol. applications of phototransformable fluorescent proteins with special emphasis on the application of tracking membrane proteins in vertebrate photoreceptor cells.
- 225Ando, R.; Hama, H.; Yamamoto-Hino, M.; Mizuno, H.; Miyawaki, A. An Optical Marker Based on the UV-Induced Green-to-Red Photoconversion of a Fluorescent Protein. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12651– 12656, DOI: 10.1073/pnas.202320599Google Scholar225An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent proteinAndo, Ryoko; Hama, Hiroshi; Yamamoto-Hino, Miki; Mizuno, Hideaki; Miyawaki, AtsushiProceedings of the National Academy of Sciences of the United States of America (2002), 99 (20), 12651-12656CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradn. with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.
- 226Patterson, G. H.; Lippincott-Schwartz, J. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 2002, 297, 1873– 1877, DOI: 10.1126/science.1074952Google Scholar226A photoactivatable GFP for selective photolabeling of proteins and cellsPatterson, George H.; Lippincott-Schwartz, JenniferScience (Washington, DC, United States) (2002), 297 (5588), 1873-1877CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradn. with 413-nm light, increases fluorescence 100 times when excited by 488-nm light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated mols. that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
- 227Zhou, X. X.; Lin, M. Z. Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications. Curr. Opin. Chem. Biol. 2013, 17, 682– 690, DOI: 10.1016/j.cbpa.2013.05.031Google Scholar227Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applicationsZhou, Xin X.; Lin, Michael Z.Current Opinion in Chemical Biology (2013), 17 (4), 682-690CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last 10 years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis-trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresoln. imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity.
- 228Shroff, H.; Galbraith, C. G.; Galbraith, J. A.; White, H.; Gillette, J.; Olenych, S.; Davidson, M. W.; Betzig, E. Dual-Color Superresolution Imaging of Genetically Expressed Probes within Individual Adhesion Complexes. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 20308– 20313, DOI: 10.1073/pnas.0710517105Google Scholar228Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexesShroff, Hari; Galbraith, Catherine G.; Galbraith, James A.; White, Helen; Gillette, Jennifer; Olenych, Scott; Davidson, Michael W.; Betzig, EricProceedings of the National Academy of Sciences of the United States of America (2007), 104 (51), 20308-20313CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Accurate detn. of the relative positions of proteins within localized regions of the cell is essential for understanding their biol. function. Although fluorescent fusion proteins are targeted with mol. precision, the position of these genetically expressed reporters is usually known only to the resoln. of conventional optics (≈200 nm). Here, the authors report the use of two-color photoactivated localization microscopy (PALM) to det. the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resoln. in whole, fixed cells of ≈20-30 nm at acquisition times of 5-30 min. The authors apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, the authors find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resoln., and speed of the technique all suggest its potential to directly visualize mol. interactions within cellular structures at the nanometer scale.
- 229Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642– 1645, DOI: 10.1126/science.1127344Google Scholar229Imaging Intracellular Fluorescent Proteins at Nanometer ResolutionBetzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.Science (Washington, DC, United States) (2006), 313 (5793), 1642-1645CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The authors introduce a method for optically imaging intracellular proteins at nanometer spatial resoln. Numerous sparse subsets of photoactivatable fluorescent protein mols. were activated, localized (to ∼2 to 25 nm), and then bleached. The aggregate position information from all subsets was then assembled into a superresoln. image. The authors used this method - termed photoactivated localization microscopy - to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, the authors imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
- 230Fernandez-Suarez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 929– 943, DOI: 10.1038/nrm2531Google Scholar230Fluorescent probes for super-resolution imaging in living cellsFernandez-Suarez, Marta; Ting, Alice Y.Nature Reviews Molecular Cell Biology (2008), 9 (12), 929-943CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resoln. imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resoln. Current research is focused on further improving spatial resoln. in an effort to reach the goal of video-rate imaging of live cells with mol. (1-5 nm) resoln. Here, the authors describe the contributions of fluorescent probes to far-field super-resoln. imaging, focusing on fluorescent proteins and org. small-mol. fluorophores. The authors describe the features of existing super-resoln. fluorophores and highlight areas of importance for future research and development.
- 231Bourgeois, D.; Adam, V. Reversible Photoswitching in Fluorescent Proteins: A Mechanistic View. IUBMB Life 2012, 64, 482– 491, DOI: 10.1002/iub.1023Google Scholar231Reversible photoswitching in fluorescent proteins: A mechanistic viewBourgeois, Dominique; Adam, VirgileIUBMB Life (2012), 64 (6), 482-491CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons Inc.)A review. Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnol. On illumination with proper actinic light, phototransformable FPs are amenable to long-lived transitions between various fluorescent or nonfluorescent states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high-d. data bio-storage, photochromic FRET, and super-resoln. microscopy by either point-scanning, structured illumination, or single mol.-based wide-field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis-trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future. © 2012 IUBMB IUBMB Life, 2012.
- 232Muranyi, W.; Malkusch, S.; Muller, B.; Heilemann, M.; Krausslich, H. G. Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail. PLoS Pathog. 2013, 9, e1003198 DOI: 10.1371/journal.ppat.1003198Google ScholarThere is no corresponding record for this reference.
- 233Muller, B.; Heilemann, M. Shedding New Light on Viruses: Super-Resolution Microscopy for Studying Human Immunodeficiency Virus. Trends Microbiol. 2013, 21, 522– 533, DOI: 10.1016/j.tim.2013.06.010Google Scholar233Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virusMuller Barbara; Heilemann MikeTrends in microbiology (2013), 21 (10), 522-33 ISSN:.For more than 70 years electron microscopy (EM) techniques have played an important role in investigating structures of enveloped viruses. By contrast, use of fluorescence microscopy (FM) methods for this purpose was limited by the fact that the size of virus particles is generally around or below the diffraction limit of light microscopy. Various super-resolution (SR) fluorescence imaging techniques developed over the past two decades bypass the diffraction limit of light microscopy, allowing visualization of subviral details and bridging the gap between conventional FM and EM methods. We summarize here findings on human immunodeficiency virus (HIV-1) obtained using SR-FM techniques. Although the number of published studies is currently limited and some of the pioneering analyses also covered methodological or descriptive aspects, recent publications clearly indicate the potential to approach open questions in HIV-1 replication from a new angle.
- 234Grove, J. Super-Resolution Microscopy: A Virus’ Eye View of the Cell. Viruses 2014, 6, 1365– 1378, DOI: 10.3390/v6031365Google Scholar234Super-resolution microscopy: a virus' eye view of the cellGrove JoeViruses (2014), 6 (3), 1365-78 ISSN:.It is difficult to observe the molecular choreography between viruses and host cell components, as they exist on a spatial scale beyond the reach of conventional microscopy. However, novel super-resolution microscopy techniques have cast aside technical limitations to reveal a nanoscale view of virus replication and cell biology. This article provides an introduction to super-resolution imaging; in particular, localisation microscopy, and explores the application of such technologies to the study of viruses and tetraspanins, the topic of this special issue.
- 235Manley, S.; Gillette, J. M.; Patterson, G. H.; Shroff, H.; Hess, H. F.; Betzig, E.; Lippincott-Schwartz, J. High-Density Mapping of Single-Molecule Trajectories with Photoactivated Localization Microscopy. Nat. Methods 2008, 5, 155– 157, DOI: 10.1038/nmeth.1176Google Scholar235High-density mapping of single-molecule trajectories with photoactivated localization microscopyManley, Suliana; Gillette, Jennifer M.; Patterson, George H.; Shroff, Hari; Hess, Harald F.; Betzig, Eric; Lippincott-Schwartz, JenniferNature Methods (2008), 5 (2), 155-157CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-mol. motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of mols., sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
- 236Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. In Vivo Cancer Targeting and Imaging with Semiconductor Quantum Dots. Nat. Biotechnol. 2004, 22, 969– 976, DOI: 10.1038/nbt994Google Scholar236In vivo cancer targeting and imaging with semiconductor quantum dotsGao, Xiaohu; Cui, Yuanyuan; Levenson, Richard M.; Chung, Leland W. K.; Nie, ShumingNature Biotechnology (2004), 22 (8), 969-976CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both s.c. injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of mol. targets in vivo.
- 237Algar, W. R.; Susumu, K.; Delehanty, J. B.; Medintz, I. L. Semiconductor Quantum Dots in Bioanalysis: Crossing the Valley of Death. Anal. Chem. 2011, 83, 8826– 8837, DOI: 10.1021/ac201331rGoogle Scholar237Semiconductor Quantum Dots in Bioanalysis: Crossing the Valley of DeathAlgar, W. Russ; Susumu, Kimihiro; Delehanty, James B.; Medintz, Igor L.Analytical Chemistry (Washington, DC, United States) (2011), 83 (23), 8826-8837CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Colloidal semiconductor quantum dots (QDs) have evolved beyond scientific novelties and are transitioning into bona fide anal. tools. We describe the burgeoning role of QDs in many different fields of bioanalyses and highlight the advantages afforded by their unique phys. and optical properties.
- 238Wegner, K. D.; Hildebrandt, N. Quantum Dots: Bright and Versatile in Vitro and in Vivo Fluorescence Imaging Biosensors. Chem. Soc. Rev. 2015, 44, 4792– 4834, DOI: 10.1039/C4CS00532EGoogle Scholar238Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensorsWegner, K. David; Hildebrandt, NikoChemical Society Reviews (2015), 44 (14), 4792-4834CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Semiconductor quantum dots (QDs) have become important fluorescent probes for in vitro and in vivo bioimaging research. Their nanoparticle surfaces for versatile bioconjugation, their adaptable photophys. properties for multiplexed detection, and their superior stability for longer investigation times are the main advantages of QDs compared to other fluorescence imaging agents. Here, we review the recent literature dealing with the design and application of QD-bioconjugates for advanced in vitro and in vivo imaging. After a short summary of QD prepn. and their most important properties, different QD-based imaging applications will be discussed from the technol. and the biol. point of view, ranging from super-resoln. microscopy and single-particle tracking over in vitro cell and tissue imaging to in vivo investigations. A substantial part of the review will focus on multifunctional applications, in which the QD fluorescence is combined with drug or gene delivery towards theranostic approaches or with complementary technologies for multimodal imaging. We also briefly discuss QD toxicity issues and give a short outlook on future directions of QD-based bioimaging.
- 239Chen, G.; Zhu, J. Y.; Zhang, Z. L.; Zhang, W.; Ren, J. G.; Wu, M.; Hong, Z. Y.; Lv, C.; Pang, D. W.; Zhao, Y. F. Transformation of Cell-Derived Microparticles into Quantum-Dot-Labeled Nanovectors for Antitumor siRNA Delivery. Angew. Chem., Int. Ed. 2015, 54, 1036– 1040, DOI: 10.1002/anie.201410223Google Scholar239Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA deliveryChen, Gang; Zhu, Jun-Yi; Zhang, Zhi-Ling; Zhang, Wei; Ren, Jian-Gang; Wu, Min; Hong, Zheng-Yuan; Lv, Cheng; Pang, Dai-Wen; Zhao, Yi-FangAngewandte Chemie, International Edition (2015), 54 (3), 1036-1040CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Cell-derived microparticles (MPs) have been recently recognized as crit. intercellular information conveyors. However, further understanding of their biol. behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomol.-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors.
- 240Rosenthal, S. J.; Chang, J. C.; Kovtun, O.; McBride, J. R.; Tomlinson, I. D. Biocompatible Quantum Dots for Biological Applications. Chem. Biol. 2011, 18, 10– 24, DOI: 10.1016/j.chembiol.2010.11.013Google Scholar240Biocompatible Quantum Dots for Biological ApplicationsRosenthal, Sandra J.; Chang, Jerry C.; Kovtun, Oleg; McBride, James R.; Tomlinson, Ian D.Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (1), 10-24CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Semiconductor quantum dots are quickly becoming a crit. diagnostic tool for discerning cellular function at the mol. level. Their high brightness, long-lasting, size-tunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biol. oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biol. compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.
- 241Jethi, L.; Mack, T. G.; Kambhampati, P. Extending Semiconductor Nanocrystals from the Quantum Dot Regime to the Molecular Cluster Regime. J. Phys. Chem. C 2017, 121, 26102– 26107, DOI: 10.1021/acs.jpcc.7b08439Google Scholar241Extending Semiconductor Nanocrystals from the Quantum Dot Regime to the Molecular Cluster RegimeJethi, Lakshay; Mack, Timothy G.; Kambhampati, PatanjaliJournal of Physical Chemistry C (2017), 121 (46), 26102-26107CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)The size-dependent optical and electronic properties of semiconductor nanocrystal (NC) were exploited over decades for various applications. This size dependence involves a transition from the regime of bulk colloids of ∼100 nm radius to quantum dots (QDs) of ∼10 nm radius, the details of which are material specific. To understand the transition from the QD (∼10 nm) to the mol. cluster regimes (∼1 nm) of nanocrystals, a set of CdSe nanocrystals with sizes 0.89-1.66 nm in radius were synthesized. As the nanocrystals become small, the surface emission strongly increases in amplitude, and the core emission broadens and red shifts. These effects are rationalized in terms of coupling to ligands via electron transfer theory. The core emission spectra arise from increased vibrational coupling of ligands for very small NC. The surface emission amplitudes arise from a size-dependent surface free energy. The transition from the QD to the mol. cluster regime is at 1.2 nm radius, in contrast to the transition from the bulk to QD transition at the Bohr radius of 5.4 nm in CdSe. These size-dependent surface electronic phenomena may be used for light emission applications.
- 242Zhou, J.; Liu, Y.; Tang, J.; Tang, W. Surface Ligands Engineering of Semiconductor Quantum Dots for Chemosensory and Biological Applications. Mater. Today 2017, 20, 360– 376, DOI: 10.1016/j.mattod.2017.02.006Google Scholar242Surface ligands engineering of semiconductor quantum dots for chemosensory and biological applicationsZhou, Jie; Liu, Yun; Tang, Jian; Tang, WeihuaMaterials Today (Oxford, United Kingdom) (2017), 20 (7), 360-376CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)Featuring size-tunable elec. and optical properties, semiconductor quantum dots (QDs) are appealing intensive interests in developing ingenious luminescent materials for chemosensory and biol. applications. The surface modification of QDs with functional ligands not only fine-tunes the physiochem. properties and fluorescence emission behaviors, but also induces the designated interplay between analytes and probes for special detn. In this review, the fundamental principles guiding the rational design of high-efficiency luminescent sensors with surface engineering are overviewed. The state-of-the-art applications of QDs-based probes are highlighted for the sensing of mol. substrates and ionic species as well as various biol. applications, with the inherent recognition mechanisms elaborated for representative cases. The challenge and future research direction in this emerging and promising research field are also discussed.
- 243Zhou, J.; Yang, Y.; Zhang, C. Y. Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 2015, 115, 11669– 11717, DOI: 10.1021/acs.chemrev.5b00049Google Scholar243Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical ApplicationZhou, Juan; Yang, Yong; Zhang, Chun-yangChemical Reviews (Washington, DC, United States) (2015), 115 (21), 11669-11717CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review.
- 244Zhang, M.; Yue, J.; Cui, R.; Ma, Z.; Wan, H.; Wang, F.; Zhu, S.; Zhou, Y.; Kuang, Y.; Zhong, Y. Bright Quantum Dots Emitting at Approximately 1,600 nm in the NIR-IIb Window for Deep Tissue Fluorescence Imaging. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 6590– 6595, DOI: 10.1073/pnas.1806153115Google Scholar244Bright quantum dots emitting at ∼1,600 nm in the NIR-IIb window for deep tissue fluorescence imagingZhang, Mingxi; Yue, Jingying; Cui, Ran; Ma, Zhuoran; Wan, Hao; Wang, Feifei; Zhu, Shoujun; Zhou, Ying; Kuang, Yun; Zhong, Yeteng; Pang, Dai-Wen; Dai, HongjieProceedings of the National Academy of Sciences of the United States of America (2018), 115 (26), 6590-6595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aq. stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in org. phase. The CdS shell plays a crit. role of protecting the lead sulfide (PbS) core from oxidn. and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aq. stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 h and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per s (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke's shift. The aq.-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.
- 245Zhao, J. Y.; Chen, G.; Gu, Y. P.; Cui, R.; Zhang, Z. L.; Yu, Z. L.; Tang, B.; Zhao, Y. F.; Pang, D. W. Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived Microvesicles. J. Am. Chem. Soc. 2016, 138, 1893– 1903, DOI: 10.1021/jacs.5b10340Google Scholar245Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived MicrovesiclesZhao, Jing-Ya; Chen, Gang; Gu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Yu, Zi-Li; Tang, Bo; Zhao, Yi-Fang; Pang, Dai-WenJournal of the American Chemical Society (2016), 138 (6), 1893-1903CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Cell-derived microvesicles (MVs) are natural carriers that can transport biol. mols. between cells, which are expected to be promising delivery vehicles for therapeutic purposes. Strategies to label MVs are very important for investigation and application of MVs. Herein, ultrasmall Mn-magnetofunctionalized Ag2Se quantum dots (Ag2Se@Mn QDs) integrated with excellent near-IR (NIR) fluorescence and magnetic resonance (MR) imaging capabilities have been developed for instant efficient labeling of MVs for their in vivo high-resoln. dual-mode tracking. The Ag2Se@Mn QDs were fabricated by controlling the reaction of Mn2+ with the Ag2Se nanocrystals having been pretreated in 80 °C NaOH soln., with an ultrasmall size of ca. 1.8 nm, water dispersibility, high NIR fluorescence quantum yield of 13.2%, and high longitudinal relaxivity of 12.87 mM-1s-1 (almost four times that of the com. contrast agent Gd-DTPA). The ultrasmall size of the Ag2Se@Mn QDs enables them to be directly and efficiently loaded into MVs by electroporation, instantly and reliably conferring both NIR fluorescence and MR traceability on MVs. Our method for labeling MVs of different origins is universal and free of unfavorable influence on intrinsic behaviors of MVs. The complementary imaging capabilities of the Ag2Se@Mn QDs have made the long-term noninvasive whole-body high-resoln. dual-mode tracking of MVs in vivo realized, by which the dynamic biodistribution of MVs has been revealed in a real-time and in situ quant. manner. This work not only opens a new window for labeling with QDs, but also facilitates greatly the investigation and application of MVs.
- 246Gu, Y. P.; Cui, R.; Zhang, Z. L.; Xie, Z. X.; Pang, D. W. Ultrasmall Near-Infrared Ag2Se Quantum Dots with Tunable Fluorescence for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134, 79– 82, DOI: 10.1021/ja2089553Google Scholar246Ultrasmall Near-Infrared Ag2Se Quantum Dots with Tunable Fluorescence for in Vivo ImagingGu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Xie, Zhi-Xiong; Pang, Dai-WenJournal of the American Chemical Society (2012), 134 (1), 79-82CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A strategy is presented that involves coupling Na2SeO3 redn. with the binding of silver ions and alanine in a quasi-biosystem to obtain ultrasmall, near-IR Ag2Se quantum dots (QDs) with tunable fluorescence at 90° in aq. soln. This strategy avoids high temps., high pressures, and org. solvents so that water-dispersible sub-3 nm Ag2Se QDs can be directly obtained. The photoluminescence of the Ag2Se QDs was size-dependent over a wavelength range from 700 to 820 nm, corresponding to sizes from 1.5 ± 0.4 to 2.4 ± 0.5 nm, with good monodispersity. The Ag2Se QDs are less cytotoxic than other nanomaterials used for similar applications. Furthermore, the NIR fluorescence of the Ag2Se QDs could penetrate through the abdominal cavity of a living nude mouse and could be detected on its back side, demonstrating the potential applications of these less toxic NIR Ag2Se QDs in bioimaging.
- 247Alivisatos, A. P.; Gu, W. W.; Larabell, C. Quantum Dots as Cellular Probes. Annu. Rev. Biomed. Eng. 2005, 7, 55– 76, DOI: 10.1146/annurev.bioeng.7.060804.100432Google Scholar247Quantum dots as cellular probesAlivisatos, A. Paul; Gu, Weiwei; Larabell, CarolynAnnual Review of Biomedical Engineering (2005), 7 (), 55-76, 3 platesCODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)A review. Robust and bright light emitters, semiconductor nanocrystals [quantum dots (QDs)] have been adopted as a new class of fluorescent labels. Six years after the first expts. of their uses in biol. applications, there have been dramatic improvements in understanding surface chem., biocompatibility, and targeting specificity. Many studies have shown the great potential of using quantum dots as new probes in vitro and in vivo. This review summarizes the recent advances of quantum dot usage at the cellular level, including immunolabeling, cell tracking, in situ hybridization, FRET, in vivo imaging, and other related technologies. Limitations and potential future uses of quantum dot probes are also discussed.
- 248Gao, X.; Yang, L.; Petros, J. A.; Marshall, F. F.; Simons, J. W.; Nie, S. In Vivo Molecular and Cellular Imaging with Quantum Dots. Curr. Opin. Biotechnol. 2005, 16, 63– 72, DOI: 10.1016/j.copbio.2004.11.003Google Scholar248In vivo molecular and cellular imaging with quantum dotsGao, Xiaohu; Yang, Lily; Petros, John A.; Marshall, Fray F.; Simons, Jonathan W.; Nie, ShumingCurrent Opinion in Biotechnology (2005), 16 (1), 63-72CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomol. and cellular imaging. In comparison with org. dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degrdn. need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of mol. targets in living cells, animal models and possibly in humans.
- 249Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science 1998, 281, 2013– 2016, DOI: 10.1126/science.281.5385.2013Google Scholar249Semiconductor nanocrystals as fluorescent biological labelsBruchez, Marcel, Jr.; Moronne, Mario; Gin, Peter; Weiss, Shimon; Alivisatos, A. PaulScience (Washington, D. C.) (1998), 281 (5385), 2013-2016CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Semiconductor nanocrystals were prepd. for use as fluorescent probes in biol. staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, sym. emission spectrum and are photochem. stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling expt. on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
- 250Smith, A. M.; Duan, H. W.; Mohs, A. M.; Nie, S. M. Bioconjugated Quantum Dots for in Vivo Molecular and Cellular Imaging. Adv. Drug Delivery Rev. 2008, 60, 1226– 1240, DOI: 10.1016/j.addr.2008.03.015Google Scholar250Bioconjugated quantum dots for in vivo molecular and cellular imagingSmith, Andrew M.; Duan, Hongwei; Mohs, Aaron M.; Nie, ShumingAdvanced Drug Delivery Reviews (2008), 60 (11), 1226-1240CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)A review. Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biol. and medicine. In comparison with org. dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small mols., QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for mol. and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicol.
- 251Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D. J.; Post, J. N.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin, T. M. Quantum Dot Ligands Provide New Insights into erbB/HER Receptor–Mediated Signal Transduction. Nat. Biotechnol. 2004, 22, 198– 203, DOI: 10.1038/nbt929Google Scholar251Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transductionLidke, Diane S.; Nagy, Peter; Heintzmann, Rainer; Arndt-Jovin, Donna J.; Post, Janine N.; Grecco, Hernan E.; Jares-Erijman, Elizabeth A.; Jovin, Thomas M.Nature Biotechnology (2004), 22 (2), 198-203CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. The authors have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (ii) continuous confocal laser scanning microscopy and flow cytometry. EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnol. developments.
- 252Srinivasan, C.; Lee, J.; Papadimitrakopoulos, F.; Silbart, L. K.; Zhao, M.; Burgess, D. J. Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots. Mol. Ther. 2006, 14, 192– 201, DOI: 10.1016/j.ymthe.2006.03.010Google Scholar252Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum DotsSrinivasan, Charudharshini; Lee, Jeunghoon; Papadimitrakopoulos, Fotios; Silbart, Lawrence K.; Zhao, Minhua; Burgess, Diane J.Molecular Therapy (2006), 14 (2), 192-201CODEN: MTOHCK; ISSN:1525-0016. (Elsevier)Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)-N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and at. force microscopy (AFM) were used to confirm the structure of QD-DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNA-reactive site of the plasmid DNA. These QD-DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ∼62%, which was comparable to the control (unconjugated) plasmid DNA.
- 253Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots. Nat. Biotechnol. 2003, 21, 41– 46, DOI: 10.1038/nbt764Google Scholar253Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dotsWu, Xingyong; Liu, Hongjian; Liu, Jianquan; Haley, Kari N.; Treadway, Joseph A.; Larson, J. Peter; Ge, Nianfeng; Peale, Frank; Bruchez, Marcel P.Nature Biotechnology (2003), 21 (1), 41-46CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small mols., can specifically and effectively label mol. targets at a subcellular level. Here we have used QDs linked to IgG (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable org. dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over org. dyes in multiplex target detection.
- 254Chen, C.; Peng, J.; Xia, H.; Wu, Q.; Zeng, L.; Xu, H.; Tang, H.; Zhang, Z.; Zhu, X.; Pang, D. Quantum-Dot-Based Immunofluorescent Imaging of HER2 and ER Provides New Insights into Breast Cancer Heterogeneity. Nanotechnology 2010, 21, 095101 DOI: 10.1088/0957-4484/21/9/095101Google Scholar254Quantum-dot-based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneityChen, Chuang; Peng, Jun; Xia, Heshun; Wu, Qiongshui; Zeng, Libo; Xu, Hao; Tang, Hongwu; Zhang, Zhiling; Zhu, Xiaobo; Pang, Daiwen; Li, YanNanotechnology (2010), 21 (9), 095101/1-095101/6CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)Breast cancer (BC) is a heterogeneous tumor, and better understanding of its heterogeneity is essential to improving treatment effect. Quantum dot (QD)-based immunofluorescent nanotechnol. (QD-IHC) for mol. pathol. has potential advantages in delineating tumor heterogeneity. This potential is explored in this paper by QD-IHC imaging of HER2 and ER. BC heterogeneity can be displayed more clearly and sensitively by QD-IHC than conventional IHC in BC tissue microarrays. Furthermore, the simultaneous imaging of ER and HER2 might help understand their interactions during the process of evolution of heterogeneous BC.
- 255Chen, C.; Xia, H. S.; Gong, Y. P.; Peng, J.; Peng, C. W.; Hu, M. B.; Zhu, X. B.; Pang, D. W.; Sun, S. R.; Li, Y. The Quantitative Detection of Total HER2 Load by Quantum Dots and the Identification of a New Subtype of Breast Cancer with Different 5-Year Prognosis. Biomaterials 2010, 31, 8818– 8825, DOI: 10.1016/j.biomaterials.2010.07.091Google Scholar255The quantitative detection of total HER2 load by quantum dots and the identification of a new subtype of breast cancer with different 5-year prognosisChen, Chuang; Xia, He-Shun; Gong, Yi-Ping; Peng, Jun; Peng, Chun-Wei; Hu, Ming-Bai; Zhu, Xiao-Bo; Pang, Dai-Wen; Sun, Sheng-Rong; Li, YanBiomaterials (2010), 31 (33), 8818-8825CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Accurate classification is fundamental for breast cancer (BC) personalized care. Current BC classification based on the either traditional morphol. staging or mol. signatures seems inefficient to reveal the"true"behaviors of invasive BC evolution. An appropriate approach combining the macro- and micro-pathol. information might be more useful academically as well as clin. Here the authors explore a holistic approach by integrating a key mol. prognostic indicator of BC, HER2, with quant. detn. using quantum dots (QDs)-based nanotechnol. and spectral anal., and a key macropathol. indicator, tumor size, resulting a new indicator, total HER2 load. This indicator might better reveal BC heterogeneity and new subtypes of BC with different 5-yr disease-free survival compared with current methods, which could be helpful in formulating a more personalized targeted therapy for BC. Furthermore, this mode integrating macro- and micro-pathol. indicators might help gain new insights into invasive BC biol. behaviors.
- 256Chen, C.; Liu, S. L.; Cui, R.; Huang, B. H.; Tian, Z. Q.; Jiang, P.; Pang, D. W.; Zhang, Z. L. Diffusion Behaviors of Water-Soluble CdSe/ZnS Core/Shell Quantum Dots Investigated by Single-Particle Tracking. J. Phys. Chem. C 2008, 112, 18904– 18910, DOI: 10.1021/jp807074tGoogle Scholar256Diffusion Behaviors of Water-Soluble CdSe/ZnS Core/Shell Quantum Dots Investigated by Single-Particle TrackingChen, Cheng; Liu, Shu-Lin; Cui, Ran; Huang, Bi-Hai; Tian, Zhi-Quan; Jiang, Peng; Pang, Dai-Wen; Zhang, Zhi-LingJournal of Physical Chemistry C (2008), 112 (48), 18904-18910CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)As is known to the authors all, quantum dots (QDs), the fluorescent semiconductor nanocrystals, have many excellent optical properties which make them attractive fluorescent tags in single-mol. tracking in live cells. Because the intracellular environment is so complex, this paper aimed at simulating the intracellular soln. environment in vitro and studied the influence of the soln. environment on the diffusion of water-sol. core/shell CdSe/ZnS QDs. Single-particle tracking (SPT) was applied to measure the diffusion coeffs. of two water-sol. core/shell QDs, CTAB-modified CdSe/ZnS QDs (CTAB-QDs) and octylamine-modified poly(acrylic acid)-modified CdSe/ZnS QDs (OPA-QDs). The exposure time was optimized to be 29.95 ms. Then the paper was focused on the effects of pH value, salt concn., and soln. viscosity on the diffusion coeffs. of the two water-sol. QDs. The pH value had a great influence on the diffusion coeff. of CTAB-QDs but little on that of OPA-QDs. The difference should be mainly due to the distinguishment of the charge and structure of surface ligands on the two water-sol. QDs. The diffusion coeff. of either CTAB-QDs or OPA-QDs was hardly affected by the salt concn. of the soln. Also, for both CTAB-QDs and OPA-QDs, the diffusion coeffs. decreased as the soln. viscosity increased, which obeyed the Stokes-Einstein relation. In summary, OPA-QDs have more promising applications in single-mol. tracking in live cells, as compared with CTAB-QDs. The obtained results would benefit the further applications of QDs in single-mol. tracking in live cells. This system could also serve as a model system for studying the diffusion behavior of nanoparticles.
- 257Gao, X.; Wang, T.; Wu, B.; Chen, J.; Chen, J.; Yue, Y.; Dai, N.; Chen, H.; Jiang, X. Quantum Dots for Tracking Cellular Transport of Lectin-Functionalized Nanoparticles. Biochem. Biophys. Res. Commun. 2008, 377, 35– 40, DOI: 10.1016/j.bbrc.2008.09.077Google Scholar257Quantum dots for tracking cellular transport of lectin-functionalized nanoparticlesGao, Xiaoling; Wang, Tao; Wu, Bingxian; Chen, Jun; Chen, Jiyao; Yue, Yang; Dai, Ning; Chen, Hongzhuan; Jiang, XinguoBiochemical and Biophysical Research Communications (2008), 377 (1), 35-40CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Inc.)Successful drug delivery by functionalized nanocarriers largely depends on their efficient intracellular transport which has not yet been fully understood. We developed a new tracking technique by encapsulating quantum dots into the core of wheat germ agglutinin-conjugated nanoparticles (WGA-NP) to track cellular transport of functionalized nanocarriers. The resulting nanoparticles showed no changes in particle size, zeta potential or biobinding activity, and the loaded probe presented excellent photostability and tracking ability. Taking advantage of these properties, cellular transport profiles of WGA-NP in Caco-2 cells was demonstrated. The cellular uptake begins with binding of WGA to its receptor at the cell surface. The subsequent endocytosis happened in a cytoskeleton-dependent manner and by means of clathrin and caveolae-mediated mechanisms. After endosome creating, transport occurs to both trans-Golgi and lysosome. Our study provides new evidences for quantum dots as a cellular tracking probe of nanocarriers and helps understand intracellular transport profile of lectin-functionalized nanoparticles.
- 258Rajan, S. S.; Liu, H. Y.; Vu, T. Q. Ligand-Bound Quantum Dot Probes for Studying the Molecular Scale Dynamics of Receptor Endocytic Trafficking in Live Cells. ACS Nano 2008, 2, 1153– 1166, DOI: 10.1021/nn700399eGoogle Scholar258Ligand-Bound Quantum Dot Probes for Studying the Molecular Scale Dynamics of Receptor Endocytic Trafficking in Live CellsRajan, Sujata Sundara; Liu, Hong Yan; Vu, Tania Q.ACS Nano (2008), 2 (6), 1153-1166CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small nos. of ligand bound-receptor complexes inside live cells is currently hampered by tech. limitations. Here, the authors develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunopptn., and immuno-colocalization, the authors illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand-receptor binding and internalization (t = 15-150 min). The unique value of these probes is illustrated by new dynamic observations: (1) that endocytosis proceeds at strikingly regulated fashion, and (2) that diffusive and active forms of transport inside cells are rapid and efficient. QDs are powerful intracellular probes that can provide biologists with new capabilities and fresh insight for studying endocytic receptor signaling events, in real time, and at the resoln. of single or small nos. of receptors in live cells.
- 259Murcia, M. J.; Minner, D. E.; Mustata, G. M.; Ritchie, K.; Naumann, C. A. Design of Quantum Dot-Conjugated Lipids for Long-Term, High-Speed Tracking Experiments on Cell Surfaces. J. Am. Chem. Soc. 2008, 130, 15054– 15062, DOI: 10.1021/ja803325bGoogle Scholar259Design of Quantum Dot-Conjugated Lipids for Long-Term, High-Speed Tracking Experiments on Cell SurfacesMurcia, Michael J.; Minner, Daniel E.; Mustata, Gina-Mirela; Ritchie, Kenneth; Naumann, Christoph A.Journal of the American Chemical Society (2008), 130 (45), 15054-15062CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking expts. on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 M mixt. of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol-2000)],are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aq. soln. is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking expts. on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-mol. tracking probes in plasma membranes of several cell types. Initial tracking expts. at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-mol. imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking expts. that used larger (40 nm) Au labels, not only push the time resoln. in long-time, continuous fluorescence-based single-mol. tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor expts.
- 260Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum Dot Bioconjugates for Imaging, Labelling and Sensing. Nat. Mater. 2005, 4, 435– 446, DOI: 10.1038/nmat1390Google Scholar260Quantum dot bioconjugates for imaging, labelling and sensingMedintz, Igor L.; Uyeda, H. Tetsuo; Goldman, Ellen R.; Mattoussi, HediNature Materials (2005), 4 (6), 435-446CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)A review. One of the fastest moving and most exciting interfaces of nanotechnol. is the use of quantum dots (QDs) in biol. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biol. investigations, in which traditional fluorescent labels based on org. mols. fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water sol. and target them to specific biomols. has led to promising applications in cellular labeling, deep-tissue imaging, assay labeling and as efficient fluorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible bioconjugation techniques. In this review, we look at current methods for prepg. QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biol. has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.
- 261Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005, 307, 538– 544, DOI: 10.1126/science.1104274Google Scholar261Quantum Dots for Live Cells, in Vivo Imaging, and DiagnosticsMichalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S.Science (Washington, DC, United States) (2005), 307 (5709), 538-544CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biol. applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biol. Recent examples of their exptl. use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-IR emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-mol. level, high-resoln. cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.
- 262Bentolila, L. A.; Ebenstein, Y.; Weiss, S. Quantum Dots for in Vivo Small-Animal Imaging. J. Nucl. Med. 2009, 50, 493– 496, DOI: 10.2967/jnumed.108.053561Google Scholar262Quantum dots for in vivo small-animal imagingBentolila, Laurent A.; Ebenstein, Yuval; Weiss, ShimonJournal of Nuclear Medicine (2009), 50 (4), 493-496CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine)A review. Nanotechnol. is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1-10 nm) made of inorg. semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the UV to the near-IR. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clin. applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.
- 263Zrazhevskiy, P.; Sena, M.; Gao, X. Designing Multifunctional Quantum Dots for Bioimaging, Detection, and Drug Delivery. Chem. Soc. Rev. 2010, 39, 4326– 4354, DOI: 10.1039/b915139gGoogle Scholar263Designing multifunctional quantum dots for bioimaging, detection, and drug deliveryZrazhevskiy, Pavel; Sena, Mark; Gao, XiaohuChemical Society Reviews (2010), 39 (11), 4326-4354CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The emerging field of bionanotechnol. aims at revolutionizing biomedical research and clin. practice via introduction of nanoparticle-based tools, expanding capabilities of existing investigative, diagnostic, and therapeutic techniques as well as creating novel instruments and approaches for addressing challenges faced by medicine. Quantum dots (QDs), semiconductor nanoparticles with unique photo-phys. properties, have become one of the dominant classes of imaging probes as well as universal platforms for engineering of multifunctional nanodevices. Possessing versatile surface chem. and superior optical features, QDs have found initial use in a variety of in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is becoming increasingly important for successful transition of this technol. from proof-of-concept studies towards real-life clin. applications. This review outlines the major design principles and criteria, from general ones to application-specific, governing the engineering of novel QD probes satisfying the increasing demands and requirements of nanomedicine and discusses the future directions of QD-focused bionanotechnol. research.
- 264Ruan, G.; Agrawal, A.; Marcus, A. I.; Nie, S. Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J. Am. Chem. Soc. 2007, 129, 14759– 14766, DOI: 10.1021/ja074936kGoogle Scholar264Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle SheddingRuan, Gang; Agrawal, Amit; Marcus, Adam I.; Nie, ShumingJournal of the American Chemical Society (2007), 129 (47), 14759-14766CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the use of Tat peptide-conjugated quantum dots (Tat-QDs) to examine the complex behavior of nanoparticle probes in live cells, a topic that is of considerable current interest in developing advanced nanoparticle agents for mol. and cellular imaging. Dynamic confocal imaging studies indicate that the peptide-conjugated QDs are internalized by macropinocytosis, a fluid-phase endocytosis process triggered by Tat-QD binding to neg. charged cell membranes. The internalized Tat-QDs are tethered to the inner vesicle surfaces and are trapped in cytoplasmic organelles. The QD loaded vesicles are actively transported by mol. machines (such as dyneins) along microtubule tracks. The destination of this active transport is an asym. perinuclear region (outside the cell nucleus) known as the microtubule organizing center (MTOC). The authors also find that Tat-QDs strongly bind to cellular membrane structures such as filopodia and that large QD-contg. vesicles are released from the tips of filopodia by vesicle shedding. These results provide new insights into the mechanisms of Tat peptide-mediated delivery as well as toward the design of functionalized nanoparticles for mol. imaging and targeted therapy.
- 265Bruchez, M. P. Quantum Dots Find Their Stride in Single Molecule Tracking. Curr. Opin. Chem. Biol. 2011, 15, 775– 780, DOI: 10.1016/j.cbpa.2011.10.011Google Scholar265Quantum dots find their stride in single molecule trackingBruchez, Marcel P.Current Opinion in Chemical Biology (2011), 15 (6), 775-780CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Thirteen years after the demonstration of quantum dots as biol. imaging agents, and nine years after the initial com. introduction of bioconjugated quantum dots, the brightness and photostability of the quantum dots has enabled a range of investigations using single mol. tracking. These materials are being routinely utilized by a no. of groups to track the dynamics of single mols. in reconstituted biophys. systems and on living cells, and are esp. powerful for investigations of single mols. over long timescales with short exposure times and high pointing accuracy. New approaches are emerging where the quantum dots are used as hard-sphere' probes for intracellular compartments. Innovations in quantum dot surface modification are poised to substantially expand the utility of these materials.
- 266Tada, H.; Higuchi, H.; Wanatabe, T. M.; Ohuchi, N. In Vivo Real-Time Tracking of Single Quantum Dots Conjugated with Monoclonal Anti-HER2 Antibody in Tumors of Mice. Cancer Res. 2007, 67, 1138– 1144, DOI: 10.1158/0008-5472.CAN-06-1185Google Scholar266In vivo Real-time Tracking of Single Quantum Dots Conjugated with Monoclonal Anti-HER2 Antibody in Tumors of MiceTada, Hiroshi; Higuchi, Hideo; Wanatabe, Tomonobu M.; Ohuchi, NoriakiCancer Research (2007), 67 (3), 1138-1144CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Studies with tracking of single nanoparticles are providing new insights into the interactions and processes involved in the transport of drug carriers in living mice. Here, the authors report the tracking of a single particle quantum dot (Qdot) conjugated with tumor-targeting antibody in tumors of living mice using a dorsal skinfold chamber and a high-speed confocal microscope with a high-sensitivity camera. Qdot labeled with the monoclonal anti-HER2 antibody was injected into mice with HER2-overexpressing breast cancer to analyze the mol. processes of its mechanistic delivery to the tumor. Movement of single complexes of the Qdot-antibody could be clearly obsd. at 30 frames/s inside the tumor through a dorsal skinfold chamber. The authors successfully identified six processes of delivery: initially in the circulation within a blood vessel, during extravasation, in the extracellular region, binding to HER2 on the cell membrane, moving from the cell membrane to the perinuclear region, and in the perinuclear region. The six processes were quant. analyzed to understand the rate-limiting constraints on Qdot-antibody delivery. The movement of the complexes at each stage was "stop-and-go.". The image anal. of the delivery processes of single particles in vivo provides valuable information on antibody-conjugated therapeutic nanoparticles, which will be useful in increasing therapeutic efficacy.
- 267Wells, N. P.; Lessard, G. A.; Werner, J. H. Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments. Anal. Chem. 2008, 80, 9830– 9834, DOI: 10.1021/ac8021899Google Scholar267Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background EnvironmentsWells, Nathan P.; Lessard, Guillaume A.; Werner, James H.Analytical Chemistry (Washington, DC, United States) (2008), 80 (24), 9830-9834CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coeff. ∼ 0.5 μm2/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than obsd. in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to det. changes in the emission lifetime as a function of position and pos. identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional vol. of a live cell for durations up to 15 s.
- 268Cui, Z. Q.; Ren, Q.; Wei, H. P.; Chen, Z.; Deng, J. Y.; Zhang, Z. P.; Zhang, X. E. Quantum Dot-Aptamer Nanoprobes for Recognizing and Labeling Influenza A Virus Particles. Nanoscale 2011, 3, 2454– 2457, DOI: 10.1039/c1nr10218dGoogle Scholar268Quantum dot-aptamer nanoprobes for recognizing and labeling influenza A virus particlesCui, Zong-Qiang; Ren, Qian; Wei, Hong-Ping; Chen, Ze; Deng, Jiao-Yu; Zhang, Zhi-Ping; Zhang, Xian-EnNanoscale (2011), 3 (6), 2454-2457CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The fluorescence labeling of viruses is a useful technol. for virus detection and imaging. By combining the excellent fluorescence properties of quantum dots (QDs) with the high affinity and specificity of aptamers, we constructed a QD-aptamer probe. The aptamer A22, against the hemagglutinin of influenza A virus, was linked to QDs, producing the QD-A22 probe. Fluorescence imaging and transmission electron microscopy showed that the QD-A22 probe could specifically recognize and label influenza A virus particles. This QD labeling technique provides a new strategy for labeling virus particles for virus detection and imaging.
- 269Saxton, M. J.; Jacobson, K. Single-Particle Tracking: Applications to Membrane Dynamics. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373– 399, DOI: 10.1146/annurev.biophys.26.1.373Google Scholar269Single-particle tracking: applications to membrane dynamicsSaxton, Michael J.; Jacobson, KenAnnual Review of Biophysics and Biomolecular Structure (1997), 26 (), 373-399CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews)Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is obsd., but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.
- 270Kusumi, A.; Sako, Y.; Yamamoto, M. Confined Lateral Diffusion of Membrane Receptors as Studied by Single Particle Tracking (Nanovid Microscopy). Effects of Calcium-Induced Differentiation in Cultured Epithelial Cells. Biophys. J. 1993, 65, 2021– 2040, DOI: 10.1016/S0006-3495(93)81253-0Google Scholar270Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cellsKusumi, Akihiro; Sako, Yasushi; Yamamoto, MutsuyaBiophysical Journal (1993), 65 (5), 2021-40CODEN: BIOJAU; ISSN:0006-3495.The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor mols. are labeled with 40 nm-φ colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resoln. of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor mols. obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were obsd.: (a) stationary mode, in which the microscopic diffusion coeff. is less than 4.6 × 10-12 cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coeff. between 4.6 × 10-12 and 1 × 10-9 cm2/s) are confined within a limited area, probably by the membrane-assocd. cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diam. (0.04-0.24 μm2 in area), in which receptor mols. are confined in the time scale of 3-30 s, and that the long-range diffusion obsd. by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of mols. in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from ∼60% to 8% (low- and high-calcium mediums, resp.).
- 271Wan, X. Y.; Zheng, L. L.; Gao, P. F.; Yang, X. X.; Li, C. M.; Li, Y. F.; Huang, C. Z. Real-Time Light Scattering Tracking of Gold Nanoparticles- Bioconjugated Respiratory Syncytial Virus Infecting HEp-2 Cells. Sci. Rep. 2015, 4, 4529, DOI: 10.1038/srep04529Google ScholarThere is no corresponding record for this reference.
- 272Marjomaki, V.; Lahtinen, T.; Martikainen, M.; Koivisto, J.; Malola, S.; Salorinne, K.; Pettersson, M.; Hakkinen, H. Site-Specific Targeting of Enterovirus Capsid by Functionalized Monodisperse Gold Nanoclusters. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 1277– 1281, DOI: 10.1073/pnas.1310973111Google Scholar272Site-specific targeting of enterovirus capsid by functionalized monodisperse gold nanoclustersMarjomaki, Varpu; Lahtinen, Tanja; Martikainen, Mari; Koivisto, Jaakko; Malola, Sami; Salorinne, Kirsi; Pettersson, Mika; Hakkinen, HannuProceedings of the National Academy of Sciences of the United States of America (2014), 111 (4), 1277-1281CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Development of precise protocols for accurate site-specific conjugation of monodisperse inorg. nanoparticles to biol. material is one of the challenges in contemporary bionanoscience and nanomedicine. We report here a successful site-specific covalent conjugation of functionalized atomically monodisperse gold clusters with 1.5-nm metal cores to viral surfaces. Water-sol. Au102(para-mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid proteins, were synthesized and conjugated to enteroviruses echovirus 1 and coxsackievirus B3. Quant. anal. of transmission electron microscopy images and the known virus structures showed high affinity and mutual ordering of the bound gold clusters on the viral surface and a clear correlation between the clusters and the targeted cysteine sites close to the viral surface. Infectivity of the viruses was not compromised by loading of several tens of gold clusters per virus. These advances allow for future investigations of the structure-function relations of enteroviruses and enterovirus-related virus-like particles, including their entry mechanisms into cells and uncoating in cellular endosomes.
- 273Wan, X. K.; Xu, W. W.; Yuan, S. F.; Gao, Y.; Zeng, X. C.; Wang, Q. M. A Near-Infrared-Emissive Alkynyl-Protected Au24 Nanocluster. Angew. Chem., Int. Ed. 2015, 54, 9683– 9686, DOI: 10.1002/anie.201503893Google Scholar273A Near-Infrared-Emissive Alkynyl-Protected Au24 NanoclusterWan, Xian-Kai; Xu, Wen Wu; Yuan, Shang-Fu; Gao, Yi; Zeng, Xiao-Cheng; Wang, Quan-MingAngewandte Chemie, International Edition (2015), 54 (33), 9683-9686CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)An alkynyl-protected Au nanocluster [Au24(C≡CPh)14(PPh3)4](SbF6)2 was prepd. by a direct redn. method. Single-crystal x-ray diffraction reveals that the mol. structure contains a Au22 core that is made of two Au13-centered cuboctahedra that share a square face. Two staple-like PhC≡CAuC≡CPh motifs are located around the center of the rod-like Au22 core. This Au24 nanocluster is highly emissive in the near-IR region with λmax = 925 nm and the nature of the HOMO-LUMO transition was studied by time-dependent DFT calcns.
- 274Jin, R.; Zeng, C.; Zhou, M.; Chen, Y. Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities. Chem. Rev. 2016, 116, 10346– 10413, DOI: 10.1021/acs.chemrev.5b00703Google Scholar274Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and OpportunitiesJin, Rongchao; Zeng, Chenjie; Zhou, Meng; Chen, YuxiangChemical Reviews (Washington, DC, United States) (2016), 116 (18), 10346-10413CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Colloidal nanoparticles are being intensely pursued in current nanoscience research. Nanochemists are often frustrated by the known fact that no two nanoparticles are the same, which precludes the deep understanding of many fundamental properties of colloidal nanoparticles in which the total structures (core plus surface) must be known. Therefore, controlling nanoparticles with at. precision and solving their total structures have long been major dreams for nanochemists. Recently, these goals are partially fulfilled in the case of gold nanoparticles, at least in the ultrasmall size regime (1-3 nm in diam., often called nanoclusters). This review summarizes the major progress in the field, including the principles that permit atomically precise synthesis, new types of at. structures, and unique phys. and chem. properties of atomically precise nanoparticles, as well as exciting opportunities for nanochemists to understand very fundamental science of colloidal nanoparticles (such as the stability, metal-ligand interfacial bonding, ligand assembly on particle surfaces, aesthetic structural patterns, periodicities, and emergence of the metallic state) and to develop a range of potential applications such as in catalysis, biomedicine, sensing, imaging, optics, and energy conversion. Although most of the research activity currently focuses on thiolate-protected gold nanoclusters, important progress also was achieved in other ligand-protected gold, silver, and bimetal (or alloy) nanoclusters. All of these types of unique nanoparticles will bring unprecedented opportunities, not only in understanding the fundamental questions of nanoparticles but also in opening up new horizons for scientific studies of nanoparticles.
- 275Zhang, L. B.; Wang, E. K. Metal Nanoclusters: New Fluorescent Probes for Sensors and Bioimaging. Nano Today 2014, 9, 132– 157, DOI: 10.1016/j.nantod.2014.02.010Google Scholar275Metal nanoclusters: New fluorescent probes for sensors and bioimagingZhang, Libing; Wang, ErkangNano Today (2014), 9 (1), 132-157CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.)A review. Fluorescent metal nanoclusters (NCs) as a new class of fluorophores have attracted more and more attention due to their unique electronic structures and the subsequent unusual phys. and chem. properties. The size of metal NCs approaches the Fermi wavelength of electrons, between metal atoms and nanoparticles, resulting in mol.-like properties including discrete energy levels, size-dependent fluorescence, good photostability and biocompatibility. These excellent properties make them ideal fluorescent probes for biol. application. Up to now, significant efforts have been devoted to the synthesis, property and application studies of gold and silver NCs. Recently, a growing no. of studies on copper and other metal clusters have also been reported. In this review article, we focus on summarizing recent advances in controllable synthesis strategies, chem. and optical properties, and sensing and imaging applications of metal NCs (mainly including Au, Ag, Cu, etc.). Finally, we conclude with a look at the future challenges and prospects of the future development of metal NCs.
- 276Shang, L.; Dong, S. J.; Nienhaus, G. U. Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications. Nano Today 2011, 6, 401– 418, DOI: 10.1016/j.nantod.2011.06.004Google Scholar276Ultra-small fluorescent metal nanoclusters: synthesis and biological applicationsShang, Li; Dong, Shaojun; Nienhaus, G. UlrichNano Today (2011), 6 (4), 401-418CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.)A review. Recent advances in nanotechnol. have given rise to a new class of fluorescent labels, fluorescent metal nanoclusters, e.g., Au and Ag. These nanoclusters are of significant interest because they provide the missing link between at. and nanoparticle behavior in metals. Composed of a few to a hundred atoms, their sizes are comparable to the Fermi wavelength of electrons, resulting in mol.-like properties including discrete electronic states and size-dependent fluorescence. Fluorescent metal nanoclusters have an attractive set of features, such as ultrasmall size, good biocompatibility and excellent photostability, making them ideal fluorescent labels for biol. applications. In this review, we summarize synthesis strategies of water-sol. fluorescent metal nanoclusters and their optical properties, highlight recent advances in their application for ultrasensitive biol. detection and fluorescent biol. imaging, and finally discuss current challenges for their potential biomedical applications.
- 277Draz, M. S.; Fang, B. A.; Li, L. J.; Chen, Z.; Wang, Y. J.; Xu, Y. H.; Yang, J.; Killeen, K.; Chen, F. F. Hybrid Nanocluster Plasmonic Resonator for Lmmunological Detection of Hepatitis B Virus. ACS Nano 2012, 6, 7634– 7643, DOI: 10.1021/nn3034056Google Scholar277Hybrid Nanocluster Plasmonic Resonator for Immunological Detection of Hepatitis B VirusDraz, Mohamed Shehata; Fang, Binbin Amanda; Li, Lanjuan; Chen, Zhi; Wang, Yingjie; Xu, Yuhong; Yang, Jun; Killeen, Kevin; Chen, Fanqing FrankACS Nano (2012), 6 (9), 7634-7643CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Approx. 88% of the world population lives in regions with intermediate to high incidence of Hepatitis B virus (HBV), yet current serol. and DNA-based detection methods have limited sensitivity and convenience. Here, the authors describe a preassembled plasmonic resonance nanocluster for HBV detection. The gold nanoparticle acceptors (AuNPs), with HBV surface antigen (HBsAg) epitope, and quantum dot (QD) donors with Fab antibody, are assembled into an immuno-mediated 3D-oriented complex with enhanced energy transfer and fluorescence quenching. The coherent plasmonic resonance between Au and QD nanoparticles is exploited to achieve improved donor-acceptor resonance within the nanocluster, which in the presence of HBV viral particles is disassembled in a highly specific manner. The nanocluster provides high detection specificity and sensitivity of HBV, with a sensitivity limit down to 1-100 viral particles per μL and to attomolar levels of HBsAg. This general platform could be used to establish multiplex diagnostic assays for a variety of other microbial pathogens.
- 278Shokri, E.; Hosseini, M.; Faridbod, F.; Rahaie, M. Rapid Pre-Symptomatic Recognition of Tristeza Viral RNA by a Novel Fluorescent Self-Dimerized DNA-Silver Nanocluster probe. RSC Adv. 2016, 6, 99437– 99443, DOI: 10.1039/C6RA15199JGoogle Scholar278Rapid pre-symptomatic recognition of tristeza viral RNA by a novel fluorescent self-dimerized DNA-silver nanocluster probeShokri, Ehsan; Hosseini, Morteza; Faridbod, Farnoush; Rahaie, MahdiRSC Advances (2016), 6 (101), 99437-99443CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Citrus tristeza virus (CTV), a pos.-strand RNA virus within the family of Closteroviridae, is distributed worldwide and causes one of the most economically important diseases of citrus. Since the CTV pathogen is easily spread by graft propagation and aphid vectors, continual monitoring of healthy seedlings and eradication of infected plants is essential for better disease management. In this study, a novel self-dimerized DNA-silver nanocluster probe was developed for the simple and rapid pre-symptomatic detection of CTV severe strains in biol. prepns. Insertion of a G rich loop maker sequence contg. internal complementary bases in the probe structure leads to the formation of a stable homodimer probe with a G-rich loop at the center that is more reactive than the formless probe. Interestingly, the results showed that the probe structure conversion between dimeric and non dimeric states upon DNA/RNA hybridization, produced an enhanced fluorescence signal allowing precise and sensitive detection of tristeza RNA. Based on the sensitivity test, CTV-RNA in the range of 5-210 nM, can be linearly detected with the detection limit of 2.5 nM. Finally, the results of our DNA-AgNCs based fluorometric assay were consistent with the results of the conventional RT-PCR.
- 279Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44, 8636– 8663, DOI: 10.1039/C5CS00607DGoogle Scholar279Metal nanoclusters: novel probes for diagnostic and therapeutic applicationsTao, Yu; Li, Mingqiang; Ren, Jinsong; Qu, XiaogangChemical Society Reviews (2015), 44 (23), 8636-8663CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Metal nanoclusters, composed of several to a few hundred metal atoms, have received worldwide attention due to their extraordinary phys. and chem. characteristics. Recently, great efforts have been devoted to the exploration of the potential diagnostic and therapeutic applications of metal nanoclusters. Here we focus on the recent advances and new horizons in this area, and introduce the rising progress on the use of metal nanoclusters for biol. anal., biol. imaging, therapeutic applications, DNA assembly and logic gate construction, enzyme mimic catalysis, as well as thermometers and pH meters. Furthermore, the future challenges in the construction of biofunctional metal nanoclusters for diagnostic and therapeutic applications are also discussed. We expect that the rapidly growing interest in metal nanocluster-based theranostic applications will certainly not only fuel the excitement and stimulate research in this highly active field, but also inspire broader concerns across various disciplines.
- 280Basle, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17, 213– 227, DOI: 10.1016/j.chembiol.2010.02.008Google Scholar280Protein Chemical Modification on Endogenous Amino AcidsBasle, Emmanuel; Joubert, Nicolas; Pucheault, MathieuChemistry & Biology (Cambridge, MA, United States) (2010), 17 (3), 213-227CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Chem. modification of protein is an arduous but fruitful task. Many chem. methods have been developed for such purpose by carefully balancing reactivity and selectivity. Now both chemists and biologists have in hand an arsenal of tools from which they can select a relevant reaction to tackle their problems. This review focuses on the various chem. transformations available for selective modification of proteins. It also provides a brief overview of some of their main applications, including detection of protein interactions, prepn. of bioconjugates, and protein microarrays.
- 281Hong, Z. Y.; Zhang, Z. L.; Tang, B.; Ao, J.; Wang, C.; Yu, C.; Pang, D. W. Equipping Inner Central Components of Influenza A Virus with Quantum Dots. Anal. Chem. 2018, 90, 14020– 14028, DOI: 10.1021/acs.analchem.8b03995Google Scholar281Equipping Inner Central Components of Influenza A Virus with Quantum DotsHong, Zheng-Yuan; Zhang, Zhi-Ling; Tang, Bo; Ao, Jian; Wang, Chuan; Yu, Cong; Pang, Dai-WenAnalytical Chemistry (Washington, DC, United States) (2018), 90 (23), 14020-14028CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Influenza A virus (IAV), a risk to public health, is enveloped and contains viral ribonucleoprotein (vRNP) complexes, where vRNP complexes are central to every aspect of the IAV life cycle. Labeling both the vRNP complexes and viral envelope with quantum dots (QDs) is conducive to achieving global long-term tracking of a single IAV infecting host cell, which has potential to provide valuable information for revealing mechanisms of IAV infection. However, even though some strategies for labeling of the viral envelope with QDs have been developed, there are few strategies for coupling of QDs to the vRNP complexes inside IAV so far. Herein, we devised a convenient electroporation-based strategy, coupled with antibody binding, to transfer green QDs-labeled nucleoprotein antibodies (GQDs-NPAb) into H1N1 and achieved the labeling of vRNP complexes with QDs [H1N1(GQDs)]. Under the optimal condition of 20 nM GQDs-NPAb and a single pulse with 20 ms duration and 750 V/cm pulse intensity, the actual efficiency of labeling is ca. 34% and H1N1(GQDs) can retain 93% infectivity. Then, dual labeling of H1N1 was realized by labeling the envelope of H1N1(GQDs) with red QDs (RQDs) via a mild and efficient hydrazine-aldehyde-based strategy. At the optimal RQDs concn. of 5 nM, the actual efficiency of dual labeling can reach to 11% and the dual-labeled H1N1 can retain 93% infectivity. Because of the similar components and structure of different IAV subtypes, this dual-labeling strategy is applicable to other subtypes of IAV, e.g., H9N2.
- 282Huisgen, R. 1.3-Dipolare Cycloadditionen Rückschau und Ausblick. Angew. Chem. 1963, 75, 604– 637, DOI: 10.1002/ange.19630751304Google Scholar2821,3-Dipolar cycloadditionsHuisgen, RolfAngewandte Chemie (1963), 75 (13), 604-37CODEN: ANCEAD; ISSN:0044-8249.cf. CA 56, 7096c. A review with 211 references.
- 283Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. 2001, 40, 2004– 2021, DOI: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5Google Scholar283Click chemistry: diverse chemical function from a few good reactionsKolb, Hartmuth C.; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2001), 40 (11), 2004-2021CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)Review with > 88 refs. Examn. of nature's favorite mols. reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds - surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon-heteroatom bonds. Even the 35 or so building blocks from which these crucial mols. are made each contain, at most, six contiguous C-C bonds, except for the three arom. amino acids. Taking a cue from nature's approach, the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compds. and combinatorial libraries through heteroatom links (C-X-C), an approach called "click chem." is addressed. Click chem. is at once defined, enabled, and constrained by a handful of nearly perfect "spring-loaded" reactions. The stringent criteria for a process to earn click chem. status are described along with examples of the mol. frameworks that are easily made using this spartan, but powerful, synthetic strategy.
- 284Nwe, K.; Brechbiel, M. W. Growing Applications of ″Click Chemistry″ for Bioconjugation in Contemporary Biomedical Research. Cancer Biother.Radiopharm. 2009, 24, 289– 302, DOI: 10.1089/cbr.2008.0626Google Scholar284Growing Applications of "Click Chemistry" for Bioconjugation in Contemporary Biomedical ResearchNwe, Kido; Brechbiel, Martin W.Cancer Biotherapy & Radiopharmaceuticals (2009), 24 (3), 289-302CODEN: CBRAFJ; ISSN:1084-9785. (Mary Ann Liebert, Inc.)A review. This update summarizes the growing application of "click" chem. in diverse areas such as bioconjugation, drug discovery, materials science, and radiochem. This update also discusses click chem. reactions that proceed rapidly with high selectivity, specificity, and yield. Two important characteristics make click chem. so attractive for assembling compds., reagents, and biomols. for preclin. and clin. applications. First, click reactions are bio-orthogonal; neither the reactants nor their product's functional groups interact with functionalized biomols. Second, the reactions proceed with ease under mild nontoxic conditions, such as at room temp. and, usually, in water. The copper-catalyzed Huisgen cycloaddn., azide-alkyne [3 + 2] dipolar cycloaddn., Staudinger ligation, and azide-phosphine ligation each possess these unique qualities. These reactions can be used to modify one cellular component while leaving others unharmed or untouched. Click chem. has found increasing applications in all aspects of drug discovery in medicinal chem., such as for generating lead compds. through combinatorial methods. Bioconjugation via click chem. is rigorously employed in proteomics and nucleic research. In radiochem., selective radiolabeling of biomols. in cells and living organisms for imaging and therapy has been realized by this technol. Bifunctional chelating agents for several radionuclides useful for positron emission tomog. and single-photon emission computed tomog. imaging have also been prepd. by using click chem. This review concludes that click chem. is not the perfect conjugation and assembly technol. for all applications, but provides a powerful, attractive alternative to conventional chem. This chem. has proven itself to be superior in satisfying many criteria (e.g., biocompatibility, selectivity, yield, stereospecificity, and so forth); thus, one can expect it will consequently become a more routine strategy in the near future for a wide range of applications.
- 285Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry. J. Am. Chem. Soc. 2008, 130, 11486– 11493, DOI: 10.1021/ja803086rGoogle Scholar285Second-Generation Difluorinated Cyclooctynes for Copper-Free Click ChemistryCodelli, Julian A.; Baskin, Jeremy M.; Agard, Nicholas J.; Bertozzi, Carolyn R.Journal of the American Chemical Society (2008), 130 (34), 11486-11493CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The 1,3-dipolar cycloaddn. of azides and activated alkynes has been used for site-selective labeling of biomols. in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3+2] cycloaddn., this method, often termed "click chem.", is currently incompatible with living systems because of the toxicity of the metal. The authors recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here the authors report a novel class of DIFO reagents for copper-free click chem. that are considerably more synthetically tractable. The new analogs maintained the same elevated rates of [3+2] cycloaddn. as the parent compd. and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chem. in the hands of biologists.
- 286Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-Free Click Chemistry for Dynamic in Vivo Imaging. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16793– 16797, DOI: 10.1073/pnas.0707090104Google Scholar286Copper-free click chemistry for dynamic in vivo imagingBaskin, Jeremy M.; Prescher, Jennifer A.; Laughlin, Scott T.; Agard, Nicholas J.; Chang, Pamela V.; Miller, Isaac A.; Lo, Anderson; Codelli, Julian A.; Bertozzi, Carolyn R.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (43), 16793-16797CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomols. such as glycans and lipids. Here, the authors report a Cu-free variant of click chem. that can label these biomols. rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The crit. reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3+2] dipolar cycloaddn. with azides installed metabolically into biomols. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, the authors studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
- 287Hoyle, C. E.; Bowman, C. N. Thiol-Ene Click Chemistry. Angew. Chem., Int. Ed. 2010, 49, 1540– 1573, DOI: 10.1002/anie.200903924Google Scholar287Thiol-Ene Click ChemistryHoyle, Charles E.; Bowman, Christopher N.Angewandte Chemie, International Edition (2010), 49 (9), 1540-1573CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Following Sharpless' visionary characterization of several idealized reactions as click reactions, the materials science and synthetic chem. communities have pursued numerous routes toward the identification and implementation of these click reactions. Herein, the authors review the radical-mediated thiol-ene reaction as one such click reaction. This reaction has all the desirable features of a click reaction, being highly efficient, simple to execute with no side products and proceeding rapidly to high yield. Further, the thiol-ene reaction is most frequently photoinitiated, particularly for photopolymns. resulting in highly uniform polymer networks, promoting unique capabilities related to spatial and temporal control of the click reaction. The reaction mechanism and its implementation in various synthetic methodologies, biofunctionalization, surface and polymer modification, and polymn. are all reviewed.
- 288Nikic, I.; Plass, T.; Schraidt, O.; Szymanski, J.; Briggs, J. A.; Schultz, C.; Lemke, E. A. Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy. Angew. Chem., Int. Ed. 2014, 53, 2245– 2249, DOI: 10.1002/anie.201309847Google Scholar288Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution MicroscopyNikic, Ivana; Plass, Tilman; Schraidt, Oliver; Szymanski, Jedrzej; Briggs, John A. G.; Schultz, Carsten; Lemke, Edward A.Angewandte Chemie, International Edition (2014), 53 (8), 2245-2249CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The growing demands of advanced fluorescence and super-resoln. microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chem. handles into proteins in living cells. Here the authors present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized org. dyes by the inverse-electron-demand Diels-Alder cycloaddn. (SPIEDAC). Furthermore, the authors fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which the authors termed selectivity enhanced (se) SPIEDAC. SeSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. The authors demonstrate the strength of the authors' method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resoln. microscopy.
- 289Salic, A.; Mitchison, T. J. A Chemical Method for Fast and Sensitive Detection of DNA Synthesis in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2415– 2420, DOI: 10.1073/pnas.0712168105Google Scholar289A chemical method for fast and sensitive detection of DNA synthesis in vivoSalic, Adrian; Mitchison, Timothy J.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (7), 2415-2420CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddn. reaction ("click" chem.). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount prepns. of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.
- 290Jao, C. Y.; Salic, A. Exploring RNA Transcription and Turnover in Vivo by Using Click Chemistry. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15779– 15784, DOI: 10.1073/pnas.0808480105Google Scholar290Exploring RNA transcription and turnover in vivo by using click chemistryJao, Cindy Y.; Salic, AdrianProceedings of the National Academy of Sciences of the United States of America (2008), 105 (41), 15779-15784CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We describe a chem. method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on av. once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddn. reaction (often referred to as "click" chem.) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.
- 291Jewett, J. C.; Bertozzi, C. R. Cu-Free Click Cycloaddition Reactions in Chemical Biology. Chem. Soc. Rev. 2010, 39, 1272– 1279, DOI: 10.1039/b901970gGoogle Scholar291Cu-free click cycloaddition reactions in chemical biologyJewett, John C.; Bertozzi, Carolyn R.Chemical Society Reviews (2010), 39 (4), 1272-1279CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Bioorthogonal chem. reactions are paving the way for new innovations in biol. These reactions possess extreme selectivity and biocompatibility, such that their participating reagents can form covalent bonds within richly functionalized biol. systems-in some cases, living organisms. This tutorial review summarizes the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddn. reactions. As we get a better idea of the powers and limitations of each bioorthogonal reaction pair, we can start using them in concert to study increasingly complex interactions in biol. settings. While the field of bioorthogonal chem. has expanded rapidly, there remains a pressing need for new reagents with fewer side reactions and increased efficiencies. Where will these new reactions come from Using history as a guide, they are found in the least likely of places, buried in a piece of purely academic scholarship that is being written up at this very moment.
- 292Rubino, F. A.; Oum, Y. H.; Rajaram, L.; Chu, Y.; Carrico, I. S. Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry. J. Visualized Exp. 2012, e4246 DOI: 10.3791/4246Google Scholar292Chemoselective modification of viral surfaces via bioorthogonal click chemistryRubino, Frederick A.; Oum, Yoon Hyeun; Rajaram, Lakshmi; Chu, Yanjie; Carrico, Isaac S.Journal of Visualized Experiments (2012), (66), E4246/1-E4246/7CODEN: JVEOA4; ISSN:1940-087X. (Journal of Visualized Experiments)The modification of virus particles has received a significant amt. of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development. Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus prodn., infectivity and cellular transduction. Using chemoselective click chem., we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible. The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chem. to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically or anal., as it allows for chemoselective ligation of targeting mols., dyes or other mols. of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness, (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access. In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purifn. of the azide-modified virus particles, an alkyne probe contg. the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE anal. is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only. In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein - strain-promoted azide-alkyne cycloaddn. (SPAAC) and copper-catalyzed azide-alkyne cycloaddn. (CuAAC) under deoxygenated atm.
- 293Oum, Y. H.; Desai, T. M.; Marin, M.; Melikyan, G. B. Click Labeling of Unnatural Sugars Metabolically Incorporated into Viral Envelope Glycoproteins Enables Visualization of Single Particle Fusion. J. Virol. Methods 2016, 233, 62– 71, DOI: 10.1016/j.jviromet.2016.02.016Google Scholar293Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusionOum, Yoon Hyeun; Desai, Tanay M.; Marin, Mariana; Melikyan, Gregory B.Journal of Virological Methods (2016), 233 (), 62-71CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with org. fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells.
- 294Huang, L. L.; Lu, G. H.; Hao, J.; Wang, H. Z.; Yin, D. L.; Xie, H. Y. Enveloped Virus Labeling via Both Intrinsic Biosynthesis and Metabolic Incorporation of Phospholipids in Host Cells. Anal. Chem. 2013, 85, 5263– 5270, DOI: 10.1021/ac4008144Google Scholar294Enveloped Virus Labeling via Both Intrinsic Biosynthesis and Metabolic Incorporation of Phospholipids in Host CellsHuang, Li-Li; Lu, Gui-Hong; Hao, Jian; Wang, Hanzhong; Yin, Du-Lin; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2013), 85 (10), 5263-5270CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)An alternative method for labeling fully replicative enveloped viruses was developed, in which both the biosynthesis and metabolic incorporation of phospholipids in host cells were simultaneously utilized to introduce an azide group to the envelope of the vaccinia virus by taking advantage of the host-derived lipid membrane formation mechanism. Such an azide group could be subsequently used to fluorescently label the envelope of the virus via a bioorthogonal reaction. Furthermore, simultaneous dual-labeling of the virus through the virus replication was realized skillfully by coupling this envelope labeling strategy with "replication-intercalation labeling" of viral nucleic acid. For the first time, it is by natural propagation of the virus in its host cells in the presence of fluorophores that simultaneous dual-labeling of living viruses can be mildly realized with high efficiency in facile and mild conditions.
- 295Hou, W.; Li, Y.; Kang, W.; Wang, X.; Wu, X.; Wang, S.; Liu, F. Real-Time Analysis of Quantum Dot Labeled Single Porcine Epidemic Diarrhea Virus Moving along the Microtubules Using Single Particle Tracking. Sci. Rep. 2019, 9, 1307, DOI: 10.1038/s41598-018-37789-9Google Scholar295Real-time analysis of quantum dot labeled single porcine epidemic diarrhea virus moving along the microtubules using single particle trackingHou Wei; Li Yangyang; Kang Wenjie; Wang Xin; Wang Shouyu; Liu Fei; Wu Xuping; Wang ShouyuScientific reports (2019), 9 (1), 1307 ISSN:.In order to study the infection mechanism of porcine epidemic diarrhea virus (PEDV), which causes porcine epidemic diarrhea, a highly contagious enteric disease, we combined quantum dot labeled method, which could hold intact infectivity of the labeled viruses to the largest extent, with the single particle tracking technique to dynamically and globally visualize the transport behaviors of PEDVs in live Vero cells. Our results were the first time to uncover the dynamic characteristics of PEDVs moving along the microtubules in the host cells. It is found that PEDVs kept restricted motion mode with a relatively stable speed in the cell membrane region; while performed a slow-fast-slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule organizing center region. In addition, the return movements of small amount of PEDVs were also observed in the live cells. Collectively, our work is crucial for understanding the movement mechanisms of PEDV in the live cells, and the proposed work also provided important references for further analysis and study on the infection mechanism of PEDVs.
- 296Lin, S.; Yan, H.; Li, L.; Yang, M.; Peng, B.; Chen, S.; Li, W.; Chen, P. R. Site-Specific Engineering of Chemical Functionalities on the Surface of Live Hepatitis D Virus. Angew. Chem., Int. Ed. 2013, 52, 13970– 13974, DOI: 10.1002/anie.201305787Google Scholar296Site-specific engineering of chemical functionalities on the surface of live hepatitis D virusLin, Shixian; Yan, Huan; Li, Lin; Yang, Maiyun; Peng, Bo; Chen, She; Li, Wenhui; Chen, Peng R.Angewandte Chemie, International Edition (2013), 52 (52), 13970-13974CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The hepatitis D virus (HDV) assembly process was engineered to accommodate pyrrolysine (Pyl)-genetic code expansion within viable human hepatocytes, which allowed the genetic and site-specific incorporation of Pyl analogs carrying various functional handles into HDV surface envelope proteins with high prodn. yield. Five recently developed Pyl analogs were used: PenK and ACPK contain an alkyne and an azide group, resp., which can participate in the CuAAC reaction for protein click chem. labeling; BCN bears a cyclooctyne moiety capable of participating in the inverse electron-demand Diels-Alder reaction and the 1,3-dipolar cycloaddn. on proteins; DiZPK possesses a photo-reactive diazirine for protein photocrosslinking; and ONBK carries a photolytically removable O-nitrobenzyloxycarbonyl group. These 5 Pyl analogs can be recognized by wild-type or mutant PylRS and were incorporated into residues located at Pre S1, Pre S2, or S domains on the HBV L protein. These HBV envelope proteins were assembled with pregenerated HDV RNA and delta antigen into infectious HDV in human Huh-7 cells and successfully labeled using the Pyl analogs.
- 297Huang, L. L.; Liu, K.; Zhang, Q.; Xu, J.; Zhao, D.; Zhu, H.; Xie, H. Y. Integrating Two Efficient and Specific Bioorthogonal Ligation Reactions with Natural Metabolic Incorporation in One Cell for Virus Dual Labeling. Anal. Chem. 2017, 89, 11620– 11627, DOI: 10.1021/acs.analchem.7b03043Google Scholar297Integrating Two Efficient and Specific Bioorthogonal Ligation Reactions with Natural Metabolic Incorporation in One Cell for Virus Dual LabelingHuang, Li-Li; Liu, Kejiang; Zhang, Qianmei; Xu, Jin; Zhao, Dongxu; Zhu, Houshun; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2017), 89 (21), 11620-11627CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Though techniques in bioorthogonal chem. and metabolic incorporation have been developed over the past decade, it remains difficult to integrate different bioorthogonal reactions or metabolic incorporations into one system. The protein and DNA metabolic incorporations were combined with two bioorthogonal reactions in one cell to develop a facile and universal method for virus dual labeling. Azide and vinyl groups were introduced into the proteins or genomes of viruses, resp., through the intrinsic biosynthesis of biomols., which were subsequently fluorescently labeled via copper-free click chem. or alkene-tetrazine ligation reactions during natural propagation process in host cells. Both the envelope viruses and the capsid viruses could be labeled, and the dual labeling efficiency was >80%. The labeled progeny virions were structurally intact and fully infectious, and their fluorescence was strong enough to track single virions.
- 298Wang, I. H.; Suomalainen, M.; Andriasyan, V.; Kilcher, S.; Mercer, J.; Neef, A.; Luedtke, N. W.; Greber, U. F. Tracking Viral Genomes in Host Cells at Single-Molecule Resolution. Cell Host Microbe 2013, 14, 468– 480, DOI: 10.1016/j.chom.2013.09.004Google Scholar298Tracking Viral Genomes in Host Cells at Single-Molecule ResolutionWang, I.-Hsuan; Suomalainen, Maarit; Andriasyan, Vardan; Kilcher, Samuel; Mercer, Jason; Neef, Anne; Luedtke, Nathan W.; Greber, Urs F.Cell Host & Microbe (2013), 14 (4), 468-480CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Viral DNA trafficking in cells has large impacts on physiol. and disease development. Current methods lack the resoln. and accuracy to visualize and quantify viral DNA trafficking at single-mol. resoln. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresoln. microscopy and copper(I)-catalyzed azide-alkyne cycloaddn. (click) reactions allowed visualization of infection at single vDNA resoln. within mammalian cells. Anal. of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.
- 299Green, N. M. Avidin and Streptavidin. Methods Enzymol; Elsevier: 1990; Vol. 184, pp 51– 67.Google ScholarThere is no corresponding record for this reference.
- 300Zhang, F.; Zheng, Z.; Liu, S. L.; Lu, W.; Zhang, Z.; Zhang, C.; Zhou, P.; Zhang, Y.; Long, G.; He, Z. Self-Biotinylation and Site-Specific Double Labeling of Baculovirus Using Quantum Dots for Single-Virus in-Situ Tracking. Biomaterials 2013, 34, 7506– 7518, DOI: 10.1016/j.biomaterials.2013.06.030Google Scholar300Self-biotinylation and site-specific double labeling of baculovirus using quantum dots for single-virus in-situ trackingZhang, Fuxian; Zheng, Zhenhua; Liu, Shu-Lin; Lu, Wen; Zhang, Zhenfeng; Zhang, Cuiling; Zhou, Peng; Zhang, Yuan; Long, Gang; He, Zhike; Pang, Dai-Wen; Hu, Qinxue; Wang, HanzhongBiomaterials (2013), 34 (30), 7506-7518CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Single-virus labeling and tracking represent a powerful tool to study virus-cell interactions. Using baculovirus as a model, here the authors developed a biochem. method for labeling both the viral envelope and the viral capsid of a virus. Viral envelope of the baculovirus AcMNPV was self-biotinylated and site-specifically conjugated with quantum dots (QDs) following one-step binding reaction, while the viral nucleocapsid was site-specifically labeled with green fluorescent protein (GFP) during viral replication. The established procedure of labeling did not affect viral infectivity, showing that the double-labeled virus retained functional structure and could be tracked for viral localization and movement in the host cells. The double-labeled virus also demonstrated the potential to be used for in-situ and real-time visualizing the internalization of a single viral particle into the host cells. Furthermore, the disassembly processes of the viral envelope and the viral nucleocapsid could be monitored for a long period of time (up to 2 h). Using the established method, several interaction details between the labeled baculoviruses and the host cells have been revealed. Given its advantages in high efficiency, high specificity, convenience and the maintenance of viral infectivity, the established approach provides a promising means for elucidating virus-cell interactions.
- 301Liu, J.; Xu, M.; Tang, B.; Hu, L.; Deng, F.; Wang, H.; Pang, D. W.; Hu, Z.; Wang, M.; Zhou, Y. Single-Particle Tracking Reveals the Sequential Entry Process of the Bunyavirus Severe Fever with Thrombocytopenia Syndrome Virus. Small 2019, 15, e1803788 DOI: 10.1002/smll.201803788Google ScholarThere is no corresponding record for this reference.
- 302Liu, J.; Yu, C.; Gui, J. F.; Pang, D. W.; Zhang, Q. Y. Real-Time Dissecting the Entry and Intracellular Dynamics of Single Reovirus Particle. Front. Microbiol. 2018, 9, 2797, DOI: 10.3389/fmicb.2018.02797Google Scholar302Real-Time Dissecting the Entry and Intracellular Dynamics of Single Reovirus ParticleLiu Jia; Gui Jian-Fang; Zhang Qi-Ya; Yu Cong; Pang Dai-WenFrontiers in microbiology (2018), 9 (), 2797 ISSN:1664-302X.Reoviruses are non-enveloped viruses with wide host range, can cause serious infections in animals, plants and microorganism, e.g., aquareovirus, which is capable of causing serious haemorrhagic in aquatic animals. To date, the entry process of aquareovirus infection remains obscure. Real-time single-virus tracking are effective tools for exploring the details in viral infection process, which are crucial for understanding the pathogenic mechanism. Here, we used quantum dots-based single particle tracking technology combined with biochemical assays and ultrastructural observation to reveal unobservable infection steps and map dynamic interactions between a reovirus, Scophthalmus maximus reovirus (SMReV), and its host cell in real time. The results showed that the single membrane-bound reovirus particle can enter into the cell within several seconds through nascent clathrin-caoted pits, and most of the particles could internalize into cytoplasm within 30 min post-infection. The specific inhibitors analysis also showed that entry of SMREV depended on clathrin-mediated endocytosis rather than cavolin-mediated endocytosis. The motion analysis of internalized single particle indicated that the reovirus initially experienced slow and directed motion in the actin-enriched cell periphery, while it underwent relatively faster and directed movement toward the cell interior, suggesting that transport of SMReV was dependent on the cytoskeleton. Further, dual-labeling of virus and cytoskeleton and inhibitor analysis both demonstrated that transport of internalized SMReV was firstly dependent on actin filaments at the cell periphery, and then on microtubules toward the cell interior. Then visualization of SMReV trafficking in the endosomes revealed that the internalized reovirus particles were sorted from early endosomes to late endosomes, then part of them were delivered to lysosome. This study for the first time revealed the entry pathway, intracellular dynamic and the infection fate of fish reovirus in host cell in real time and in situ, which provided new insights into the infection mechanism of non-enveloped viruses.
- 303Dixit, S. K.; Goicochea, N. L.; Daniel, M. C.; Murali, A.; Bronstein, L.; De, M.; Stein, B.; Rotello, V. M.; Kao, C. C.; Dragnea, B. Quantum Dot Encapsulation in Viral Capsids. Nano Lett. 2006, 6, 1993– 1999, DOI: 10.1021/nl061165uGoogle Scholar303Quantum Dot Encapsulation in Viral CapsidsDixit, Suraj K.; Goicochea, Nancy L.; Daniel, Marie-Christine; Murali, Ayaluru; Bronstein, Lyudmila; De, Mrinmoy; Stein, Barry; Rotello, Vincent M.; Kao, C. Cheng; Dragnea, BogdanNano Letters (2006), 6 (9), 1993-1999CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Incorporation of CdSe/ZnS semiconductor quantum dots (QDs) into viral particles provides a new paradigm for the design of intracellular microscopic probes and vectors. Several strategies for the incorporation of QDs into viral capsids were explored; those functionalized with poly(ethylene glycol) (PEG) can be self-assembled into viral particles with minimal release of photoreaction products and enhanced stability against prolonged irradn.
- 304Huang, B. H.; Lin, Y.; Zhang, Z. L.; Zhuan, F.; Liu, A. A.; Xie, M.; Tian, Z. Q.; Zhang, Z.; Wang, H.; Pang, D. W. Surface Labeling of Enveloped Viruses Assisted by Host Cells. ACS Chem. Biol. 2012, 7, 683– 688, DOI: 10.1021/cb2001878Google Scholar304Surface Labeling of Enveloped Viruses Assisted by Host CellsHuang, Bi-Hai; Lin, Yi; Zhang, Zhi-Ling; Zhuan, Fangfang; Liu, An-An; Xie, Min; Tian, Zhi-Quan; Zhang, Zhenfeng; Wang, Hanzhong; Pang, Dai-WenACS Chemical Biology (2012), 7 (4), 683-688CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Labeling of virus opens new pathways for the understanding of viruses themselves and facilitates the utilization of viruses in modern biol., medicine, and materials. Based on the characteristic that viruses hijack their host cellular machineries to survive and reproduce themselves, a host-cell-assisted strategy is proposed to label enveloped viruses. By simply feeding Vero cells with com. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotin-Cap-PE), the authors obtained biotinylated Vero cells whose membrane systems were modified with biotin. Subsequently, pseudorabies viruses (PrV) were cultivated in the biotinylated Vero cells, and the PrV progenies were spontaneously labeled with Biotin-Cap-PE during viral natural assembly process. Since the viral natural assembly process was employed for the labeling, potential threats of genetic engineering and difficulties in keeping viral natural bioactivity were avoided. Importantly, this labeling strategy for enveloped virus greatly reduces the tech. complexity and allows researchers from different backgrounds to apply it for their specified demands.
- 305You, J. O.; Liu, Y. S.; Liu, Y. C.; Joo, K. I.; Peng, C. A. Incorporation of Quantum Dots on Virus in Polycationic Solution. Int. J. Nanomedicine 2006, 1, 59– 64, DOI: 10.2147/nano.2006.1.1.59Google Scholar305Incorporation of quantum dots on virus in polycationic solutionYou, Jin-Oh; Liu, Yu-San; Liu, Yu-Chuan; Joo, Kye-Il; Peng, Ching-AnInternational Journal of Nanomedicine (2006), 1 (1), 59-64CODEN: IJNNHQ; ISSN:1176-9114. (Dove Medical Press (NZ) Ltd.)Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biol. imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both neg. charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examg. the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 h, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.
- 306Chen, Y. H.; Wang, C. H.; Chang, C. W.; Peng, C. A. In Situ Formation of Viruses Tagged with Quantum Dots. Integr. Biol. 2010, 2, 258– 264, DOI: 10.1039/b926852aGoogle Scholar306In situ formation of viruses tagged with quantum dotsChen, Yu-Hao; Wang, Chung-Hao; Chang, Chia-Wei; Peng, Ching-AnIntegrative Biology (2010), 2 (5-6), 258-264CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)Quantum dots (QDs) have great potential for applications in bio-related fields, due to their high photoluminescence, photochem. stability and size-dependent emission. QDs used for the construction of QD-virus hybrids can be harnessed as an imaging probe to reveal viral infection pathways and screen antiviral agents. In the study, human embryonic kidney (HEK) 293T cells were transfected with three plasmids, pSIN-EGFP, pMDG, and p8.91, to produce lentiviruses which can make infected cells express enhanced green fluorescent protein (EGFP). The QDs employed were CdSe-ZnS semiconductor nanocrystals emitting red fluorescence. The QD-virus hybrids, constructed as lentiviruses, were budding from the membrane surface of HEK 293T producer cells on which QDs encapsulated with alkylated chitosan (Chitosan-QDs) were pre-adsorbed via electrostatic attraction force. Such in situ formation of QD-virus hybrids was confirmed by TEM micrographs indicating the lentivirus was capped with chitosan-modified QDs. To further illustrate the effectiveness (i.e., infectivity and photoluminescence) of the constructed QD-virus hybrids, NIH 3T3 cells were infected with the in situ fabricated QD-virus hybrids. Our results showed QDs were indeed entering NIH 3T3 cells along with lentiviruses as hybrids. Moreover, photoluminescence and infectivity of QD-virus hybrids remained intact, as compared to QDs and lentivirus alone. The unique approach of constructing QD-virus hybrids taking advantage of the viral budding process offers a feasible tool to create enveloped virus incorporated with nanomaterials for the study of fundamental and applied virol.
- 307Li, F.; Zhang, Z. P.; Peng, J.; Cui, Z. Q.; Pang, D. W.; Li, K.; Wei, H. P.; Zhou, Y. F.; Wen, J. K.; Zhang, X. E. Imaging Viral Behavior in Mammalian Cells with Self-Assembled Capsid-Quantum-Dot Hybrid Particles. Small 2009, 5, 718– 726, DOI: 10.1002/smll.200801303Google Scholar307Imaging viral behavior in mammalian cells with self-assembled capsid-quantum-dot hybrid particlesLi, Feng; Zhang, Zhi-Ping; Peng, Jun; Cui, Zong-Qiang; Pang, Dai-Wen; Li, Ke; Wei, Hong-Ping; Zhou, Ya-Feng; Wen, Ji-Kai; Zhang, Xian-EnSmall (2009), 5 (6), 718-726CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long-term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD-contg. virus-like particles (VLPs) of simian virus 40 (SV40), termed SVLP-QDs, are constructed by in vitro self-assembly of the major capsid protein of SV40. SVLP-QDs show homogeneity in size (≈ 24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP-QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.
- 308Johnson, H. E.; Haugh, J. M. Quantitative Analysis of Phosphoinositide 3-Kinase (PI3K) Signaling Using Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr. Protoc. Cell Biol. 2013, 61, 14.14.1– 14.14.24, DOI: 10.1002/0471143030.cb1414s61Google ScholarThere is no corresponding record for this reference.
- 309Chen, I.; Ting, A. Y. Site-Specific Labeling of Proteins with Small Molecules in Live Cells. Curr. Opin. Biotechnol. 2005, 16, 35– 40, DOI: 10.1016/j.copbio.2004.12.003Google Scholar309Site-specific labeling of proteins with small molecules in live cellsChen, Irwin; Ting, Alice Y.Current Opinion in Biotechnology (2005), 16 (1), 35-40CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. The principal bottleneck for the utilization of small-mol. probes in live cells is the shortage of methodologies for targeting them with very high specificity to biol. mols. or compartments of interest. Recently developed methods for labeling proteins with small-mol. probes in cells employ special protein or peptide handles that recruit small-mol. ligands, harness enzymes to catalyze small-mol. conjugation or hijack the cell's protein translation machinery.
- 310Gong, Y. K.; Pan, L. F. Recent Advances in Bioorthogonal Reactions for Site-Specific Protein Labeling and Engineering. Tetrahedron Lett. 2015, 56, 2123– 2132, DOI: 10.1016/j.tetlet.2015.03.065Google Scholar310Recent advances in bioorthogonal reactions for site-specific protein labeling and engineeringGong, Yukang; Pan, LifengTetrahedron Letters (2015), 56 (17), 2123-2132CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)A review. In the past two decades, with the rapid development of chem. biol., tremendous small-mol. based toolkits were created by org. chemists, and were widely used to study and manipulate proteins to dissect their complicated biol. functions. This review summarizes some recent progresses of bioorthogonal reactions for site-specific protein labeling and engineering, and highlights the powers of using these methods to study the biol. functions of some proteins.
- 311Zhang, G.; Zheng, S. Q.; Liu, H. P.; Chen, P. R. Illuminating Biological Processes through Site-Specific Protein Labeling. Chem. Soc. Rev. 2015, 44, 3405– 3417, DOI: 10.1039/C4CS00393DGoogle Scholar311Illuminating biological processes through site-specific protein labelingZhang, Gong; Zheng, Siqi; Liu, Haiping; Chen, Peng R.Chemical Society Reviews (2015), 44 (11), 3405-3417CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biol. setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biol. problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biol. processes.
- 312Jing, C.; Cornish, V. W. Chemical Tags for Labeling Proteins inside Living Cells. Acc. Chem. Res. 2011, 44, 784– 792, DOI: 10.1021/ar200099fGoogle Scholar312Chemical Tags for Labeling Proteins Inside Living CellsJing, Chaoran; Cornish, Virginia W.Accounts of Chemical Research (2011), 44 (9), 784-792CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, the challenge of understanding how macromol. machines, signaling pathways, and other biol. networks operate in the complex environment of the living cell must now be faced. The fluorescent proteins (FPs) revolutionized the ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biol., they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chem. methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chem. tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chem. tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an org. fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chem. tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, the authors focus on the development and application of chem. tags for studying protein function within living cells. Thus, in the authors' overview of different chem. tagging strategies and technologies, the authors emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. The authors highlight recent applications in which the chem. tags have enabled sophisticated biophys. measurements that would be difficult or even impossible with FPs. Finally, the authors conclude by looking forward to (i) the development of high-photon-output chem. tags compatible with living cells to enable high-resoln. imaging, (ii) the realization of the potential of the chem. tags to significantly reduce tag size, and (iii) the exploitation of the modular chem. tag label to go beyond fluorescent imaging.
- 313Klein, T.; Loschberger, A.; Proppert, S.; Wolter, S.; van de Linde, S.; Sauer, M. Live-Cell dSTORM with SNAP-Tag Fusion Proteins. Nat. Methods 2011, 8, 7– 9, DOI: 10.1038/nmeth0111-7bGoogle Scholar313Live-cell dSTORM with SNAP-tag fusion proteinsKlein, Teresa; Loeschberger, Anna; Proppert, Sven; Wolter, Steve; van de Linde, Sebastian; Sauer, MarkusNature Methods (2011), 8 (1), 7-9CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)In the Sept. 2010 issue of Nature Methods, live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins was demonstrated using a trimethoprim chem. tag (TMP tag) for genetic encoding with photo stable std. fluorophores. In line with this, this study reported that live-cell dSTORM of core histone H2B proteins in different eukaryotic cell lines is possible using com. available SNAP tags as well. Protein-specific labeling was based on the irreversible reaction of human O6-alkylguanine-DNA alkyltransferase with the rhodamine O6-benzylguanine derivs. SNAP-Cell 505 (rhodamine green) and SNAP-Cell TMR-Star (tetramethylrhodamine) (New England BioLabs). Data generally suggest that dSTORM used in combination with std. chem. tags and conventional synthetic org. fluorophores is a simple method for live-cell super-resoln. imaging with high spatiotemporal resoln. that can be advantageously combined with photoactivatable fluorescent proteins for multicolor applications. Notably, the highly reversible and reliable photoswitching process of rhodamine and oxazine fluorophores in the cellular environment enables reversible photoswitching of most com. org. fluorophores in living cells without any additives.
- 314Chen, Z.; Cornish, V. W.; Min, W. Chemical Tags: Inspiration for Advanced Imaging Techniques. Curr. Opin. Chem. Biol. 2013, 17, 637– 643, DOI: 10.1016/j.cbpa.2013.05.018Google Scholar314Chemical tags: inspiration for advanced imaging techniquesChen, Zhixing; Cornish, Virginia W.; Min, WeiCurrent Opinion in Chemical Biology (2013), 17 (4), 637-643CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)This review summarizes recent applications of chem. tags in conjunction with advanced bio-imaging techniques including single-mol. fluorescence, spatiotemporally resolved ensemble microscopy techniques, and imaging modalities beyond fluorescence. The authors aim to illustrate the unique advantages of chem. tags in facilitating contemporary microscopy to address biol. problems that are difficult or near impossible to approach otherwise. The authors hope the authors' review will inspire more innovative applications enabled by the mingling of these two growing fields.
- 315Wombacher, R.; Cornish, V. W. Chemical Tags: Applications in Live Cell Fluorescence Imaging. J. Biophotonics 2011, 4, 391– 402, DOI: 10.1002/jbio.201100018Google Scholar315Chemical tags: Applications in live cell fluorescence imagingWombacher, Richard; Cornish, Virginia W.Journal of Biophotonics (2011), 4 (6), 391-402CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biol. processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chem. biol. techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chem. tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected expts. in which chem. tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
- 316Specht, E. A.; Braselmann, E.; Palmer, A. E. A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging. Annu. Rev. Physiol. 2017, 79, 93– 117, DOI: 10.1146/annurev-physiol-022516-034055Google Scholar316A Critical and Comparative Review of Fluorescent Tools for Live-Cell ImagingSpecht, Elizabeth A.; Braselmann, Esther; Palmer, Amy E.Annual Review of Physiology (2017), 79 (), 93-117CODEN: ARPHAD; ISSN:0066-4278. (Annual Reviews)Fluorescent tools have revolutionized our ability to probe biol. dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-mol. dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small mols., and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell anal. and esp. live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.
- 317Eckhardt, M.; Anders, M.; Muranyi, W.; Heilemann, M.; Krijnse-Locker, J.; Muller, B. A SNAP-Tagged Derivative of HIV-1--A Versatile Tool to Study Virus-Cell Interactions. PLoS One 2011, 6, e22007 DOI: 10.1371/journal.pone.0022007Google Scholar317A SNAP-tagged derivative of HIV-1 - a versatile tool to study virus-cell interactionsEckhardt, Manon; Anders, Maria; Muranyi, Walter; Heilemann, Mike; Krijnse-Locker, Jacomine; Mueller, BarbaraPLoS One (2011), 6 (7), e22007CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Fluorescently labeled human immunodeficiency virus (HIV) derivs., combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochem. properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate mols. in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV deriv. HIVSNAP, which carries the SNAP-tag as an addnl. domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the anal. of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.
- 318Hanne, J.; Gottfert, F.; Schimer, J.; Anders-Osswein, M.; Konvalinka, J.; Engelhardt, J.; Muller, B.; Hell, S. W.; Krausslich, H. G. Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation. ACS Nano 2016, 10, 8215– 8222, DOI: 10.1021/acsnano.6b03850Google Scholar318Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic MaturationHanne, Janina; Goettfert, Fabian; Schimer, Jiri; Anders-Oesswein, Maria; Konvalinka, Jan; Engelhardt, Johann; Mueller, Barbara; Hell, Stefan W.; Kraeusslich, Hans-GeorgACS Nano (2016), 10 (9), 8215-8222CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomog. provided structures of immature and mature HIV-1 with a diam. of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, the authors employed super-resoln. STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resoln. microscopy. This study shows the rearrangement of subviral structures in a super-resoln. light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biol. process by an optical switch.
- 319Sun, X.; Zhang, A.; Baker, B.; Sun, L.; Howard, A.; Buswell, J.; Maurel, D.; Masharina, A.; Johnsson, K.; Noren, C. J. Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging. ChemBioChem 2011, 12, 2217– 2226, DOI: 10.1002/cbic.201100173Google Scholar319Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence ImagingSun, Xiaoli; Zhang, Aihua; Baker, Brenda; Sun, Luo; Howard, Angela; Buswell, John; Maurel, Damien; Masharina, Anastasiya; Johnsson, Kai; Noren, Christopher J.; Xu, Ming-Qun; Correa, Ivan R.ChemBioChem (2011), 12 (14), 2217-2226CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The ability to specifically attach chem. probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of exptl. settings. However, a potential drawback of detection using chem. probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report the authors present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivs. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. The authors describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addn., the authors have characterized a fast-labeling variant of SNAP-tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
- 320Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Labeling of Fusion Proteins with Synthetic Fluorophores in Live Cells. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9955– 9959, DOI: 10.1073/pnas.0401923101Google Scholar320Labeling of fusion proteins with synthetic fluorophores in live cellsKeppler, Antje; Pick, Horst; Arrivoli, Claudio; Vogel, Horst; Johnsson, KaiProceedings of the National Academy of Sciences of the United States of America (2004), 101 (27), 9955-9959CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A general approach for the sequential labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor anal. of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.
- 321Lavis, L. D. Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines. Annu. Rev. Biochem. 2017, 86, 825– 843, DOI: 10.1146/annurev-biochem-061516-044839Google Scholar321Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and RhodaminesLavis, Luke D.Annual Review of Biochemistry (2017), 86 (), 825-843CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)Small-mol. fluorophores, such as fluorescein and rhodamine derivs., are crit. tools in modern biochem. and biol. research. The field of chem. dyes is old; colored mols. were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochem. and biol. The application of modern chem., biochem., mol. genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This crit. review focuses on an important example of chem. biol.-the melding of old and new chem. knowledge-leading to useful mols. for advanced biochem. and biol. expts.
- 322Ross-Thriepland, D.; Mankouri, J.; Harris, M. Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes. J. Virol. 2015, 89, 3123– 3135, DOI: 10.1128/JVI.02995-14Google Scholar322Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexesRoss-Thriepland, Douglas; Mankouri, Jamel; Harris, MarkJournal of Virology (2015), 89 (6), 3123-3135CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold redn. in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.
- 323Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.; Kindermann, M.; Beaufils, F.; Johnsson, K. An Engineered Protein Tag for Multiprotein Labeling in Living Cells. Chem. Biol. 2008, 15, 128– 136, DOI: 10.1016/j.chembiol.2008.01.007Google Scholar323An Engineered Protein Tag for Multiprotein Labeling in Living CellsGautier, Arnaud; Juillerat, Alexandre; Heinis, Christian; Correa, Ivan Reis; Kindermann, Maik; Beaufils, Florent; Johnsson, KaiChemistry & Biology (Cambridge, MA, United States) (2008), 15 (2), 128-136CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivs. bearing a chem. probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivs. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different mol. probes in living cells. We furthermore show simultaneous pulse-chase expts. to visualize different generations of two different proteins in one sample.
- 324Liu, A. A.; Zhang, Z.; Sun, E. Z.; Zheng, Z.; Zhang, Z. L.; Hu, Q.; Wang, H.; Pang, D. W. Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy. ACS Nano 2016, 10, 1147– 1155, DOI: 10.1021/acsnano.5b06438Google Scholar324Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific Halo Tag Labeling StrategyLiu, An-An; Zhang, Zhenfeng; Sun, En-Ze; Zheng, Zhenhua; Zhang, Zhi-Ling; Hu, Qinxue; Wang, Hanzhong; Pang, Dai-WenACS Nano (2016), 10 (1), 1147-1155CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Real-time, long-term, single-particle tracking (SPR) provides an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, the authors established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technol. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT expts. without further modification. Thus, virus infectivity, which is crit. for the visualization and anal. of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.
- 325Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3, 373– 382, DOI: 10.1021/cb800025kGoogle Scholar325HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein AnalysisLos, Georgyi V.; Encell, Lance P.; McDougall, Mark G.; Hartzell, Danette D.; Karassina, Natasha; Zimprich, Chad; Wood, Monika G.; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.ACS Chemical Biology (2008), 3 (6), 373-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in soln., in living cells, or in chem. fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful mols., such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiol. conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple mol. processes assocd. with NF-κB-mediated cellular physiol., including imaging of subcellular protein translocation and capture of protein-protein and protein-DNA complexes.
- 326Chen, Z.; Jing, C.; Gallagher, S. S.; Sheetz, M. P.; Cornish, V. W. Second-Generation Covalent TMP-Tag for Live Cell Imaging. J. Am. Chem. Soc. 2012, 134, 13692– 13699, DOI: 10.1021/ja303374pGoogle Scholar326Second-Generation Covalent TMP-Tag for Live Cell ImagingChen, Zhixing; Jing, Chaoran; Gallagher, Sarah S.; Sheetz, Michael P.; Cornish, Virginia W.Journal of the American Chemical Society (2012), 134 (33), 13692-13699CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with org. fluorophores that have improved brightness and other specialized properties. Recently, the authors successfully rendered the their TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here the authors report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, the authors designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chem. synthesis for routine prodn. of a variety of A-TMP-probe v2.0 labels. The authors then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, the authors demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited no. of chem. tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and org. chem. as tools not only for chem. tag engineering but also more broadly for synthetic biol.
- 327Gallagher, S. S.; Jing, C.; Peterka, D. S.; Konate, M.; Wombacher, R.; Kaufman, L. J.; Yuste, R.; Cornish, V. W. A Trimethoprim-Based Chemical Tag for Live Cell Two-Photon Imaging. ChemBioChem 2010, 11, 782– 784, DOI: 10.1002/cbic.200900731Google Scholar327A Trimethoprim-Based Chemical Tag for Live Cell Two-Photon ImagingGallagher, Sarah S.; Jing, Chaoran; Peterka, Darcy S.; Konate, Mariam; Wombacher, Richard; Kaufman, Laura J.; Yuste, Rafael; Cornish, Virginia W.ChemBioChem (2010), 11 (6), 782-784CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)TMP-BC575 is a viable tool for imaging proteins in live cells by using two-photon microscopy. This two-photon fluorophore expands the TMP-tag tool kit, adding to the value of this modular-labeling technol. A protein of interest can be tagged with Escherichia coli dihydrofolate reductase (eDHFR), and different labels can then be swapped in, allowing the protein to be readily analyzed by multiple techniques. While cell permeability and lipid partitioning appear to be tag dependent, these expts. suggest BC575 might also be compatible with other chem. tags. Given its broad excitation maxima and distinct emission wavelength, TMP-BC575 offers an alternative to other fluorophores for multicolor two-photon imaging with enhanced green fluorescent protein (EGFP).
- 328Wombacher, R.; Heidbreder, M.; van de Linde, S.; Sheetz, M. P.; Heilemann, M.; Cornish, V. W.; Sauer, M. Live-Cell Super-Resolution Imaging with Trimethoprim Conjugates. Nat. Methods 2010, 7, 717– 719, DOI: 10.1038/nmeth.1489Google Scholar328Live-cell super-resolution imaging with trimethoprim conjugatesWombacher, Richard; Heidbreder, Meike; van de Linde, Sebastian; Sheetz, Michael P.; Heilemann, Mike; Cornish, Virginia W.; Sauer, MarkusNature Methods (2010), 7 (9), 717-719CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The spatiotemporal resoln. of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chem. tag that allows proteins to be labeled with an org. fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at ∼ 20 nm resoln.
- 329Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W. In Vivo Protein Labeling with Trimethoprim Conjugates: A Flexible Chemical Tag. Nat. Methods 2005, 2, 255– 257, DOI: 10.1038/nmeth749Google Scholar329In vivo protein labeling with trimethoprim conjugates: a flexible chemical tagMiller, Lawrence W.; Cai, Yunfei; Sheetz, Michael P.; Cornish, Virginia W.Nature Methods (2005), 2 (4), 255-257CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The introduction of green fluorescent protein and its variants (GFPs) has allowed protein anal. at the level of the cell. Now, chem. methods are needed to label proteins in vivo with a wider variety of functionalities so that mechanistic questions about protein function in the complex cellular environment can be addressed. Here we demonstrate that trimethoprim derivs. can be used to selectively tag Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins in wild-type mammalian cells with minimal background and fast kinetics.
- 330Rudner, L.; Nydegger, S.; Coren, L. V.; Nagashima, K.; Thali, M.; Ott, D. E. Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling. J. Virol. 2005, 79, 4055– 4065, DOI: 10.1128/JVI.79.7.4055-4065.2005Google Scholar330Dynamic fluorescent imaging of Human immunodeficiency virus type 1 Gag in live cells by biarsenical labelingRudner, Lynnie; Nydegger, Sascha; Coren, Lori V.; Nagashima, Kunio; Thali, Markus; Ott, David E.Journal of Virology (2005), 79 (7), 4055-4065CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compds. FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examd. Fluorescent two-color anal. of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristoylation mutant obsd. a diffuse signal throughout the cell, consistent with the role of myristoylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concs. in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.
- 331Das, S. C.; Panda, D.; Nayak, D.; Pattnaik, A. K. Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells. J. Virol. 2009, 83, 2611– 2622, DOI: 10.1128/JVI.01668-08Google Scholar331Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged M protein allows dynamic imaging of M protein and virus uncoating in infected cellsDas, Subash C.; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K.Journal of Virology (2009), 83 (6), 2611-2622CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addn. to wild-type (wt.) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt. VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent assocn. of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 h. Using dually fluorescent VSV, we detd. that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approx. 28 min.
- 332Li, Y.; Lu, X.; Li, J.; Berube, N.; Giest, K. L.; Liu, Q.; Anderson, D. H.; Zhou, Y. Genetically Engineered, Biarsenically Labeled Influenza Virus Allows Visualization of Viral NS1 Protein in Living Cells. J. Virol. 2010, 84, 7204– 7213, DOI: 10.1128/JVI.00203-10Google Scholar332Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cellsLi, Yang; Lu, Xinya; Li, Junwei; Berube, Nathalie; Giest, Kerri-Lane; Liu, Qiang; Anderson, Deborah H.; Zhou, YanJournal of Virology (2010), 84 (14), 7204-7213CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.
- 333Martin, B. R.; Giepmans, B. N.; Adams, S. R.; Tsien, R. Y. Mammalian Cell-Based Optimization of the Biarsenical-Binding Tetracysteine Motif for Improved Fluorescence and Affinity. Nat. Biotechnol. 2005, 23, 1308– 1314, DOI: 10.1038/nbt1136Google Scholar333Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinityMartin, Brent R.; Giepmans, Ben N. G.; Adams, Stephen R.; Tsien, Roger Y.Nature Biotechnology (2005), 23 (10), 1308-1314CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dil. proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concns. of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.
- 334Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications. J. Am. Chem. Soc. 2002, 124, 6063– 6076, DOI: 10.1021/ja017687nGoogle Scholar334New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applicationsAdams, Stephen R.; Campbell, Robert E.; Gross, Larry A.; Martin, Brent R.; Walkup, Grant K.; Yao, Yong; Llopis, Juan; Tsien, Roger Y.Journal of the American Chemical Society (2002), 124 (21), 6063-6076CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein deriv. with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissocn. consts. (∼10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed α-helix. Many analogs of FlAsH have been synthesized, including ReAsH, a resorufin deriv. excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatog., fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
- 335Zheng, L. L.; Li, C. M.; Zhen, S. J.; Li, Y. F.; Huang, C. Z. His-Tag Based in Situ Labelling of Progeny Viruses for Real-Time Single Virus Tracking in Living Cells. Nanoscale 2016, 8, 18635– 18639, DOI: 10.1039/C6NR05806JGoogle Scholar335His-tag based in situ labelling of progeny viruses for real-time single virus tracking in living cellsZheng, Lin Ling; Li, Chun Mei; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng ZhiNanoscale (2016), 8 (44), 18635-18639CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)Tracking virus infection events in live cells is useful for understanding the mechanism of virus infection, and fluorescent labeling is a crit. step. Herein a noninvasive strategy for labeling viruses with His-tags was developed by in situ modifying the cell surface proteins with polypeptides contg. His-tags during progeny virus assembly. The His-tagged viruses were further conjugated with Ni2+-nitrilotriacetate complex modified quantum dots, and retained their infectivity for real-time single virus tracking in living cells.
- 336Guignet, E. G.; Hovius, R.; Vogel, H. Reversible Site-Selective Labeling of Membrane Proteins in Live Cells. Nat. Biotechnol. 2004, 22, 440– 444, DOI: 10.1038/nbt954Google Scholar336Reversible site-selective labeling of membrane proteins in live cellsGuignet, Emmanuel G.; Hovius, Ruud; Vogel, HorstNature Biotechnology (2004), 22 (4), 440-444CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Chem. and biol. labeling is fundamental for the elucidation of the function of proteins within biochem. cellular networks. In particular, fluorescent probes allow detection of mol. interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resoln. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small mol. probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each contg. a polyhistidine sequence. We investigated the ionotropic 5HT3 serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.
- 337Chen, I.; Howarth, M.; Lin, W.; Ting, A. Y. Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes Using Biotin Ligase. Nat. Methods 2005, 2, 99– 104, DOI: 10.1038/nmeth735Google Scholar337Site-specific labeling of cell surface proteins with biophysical probes using biotin ligaseChen, Irwin; Howarth, Mark; Lin, Weiying; Ting, Alice Y.Nature Methods (2005), 2 (2), 99-104CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophys. probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized mols. We demonstrate this two-stage protein labeling methodol. on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.
- 338Howarth, M.; Ting, A. Y. Imaging Proteins in Live Mammalian Cells with Biotin Ligase and Monovalent Streptavidin. Nat. Protoc. 2008, 3, 534– 545, DOI: 10.1038/nprot.2008.20Google Scholar338Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidinHowarth, Mark; Ting, Alice Y.Nature Protocols (2008), 3 (3), 534-545CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a simple and efficient way to label specific cell surface proteins with biophys. probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the crosslinking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.
- 339Uttamapinant, C.; White, K. A.; Baruah, H.; Thompson, S.; Fernandez-Suarez, M.; Puthenveetil, S.; Ting, A. Y. A Fluorophore Ligase for Site-Specific Protein Labeling inside Living Cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 10914– 10919, DOI: 10.1073/pnas.0914067107Google Scholar339A fluorophore ligase for site-specific protein labeling inside living cellsUttamapinant, Chayasith; White, Katharine A.; Baruah, Hemanta; Thompson, Samuel; Fernandez-Subrez, Marta; Puthenveetil, Sujiet; Ting, Alice Y.Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (24), 10914-10919, S10914/1-S10914/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Biol. microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LpIA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LpIA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.
- 340Fernandez-Suarez, M.; Baruah, H.; Martinez-Hernandez, L.; Xie, K. T.; Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Redirecting Lipoic Acid Ligase for Cell Surface Protein Labeling with Small-Molecule Probes. Nat. Biotechnol. 2007, 25, 1483– 1487, DOI: 10.1038/nbt1355Google Scholar340Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probesFernandez-Suarez, Marta; Baruah, Hemanta; Martinez-Hernandez, Laura; Xie, Kathleen T.; Baskin, Jeremy M.; Bertozzi, Carolyn R.; Ting, Alice Y.Nature Biotechnology (2007), 25 (12), 1483-1487CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissoc. from proteins after internalization. Here, we report technol. for covalent, specific tagging of cellular proteins with chem. probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodol. should provide general access to biochem. and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.
- 341Schumacher, D.; Helma, J.; Mann, F. A.; Pichler, G.; Natale, F.; Krause, E.; Cardoso, M. C.; Hackenberger, C. P.; Leonhardt, H. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase. Angew. Chem., Int. Ed. 2015, 54, 13787– 13791, DOI: 10.1002/anie.201505456Google Scholar341Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine LigaseSchumacher, Dominik; Helma, Jonas; Mann, Florian A.; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M. Cristina; Hackenberger, Christian P. R.; Leonhardt, HeinrichAngewandte Chemie, International Edition (2015), 54 (46), 13787-13791CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A novel chemoenzymic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivs. as small bioorthogonal handles to proteins contg. a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochem., cell biol., and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.
- 342Schumacher, D.; Lemke, O.; Helma, J.; Gerszonowicz, L.; Waller, V.; Stoschek, T.; Durkin, P. M.; Budisa, N.; Leonhardt, H.; Keller, B. G. Broad Substrate Tolerance of Tubulin Tyrosine Ligase Enables One-Step Site-Specific Enzymatic Protein Labeling. Chem. Sci. 2017, 8, 3471– 3478, DOI: 10.1039/C7SC00574AGoogle Scholar342Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labelingSchumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M.; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G.; Hackenberger, Christian P. R.Chemical Science (2017), 8 (5), 3471-3478CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking expts. and all-atom mol. dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.
- 343Zhou, Z.; Cironi, P.; Lin, A. J.; Xu, Y.; Hrvatin, S.; Golan, D. E.; Silver, P. A.; Walsh, C. T.; Yin, J. Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases. ACS Chem. Biol. 2007, 2, 337– 346, DOI: 10.1021/cb700054kGoogle Scholar343Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl TransferasesZhou, Zhe; Cironi, Pablo; Lin, Alison J.; Xu, Yangqing; Hrvatin, Sinisa; Golan, David E.; Silver, Pamela A.; Walsh, Christopher T.; Yin, JunACS Chemical Biology (2007), 2 (5), 337-346CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), resp. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small mols. of diverse structures to the target proteins with high efficiency and specificity.
- 344Yin, J.; Straight, P. D.; McLoughlin, S. M.; Zhou, Z.; Lin, A. J.; Golan, D. E.; Kelleher, N. L.; Kolter, R.; Walsh, C. T. Genetically Encoded Short Peptide Tag for Versatile Protein Labeling by Sfp Phosphopantetheinyl Transferase. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 15815– 15820, DOI: 10.1073/pnas.0507705102Google Scholar344Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferaseYin, Jun; Straight, Paul D.; McLoughlin, Shaun M.; Zhou, Zhe; Lin, Alison J.; Golan, David E.; Kelleher, Neil L.; Kolter, Roberto; Walsh, Christopher T.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (44), 15815-15820CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyltransferase-catalyzed protein labeling by small mol.-CoA conjugates. The authors name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the first Ser residue, and the ybbR tag was found to have a strong tendency for adopting an α-helical conformation in soln. Here the authors demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-mol. probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.
- 345Crivat, G.; Taraska, J. W. Imaging Proteins inside Cells with Fluorescent Tags. Trends Biotechnol. 2012, 30, 8– 16, DOI: 10.1016/j.tibtech.2011.08.002Google Scholar345Imaging proteins inside cells with fluorescent tagsCrivat, Georgeta; Taraska, Justin W.Trends in Biotechnology (2012), 30 (1), 8-16CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. Watching biol. mols. provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four std. genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.
- 346Hoehnel, S.; Lutolf, M. P. Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag Technology. Bioconjugate Chem. 2015, 26, 1678– 1686, DOI: 10.1021/acs.bioconjchem.5b00268Google Scholar346Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag TechnologyHoehnel, S.; Lutolf, M. P.Bioconjugate Chemistry (2015), 26 (8), 1678-1686CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Juxtacrine or contact-dependent signaling is a major form of cell communication in multicellular organisms. The involved cell-cell and cell-extracellular-matrix (ECM) interactions are crucial for the organization and maintenance of tissue architecture and function. However, because cell-cell contacts are relatively weak, it is difficult to isolate interacting cells in their native state to study, for example, how specific cell types interact with others (e.g., stem cells with niche cells) or where they locate within tissues to execute specific tasks. To achieve this, the authors propose artificial in situ cell-to-cell linking systems that are based on SNAP-tag and CLIP-tag, engineered mutants of the human O6-alkylguanine-DNA alkyltransferase. SNAP-tag can be used to efficiently and covalently tether cells to poly(ethylene glycol) (PEG)-based hydrogel surfaces that have been functionalized with the SNAP-tag substrate benzylguanine (BG). Furthermore, using PEG-based spherical microgels as an artificial cell model, the authors provide proof-of-principle for inducing clustering that mimics cell-cell pairing.
- 347Liss, V.; Barlag, B.; Nietschke, M.; Hensel, M. Self-Labelling Enzymes as Universal Tags for Fluorescence Microscopy, Super-Resolution Microscopy and Rectron Microscopy. Sci. Rep. 2016, 5, 17740, DOI: 10.1038/srep17740Google ScholarThere is no corresponding record for this reference.
- 348Jing, C.; Cornish, V. W. A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell Imaging. ACS Chem. Biol. 2013, 8, 1704– 1712, DOI: 10.1021/cb300657rGoogle Scholar348A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell ImagingJing, Chaoran; Cornish, Virginia W.ACS Chemical Biology (2013), 8 (8), 1704-1712CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Developed to complement the use of fluorescent proteins in live cell imaging, chem. tags enjoy the benefit of modular incorporation of org. fluorophores, opening the possibility of high photon output and special photophys. properties. However, the theor. challenge in using chem. tags as opposed to fluorescent proteins for high-resoln. imaging is background noise from unbound and/or nonspecifically bound ligand-fluorophore. The authors envisioned the authors could overcome this limit by engineering fluorogenic trimethoprim-based chem. tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR)-tagged protein displaces the quencher. Thus, the authors began by building a nonfluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here the authors take the final step and render the covalent TMP-tag fluorogenic. In brief, the authors designed a trimeric TMP-fluorophore-quencher mol. (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. The authors present the in vitro expts. showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only the authors' initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins but also less abundant, more dynamic cytoplasmic proteins. These results suggest that the fluorogenic TMP-tag can significantly impact high-resoln. live cell imaging and further establish the potential of proximity-induced reactivity and org. chem. more broadly as part of the growing toolbox for synthetic biol. and cell engineering.
- 349Lai, Y. T.; Chang, Y. Y.; Hu, L.; Yang, Y.; Chao, A.; Du, Z. Y.; Tanner, J. A.; Chye, M. L.; Qian, C.; Ng, K. M. Rapid Labeling of Intracellular His-Tagged Proteins in Living Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 2948– 2953, DOI: 10.1073/pnas.1419598112Google Scholar349Rapid labeling of intracellular His-tagged proteins in living cellsLai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A.; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, HongzheProceedings of the National Academy of Sciences of the United States of America (2015), 112 (10), 2948-2953CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Small mol.-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purifn., there is significant interest in fluorescent Ni2+-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.
- 350Stephanopoulos, N.; Francis, M. B. Choosing an Effective Protein Bioconjugation Strategy. Nat. Chem. Biol. 2011, 7, 876– 884, DOI: 10.1038/nchembio.720Google Scholar350Choosing an effective protein bioconjugation strategyStephanopoulos, Nicholas; Francis, Matthew B.Nature Chemical Biology (2011), 7 (12), 876-884CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. The collection of chem. techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biol. understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate prodn., and a no. of techniques can be used to label a single protein in a complex biol. mixt. This review examines the way in which exptl. circumstances influence one's selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.
- 351Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7583– 7588, DOI: 10.1073/pnas.0503125102Google Scholar351Targeting quantum dots to surface proteins in living cells with biotin ligaseHowarth, Mark; Takao, Keizo; Hayashi, Yasunori; Ting, Alice Y.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (21), 7583-7588CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.
- 352Cohen, J. D.; Zou, P.; Ting, A. Y. Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone Formation. ChemBioChem 2012, 13, 888– 894, DOI: 10.1002/cbic.201100764Google Scholar352Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone FormationCohen, Justin D.; Zou, Peng; Ting, Alice Y.ChemBioChem (2012), 13 (6), 888-894CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for Alexa Fluor 568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, the authors labeled the low-d. lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-mol. imaging and tracking over time.
- 353Lotze, J.; Reinhardt, U.; Seitz, O.; Beck-Sickinger, A. G. Peptide-Tags for Site-Specific Protein Labelling in Vitro and in Vivo. Mol. BioSyst. 2016, 12, 1731– 1745, DOI: 10.1039/C6MB00023AGoogle Scholar353Peptide-tags for site-specific protein labelling in vitro and in vivoLotze, Jonathan; Reinhardt, Ulrike; Seitz, Oliver; Beck-Sickinger, Annette G.Molecular BioSystems (2016), 12 (6), 1731-1745CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Peptide-tag based labeling can be achieved by (i) enzymes (ii) recognition of metal ions or small mols. and (iii) peptide-peptide interactions and enables site-specific protein visualization to investigate protein localization and trafficking.
- 354Sunbul, M.; Yen, M.; Zou, Y.; Yin, J. Enzyme Catalyzed Site-Specific Protein Labeling and Cell Imaging with Quantum Dots. Chem. Commun. 2008, 5927– 5929, DOI: 10.1039/b812162aGoogle Scholar354Enzyme catalyzed site-specific protein labeling and cell imaging with quantum dotsSunbul, Murat; Yen, Michelle; Zou, Yekui; Yin, JunChemical Communications (Cambridge, United Kingdom) (2008), (45), 5927-5929CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)We have developed an efficient method for one-step covalent labeling of cell surface proteins with quantum dots based on enzyme catalyzed site-specific modification of short peptide tags.
- 355Zhang, Y.; Ke, X.; Zheng, Z.; Zhang, C.; Zhang, Z.; Zhang, F.; Hu, Q.; He, Z.; Wang, H. Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus Infection. ACS Nano 2013, 7, 3896– 3904, DOI: 10.1021/nn305189nGoogle Scholar355Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus InfectionZhang, Yuan; Ke, Xianliang; Zheng, Zhenhua; Zhang, Cuiling; Zhang, Zhenfeng; Zhang, Fuxian; Hu, Qinxue; He, Zhike; Wang, HanzhongACS Nano (2013), 7 (5), 3896-3904CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Utilization of quantum dots (QDs) for single virus tracking has attracted growing interest. Through modification of viral surface proteins, viruses can be labeled with various functionalized QDs and used for tracking the routes of viral infections. However, incorporation of QDs on the viral surface may affect the efficiency of viral entry and alter virus-cell interactions. Here, we describe that QDs can be encapsulated into the capsid of vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus (PTLV) in living cells without modification of the viral surface. QDs conjugated with modified genomic RNAs (gRNAs), which contain a packaging signal (Psi) sequence for viral genome encapsulation, can be packaged into virions together with the gRNAs. QD-contg. PTLV demonstrated similar entry efficiency as the wild-type PTLV. After infection, QD signals entered the Rab5+ endosome and then moved to the microtubule organizing center of the infected cells in a microtubule-dependent manner. Findings in this study are consistent with previously reported infection routes of VSV and VSV-G pseudotyped lentivirus, indicating that our established QD packaging approach can be used for enveloped virus labeling and tracking.
- 356Molenaar, C.; Marras, S. A.; Slats, J. C.; Truffert, J. C.; Lemaitre, M.; Raap, A. K.; Dirks, R. W.; Tanke, H. J. Linear 2’ O-Methyl RNA Probes for the Visualization of RNA in Living Cells. Nucleic Acids Res. 2001, 29, E89– 9, DOI: 10.1093/nar/29.17.e89Google Scholar356Linear 2' O-methyl RNA probes for the visualization of RNA in living cellsMolenaar, C.; Marras, S. A.; Slats, J. C. M.; Truffert, J.-C.; Lemaitre, M.; Raap, A. K.; Dirks, R. W.; Tanke, H. J.Nucleic Acids Research (2001), 29 (17), e89/1-e89/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)U1snRNA, U3snRNA, 28S rRNA, poly(A) RNA and a specific mRNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Me oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic rRNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of mol. beacons, which due to their structure should theor. fluoresce only upon hybridization. No improvements were achieved however with the mol. beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.
- 357Ma, Y.; Mao, G.; Huang, W.; Wu, G.; Yin, W.; Ji, X.; Deng, Z.; Cai, Z.; Zhang, X. E.; He, Z. Quantum Dot Nanobeacons for Single RNA Labeling and Imaging. J. Am. Chem. Soc. 2019, 141, 13454– 13458, DOI: 10.1021/jacs.9b04659Google Scholar357Quantum Dot Nanobeacons for Single RNA Labeling and ImagingMa, Yingxin; Mao, Guobin; Huang, Weiren; Wu, Guoqiang; Yin, Wen; Ji, Xinghu; Deng, Zishi; Cai, Zhiming; Zhang, Xian-En; He, Zhike; Cui, ZongqiangJournal of the American Chemical Society (2019), 141 (34), 13454-13458CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Detection and imaging RNAs in live cells is in high demand. Methodol. for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate co-modified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Addnl., QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being esp. meaningful for tracking of individual RNAs in live cells.
- 358Ortega-Arroyo, J.; Kukura, P. Interferometric Scattering Microscopy (iSCAT): New Frontiers in Ultrafast and Ultrasensitive Optical Microscopy. Phys. Chem. Chem. Phys. 2012, 14, 15625– 15636, DOI: 10.1039/c2cp41013cGoogle Scholar358Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopyOrtega-Arroyo, Jaime; Kukura, PhilippPhysical Chemistry Chemical Physics (2012), 14 (45), 15625-15636CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)A review. Optical microscopes have for centuries been window to the microscopic world. The advent of single-mol. optics over the past few decades has ushered in a new era in optical imaging, partly because it has enabled the observation of motion and more recently structure on the nanoscopic scale through the development of super-resoln. techniques. The large majority of these studies have relied on the efficient detection of fluorescence as the basis of single-mol. sensitivity. Despite the many advantages of using single emitters as light sources, the intensity and duration of their emission impose fundamental limits on the imaging speed and precision for tracking studies. Here, the potential of a novel imaging technique based on interferometric scattering (iSCAT) that pushes both the sensitivity and time resoln. far beyond what is currently achievable by single-emitter-based approaches are discussed. The authors present recent results that demonstrate single-mol. sensitivity and imaging speeds on the microsecond timescale.
- 359Kukura, P.; Ewers, H.; Muller, C.; Renn, A.; Helenius, A.; Sandoghdar, V. High-Speed Nanoscopic Tracking of the Position and Orientation of a Single Virus. Nat. Methods 2009, 6, 923– 927, DOI: 10.1038/nmeth.1395Google Scholar359High-speed nanoscopic tracking of the position and orientation of a single virusKukura, Philipp; Ewers, Helge; Mueller, Christian; Renn, Alois; Helenius, Ari; Sandoghdar, VahidNature Methods (2009), 6 (12), 923-927CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the mol. dynamics of early virus-host couplings, which are important for cell infection. Here, we present a colocalization methodol. that combines scattering interferometry and single-mol. fluorescence microscopy to visualize both position and orientation of single quantum dot-labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resoln., we obsd. sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions sepd. by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biol. nano-objects.
- 360Renz, M. Fluorescence Microscopy-A Historical and Technical Perspective. Cytometry, Part A 2013, 83, 767– 779, DOI: 10.1002/cyto.a.22295Google Scholar360Fluorescence microscopy-a historical and technical perspectiveRenz MalteCytometry. Part A : the journal of the International Society for Analytical Cytology (2013), 83 (9), 767-79 ISSN:.For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Recent developments improved both visualization and quantification by fluorescence microscopy imaging and established a methodology of fluorescence microscopy. By outlining basic principles and their historical development, I seek to provide insight into and understanding of the ever-growing tools of fluorescence microscopy. Thereby, this synopsis may help the interested researcher to choose a fluorescence microscopic method capable of addressing a specific scientific question.
- 361Lorenz, K. S.; Salama, P.; Dunn, K. W.; Delp, E. J. Digital Correction of Motion Artefacts in Microscopy Image Sequences Collected from Living Animals Using Rigid and Nonrigid Registration. J. Microsc. 2012, 245, 148– 160, DOI: 10.1111/j.1365-2818.2011.03557.xGoogle Scholar361Digital correction of motion artefacts in microscopy image sequences collected from living animals using rigid and nonrigid registrationLorenz K S; Salama P; Dunn K W; Delp E JJournal of microscopy (2012), 245 (2), 148-60 ISSN:.Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artefacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and nonrigid components. The rigid registration component corrects global image translations, whereas the nonrigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung and salivary gland of living rodents.
- 362Combs, C. A.; Shroff, H. Fluorescence Microscopy: A Concise Guide to Current Imaging Methods. Curr. Protoc. Neurosci. 2017, 79, 2.1.1– 2.1.25, DOI: 10.1002/cpns.29Google ScholarThere is no corresponding record for this reference.
- 363Masters, B. R. Fluorescence Microscopy: from Principles to Biological Applications. J. Biomed. Opt. 2014, 19, 049901 DOI: 10.1117/1.JBO.19.4.049901Google ScholarThere is no corresponding record for this reference.
- 364Ma, Y. Y.; Wang, X.; Liu, H.; Wei, L.; Xiao, L. H. Recent Advances in Optical Microscopic Methods for Single-Particle Tracking in Biological Samples. Anal. Bioanal. Chem. 2019, 411, 4445– 4463, DOI: 10.1007/s00216-019-01638-zGoogle Scholar364Recent advances in optical microscopic methods for single-particle tracking in biological samplesMa, Yuanyuan; Wang, Xiao; Liu, Hua; Wei, Lin; Xiao, LehuiAnalytical and Bioanalytical Chemistry (2019), 411 (19), 4445-4463CODEN: ABCNBP; ISSN:1618-2642. (Springer)A review. With the rapid development of optical microscopic techniques, explorations on the chem. and biol. properties of target objects in biol. samples at single-mol./particle level have received great attention recently. In the past decades, various powerful techniques have been developed for single-particle tracking (SPT) in biol. samples. In this review, we summarize the commonly used optical microscopic methods for SPT, such as total internal reflection fluorescence microscopy (TIRFM), super-resoln. fluorescence microscopy (SRM), dark-field optical microscopy (DFM), total internal reflection scattering microscopy (TIRSM), and differential interference contrast microscopy (DICM). We then discuss the image processing and data anal. methods, including particle localization, trajectory reconstruction, and diffusion behavior anal. The application of SPT on the cell membrane, within the cell, and the cellular invading process of viruses are introduced. Finally, the challenges and prospects of optical microscopic technologies for SPT are delineated. [Figure not available: see fulltext.].
- 365Deschout, H.; Cella Zanacchi, F.; Mlodzianoski, M.; Diaspro, A.; Bewersdorf, J.; Hess, S. T.; Braeckmans, K. Precisely and Accurately Localizing Single Emitters in Fluorescence Microscopy. Nat. Methods 2014, 11, 253– 266, DOI: 10.1038/nmeth.2843Google Scholar365Precisely and accurately localizing single emitters in fluorescence microscopyDeschout, Hendrik; Zanacchi, Francesca Cella; Mlodzianoski, Michael; Diaspro, Alberto; Bewersdorf, Joerg; Hess, Samuel T.; Braeckmans, KevinNature Methods (2014), 11 (3), 253-266CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Methods based on single-mol. localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of mols. and nanoparticles and contributed to the recent revolution in super-resoln. localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing no. of researchers using single-particle tracking and super-resoln. localization microscopy.
- 366Lichtman, J. W.; Conchello, J. A. Fluorescence Microscopy. Nat. Methods 2005, 2, 910– 919, DOI: 10.1038/nmeth817Google Scholar366Fluorescence microscopyLichtman, Jeff W.; Conchello, Jose-AngelNature Methods (2005), 2 (12), 910-919CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Although fluorescence microscopy permeates all of cell and mol. biol., most biologists have little experience with the underlying photophys. phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Addnl., fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.
- 367Beier, H. T.; Ibey, B. L. Experimental Comparison of the High-Speed Imaging Performance of an EM-CCD and sCMOS Camera in a Dynamic Live-Cell Imaging Test Case. PLoS One 2014, 9, e84614 DOI: 10.1371/journal.pone.0084614Google Scholar367Experimental comparison of the high-speed imaging performance of an EM-CCD and sCMOS camera in a dynamic live-cell imaging test caseBeier, Hope T.; Ibey, Bennett L.PLoS One (2014), 9 (1), e84614/1-e84614/6, 6 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The study of living cells may require advanced imaging techniques to track weak and rapidly changing signals. Fundamental to this need is the recent advancement in camera technol. Two camera types, specifically sCMOS and EM-CCD, promise both high signal-to-noise and high speed (>100 fps), leaving researchers with a crit. decision when detg. the best technol. for their application. In this article, we compare two cameras using a live-cell imaging test case in which small changes in cellular fluorescence must be rapidly detected with high spatial resoln. The EM-CCD maintained an advantage of being able to acquire discernible images with a lower no. of photons due to its EM-enhancement. However, if high-resoln. images at speeds approaching or exceeding 1000 fps are desired, the flexibility of the full-frame imaging capabilities of sCMOS is superior.
- 368Long, F.; Zeng, S.; Huang, Z. L. Localization-Based Super-Resolution Microscopy with an sCMOS Camera Part II: Experimental Methodology for Comparing sCMOS with EMCCD Cameras. Opt. Express 2012, 20, 17741– 17759, DOI: 10.1364/OE.20.017741Google Scholar368Localization-based super-resolution microscopy with an sCMOS camera part II: experimental methodology for comparing sCMOS with EMCCD camerasLong Fan; Zeng Shaoqun; Huang Zhen-LiOptics express (2012), 20 (16), 17741-59 ISSN:.Nowadays, there is a hot debate among industry and academic researchers that whether the newly developed scientific-grade Complementary Metal Oxide Semiconductor (sCMOS) cameras could become the image sensors of choice in localization-based super-resolution microscopy. To help researchers find answers to this question, here we reported an experimental methodology for quantitatively comparing the performance of low-light cameras in single molecule detection (characterized via image SNR) and localization (via localization accuracy). We found that a newly launched sCMOS camera can present superior imaging performance than a popular Electron Multiplying Charge Coupled Device (EMCCD) camera in a signal range (15-12000 photon/pixel) more than enough for typical localization-based super-resolution microscopy.
- 369Fullerton, S. A Guide to Choosing and Using Scientific Imaging Cameras. Laser Focus World 2014, 50, 37– 40Google ScholarThere is no corresponding record for this reference.
- 370Ivanchenko, S.; Godinez, W. J.; Lampe, M.; Krausslich, H. G.; Eils, R.; Rohr, K.; Brauchle, C.; Muller, B.; Lamb, D. C. Dynamics of HIV-1 Assembly and Release. PLoS Pathog. 2009, 5, e1000652 DOI: 10.1371/journal.ppat.1000652Google Scholar370Dynamics of HIV-1 assembly and releaseIvanchenko Sergey; Godinez William J; Lampe Marko; Krausslich Hans-Georg; Eils Roland; Rohr Karl; Brauchle Christoph; Muller Barbara; Lamb Don CPLoS pathogens (2009), 5 (11), e1000652 ISSN:.Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of approximately 5 x 10(-3) s(-1), corresponding to 8-9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at approximately 1,500+/-700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.
- 371Axelrod, D. Cell-Substrate Contacts Illuminated by Total Internal Reflection Fluorescence. J. Cell Biol. 1981, 89, 141– 145, DOI: 10.1083/jcb.89.1.141Google Scholar371Cell-substrate contacts illuminated by total internal reflection fluorescenceAxelrod DThe Journal of cell biology (1981), 89 (1), 141-5 ISSN:0021-9525.A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.
- 372Axelrod, D. Total Internal Reflection Fluorescence Microscopy in Cell Biology. Traffic 2001, 2, 764– 774, DOI: 10.1034/j.1600-0854.2001.21104.xGoogle Scholar372Total internal reflection fluorescence microscopy in cell biologyAxelrod, DanielTraffic (Copenhagen, Denmark) (2001), 2 (11), 764-774CODEN: TRAFFA; ISSN:1398-9219. (Munksgaard International Publishers Ltd.)A review. Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochem. kinetics and single biomol. dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the phys. basis for the technique and the various ways to implement total internal reflection fluorescence in a std. fluorescence microscope.
- 373Reichert, W. M.; Truskey, G. A. Total Internal Reflection Fluorescence (TIRF) Microscopy. I. Modelling Cell Contact Region Fluorescence. J. Cell Sci. 1990, 96, 219– 230Google Scholar373Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescenceReichert W M; Truskey G AJournal of cell science (1990), 96 ( Pt 2) (), 219-30 ISSN:0021-9533.Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.
- 374Johnson, C. Spectroscopy and Dynamics of Single Molecules: Methods and Applications; Elsevier: 2019.Google ScholarThere is no corresponding record for this reference.
- 375Tokunaga, M.; Kitamura, K.; Saito, K.; Iwane, A. H.; Yanagida, T. Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy. Biochem. Biophys. Res. Commun. 1997, 235, 47– 53, DOI: 10.1006/bbrc.1997.6732Google Scholar375Single molecule imaging of fluorophores and enzymic reactions achieved by objective-type total internal reflection fluorescence microscopyTokunaga, Makio; Kitamura, Kazuo; Saito, Kiwamu; Iwane, Atsuko Hikikoshi; Yanagida, ToshioBiochemical and Biophysical Research Communications (1997), 235 (1), 47-53CODEN: BBRCA9; ISSN:0006-291X. (Academic)Imaging of single fluorescence mols. has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single mols. of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 ×, Na 1.4) with ordinary coverslips and immersion oil. This method allowed visualization of single mols. under scanning probe microscopes. Taking advantage of the technique of single mol. imaging, individual ATP turnovers have been visualized with a fluorescent ATP analog, Cy3-ATP, using a simple exptl. strategy. Clear on/off signals were obtained that correspond to the assocn. and dissocn. of single Cy3-ATP/ADP mols. with a single myosin head mol. This method will allow a variety of single-mol. assays of biomol. functions to be performed using fluorescently labeled substrates, ligands, messengers, and biol. active mols. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single mols.
- 376Parhamifar, L.; Moghimi, S. M. Total Internal Reflection Fluorescence (TIRF) Microscopy for Real-Time Imaging of Nanoparticle-Cell Plasma Membrane Interaction. Methods Mol. Biol. 2012, 906, 473– 482, DOI: 10.1007/978-1-61779-953-2_38Google Scholar376Total internal reflection fluorescence (TIRF) microscopy for real-time imaging of nanoparticle-cell plasma membrane interactionParhamifar, Ladan; Moghimi, S. MoeinMethods in Molecular Biology (New York, NY, United States) (2012), 906 (Nanoparticles in Biology and Medicine), 473-482CODEN: MMBIED; ISSN:1064-3745. (Springer)Nanoparticulate systems are widely used for site-specific drug and gene delivery as well as for medical imaging. The mode of nanoparticle-cell interaction may have a significant effect on the pathway of nanoparticle internalization and subsequent intracellular trafficking. Total internal reflection fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane.
- 377Fish, K. N. Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr. Protoc. Cytom. 2009, 12, 12– 18, DOI: 10.1002/0471142956.cy1218s50Google ScholarThere is no corresponding record for this reference.
- 378Mattheyses, A. L.; Simon, S. M.; Rappoport, J. Z. Imaging with Total Internal Reflection Fluorescence Microscopy for the Cell Biologist. J. Cell Sci. 2010, 123, 3621– 3628, DOI: 10.1242/jcs.056218Google Scholar378Imaging with total internal reflection fluorescence microscopy for the cell biologistMattheyses, Alexa L.; Simon, Sanford M.; Rappoport, Joshua Z.Journal of Cell Science (2010), 123 (21), 3621-3628CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)A review. Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biol. applications, and is particularly well suited to anal. of the localization and dynamics of mols. and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ∼100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid expsoure times. In this Commentary, we discuss the applications of TIRF to the study of cell biol., the phys. basis of TIRF, exptl. setup and troubleshooting.
- 379Johnson, D. S.; Jaiswal, J. K.; Simon, S. Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events. Curr. Protoc. Cytom. 2012, 12, 12– 29, DOI: 10.1002/0471142956.cy1229s61Google ScholarThere is no corresponding record for this reference.
- 380Ewers, H.; Schelhaas, M. Analysis of Virus Entry and Cellular Membrane Dynamics by Single Particle Tracking. Methods Enzymol. 2012, 506, 63– 80, DOI: 10.1016/B978-0-12-391856-7.00028-7Google Scholar380Analysis of virus entry and cellular membrane dynamics by single particle trackingEwers, Helge; Schelhaas, MarioMethods in Enzymology (2012), 506 (Imaging and Spectroscopic Analysis of Living Cells), 63-80CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. Viruses have evolved to mimic cellular ligands in order to gain access to their host cells for replication. Since viruses are simple in structure, they rely on host cells for all their transportation needs. Following single virus particles during the initial phase of infection, i.e., virus entry into target cells, can reveal crucial information on the mechanism of pathogen infections and likewise cellular transport and membrane dynamics. Here, we give an overview on how to fluorescently label virus particles for live cell microscopy, and on how virus entry can be analyzed by single particle tracking expts. Highlighted are strategies, on how to chem. introduce fluorophores into virions, and on how to ext. quant. information from live cell data.
- 381Tokunaga, M.; Imamoto, N.; Sakata-Sogawa, K. Highly Inclined Thin Illumination Enables Clear Single-Molecule Imaging in Cells. Nat. Methods 2008, 5, 159– 161, DOI: 10.1038/nmeth1171Google Scholar381Highly inclined thin illumination enables clear single-molecule imaging in cellsTokunaga, Makio; Imamoto, Naoko; Sakata-Sogawa, KumikoNature Methods (2008), 5 (2), 159-161CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe a simple illumination method of fluorescence microscopy for mol. imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-mol. images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify mol. dynamics, interactions and kinetics in cells for mol. systems biol.
- 382Schmidt, F. I.; Kuhn, P.; Robinson, T.; Mercer, J.; Dittrich, P. S. Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times. Biophys. J. 2013, 105, 420– 431, DOI: 10.1016/j.bpj.2013.06.016Google Scholar382Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag TimesSchmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.Biophysical Journal (2013), 105 (2), 420-431CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Recent studies revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. The authors developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which the authors analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concns. of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addn., EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiol. pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further studies into a potential VACV viroporin are justified. The authors' findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.
- 383Naredi-Rainer, N.; Prescher, J.; Hartschuh, A.; Lamb, D. C. Confocal Microscopy. Fluorescence Microscopy 2013, 1075, 175– 213, DOI: 10.1002/9783527671595.ch5Google ScholarThere is no corresponding record for this reference.
- 384Minsky, M. Memoir on Inventing the Confocal Scanning Microscope. Scanning 1988, 10, 128– 138, DOI: 10.1002/sca.4950100403Google ScholarThere is no corresponding record for this reference.
- 385Graf, R.; Rietdorf, J.; Zimmermann, T. Live Cell Spinning Disk Microscopy. Adv. Biochem. Eng./Biotechnol. 2005, 95, 57– 75, DOI: 10.1007/b102210Google Scholar385Live cell spinning disk microscopyGraf Ralph; Rietdorf Jens; Zimmermann TimoAdvances in biochemical engineering/biotechnology (2005), 95 (), 57-75 ISSN:0724-6145.In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. Confocal laser scanning microscopy is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi-beam scanning is much faster. Spinning disk confocal microscopes are therefore very suited for fast in vivo imaging. The principles of spinning disk microscopy will be explained in this chapter and a thorough comparison of the performance of single beam and multi-beam scanning systems is made and illustrated with an example of in vivo imaging in Dictyostelium discoideum.
- 386Egger, M. D.; Petran, M. New Reflected-Light Microscope for Viewing Unstained Brain and Ganglion Cells. Science 1967, 157, 305– 307, DOI: 10.1126/science.157.3786.305Google Scholar386New reflected-light microscope for viewing unstained brain and ganglion cellsEgger M D; Petran MScience (New York, N.Y.) (1967), 157 (3786), 305-7 ISSN:0036-8075.We have designed and constructed a new type of reflected-light microscope to form images including only light reflected near the plane of the object. This selectivity of image formation is based on a mechanical flying-spot technique. Objects difficult or impossible to see with earlier microscopes, such as unstained cells and cell processes in brains of living salamanders and in excised dorsal root ganglia of frogs, have been observed routinely with this microscope.
- 387Andersson, S. B. Tracking a Single Fluorescent Molecule with a Confocal Microscope. Appl. Phys. B: Lasers Opt. 2005, 80, 809– 816, DOI: 10.1007/s00340-005-1801-xGoogle Scholar387Tracking a single fluorescent molecule with a confocal microscopeAndersson, S. B.Applied Physics B: Lasers and Optics (2005), 80 (7), 809-816CODEN: APBOEM; ISSN:0946-2171. (Springer GmbH)We consider the problem of tracking a single fluorescent mol. in both two and three dimensions using a confocal laser scanning microscope. An est. of the position of the mol. is generated from the measured fluorescence signal through the use of parameter estn. theory. This est. is used in a nonlinear controller designed both to track the position of the mol. and to provide good measurements for use in the estn. algorithm. The performance of the approach is investigated through numerical simulation for mols. undergoing diffusion and directed transport and the capabilities of the controller relative to exptl. limitations are discussed.
- 388Han, J. J.; Kiss, C.; Bradbury, A. R. M.; Werner, J. H. Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three Dimensions. ACS Nano 2012, 6, 8922– 8932, DOI: 10.1021/nn302912jGoogle Scholar388Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three DimensionsHan, Jason J.; Kiss, Csaba; Bradbury, Andrew R. M.; Werner, James H.ACS Nano (2012), 6 (10), 8922-8932CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors demonstrate following individual fluorescent protein constructs and individual org. dyes as they diffuse in 3-D in soln. at rates up to 1 μm2/s over distances of several micrometers in X, Y, and Z. The authors' 3-D tracking method is essentially a stage scanning confocal microscope that uses a unique spatial filter geometry and active feedback 200 times/s to follow fast 3-D motion. Here the authors detail simulations used to find optimal feedback parameters for following individual fluorescent proteins in 3-D and show that a wide range of parameters are capable of following individual proteins diffusing at 1 μm2/s rates. In addn., exptl. through 3-D single-mol. tracking of a protein oligomer series (monomer, dimer, and tetramer) of the fluorescent protein Azami Green, the protein oligomerization state can be detd. The authors also perform time-resolved spectroscopy (photon pair correlation measurements) during the measured 3-D trajectories. The photon pair correlation measurements show clear fluorescence photon antibunching, demonstrating that the trajectories are of single fluorescent mols. The rates of single-mol. diffusive motion the authors follow (∼1 μm2/s) are comparable to or faster than many intracellular transport processes.
- 389Krzic, U.; Gunther, S.; Saunders, T. E.; Streichan, S. J.; Hufnagel, L. Multiview Light-Sheet Microscope for Rapid in toto Imaging. Nat. Methods 2012, 9, 730– 733, DOI: 10.1038/nmeth.2064Google Scholar389Multiview light-sheet microscope for rapid in toto imagingKrzic, Uros; Gunther, Stefan; Saunders, Timothy E.; Streichan, Sebastian J.; Hufnagel, LarsNature Methods (2012), 9 (7_part1), 730-733CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biol. specimens with subcellular resoln. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.
- 390Vettenburg, T.; Dalgarno, H. I. C.; Nylk, J.; Coll-Llado, C.; Ferrier, D. E. K.; Cizmar, T.; Gunn-Moore, F. J.; Dholakia, K. Light-Sheet Microscopy Using An Airy Beam. Nat. Methods 2014, 11, 541– 544, DOI: 10.1038/nmeth.2922Google Scholar390Light-sheet microscopy using an Airy beamVettenburg, Tom; Dalgarno, Heather I. C.; Nylk, Jonathan; Coll-Llado, Clara; Ferrier, David E. K.; Cizmar, Tomas; Gunn-Moore, Frank J.; Dholakia, KishanNature Methods (2014), 11 (5), 541-544CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resoln. We show that the Airy beam innately yields high contrast and resoln. up to a tenfold larger FOV. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beam's characteristic asym. excitation pattern results in all fluorescence contributing pos. to the contrast, enabling a step change for light-sheet microscopy.
- 391Pitrone, P. G.; Schindelin, J.; Stuyvenberg, L.; Preibisch, S.; Weber, M.; Eliceiri, K. W.; Huisken, J.; Tomancak, P. OpenSPIM: An Open-Access Light-Sheet Microscopy Platform. Nat. Methods 2013, 10, 598– 599, DOI: 10.1038/nmeth.2507Google Scholar391OpenSPIM: an open-access light-sheet microscopy platformPitrone, Peter G.; Schindelin, Johannes; Stuyvenberg, Luke; Preibisch, Stephan; Weber, Michael; Eliceiri, Kevin W.; Huisken, Jan; Tomancak, PavelNature Methods (2013), 10 (7), 598-599CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)An open hardware and software platform for constructing a customizable microscope for selective-plane illumination microscopy (SPIM) is presented.
- 392Li, Y.; Hu, Y.; Cang, H. Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living Cells. J. Phys. Chem. B 2013, 117, 15503– 15511, DOI: 10.1021/jp405380gGoogle Scholar392Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living CellsLi, Yu; Hu, Ying; Cang, HuJournal of Physical Chemistry B (2013), 117 (49), 15503-15511CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Single particle tracking is a powerful tool to study single mol. dynamics in living biol. samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single mols. on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF mols. on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temp., the av. diffusion coeff. of EGF on A549 cells was measured to be 0.13 μm2/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coeffs. and an increase of the av. diffusion coeff. at room temp. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole lifetime of a particle from binding till photobleaching or internalization.
- 393Keller, P. J.; Schmidt, A. D.; Wittbrodt, J.; Stelzer, E. H. Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy. Science 2008, 322, 1065– 1069, DOI: 10.1126/science.1162493Google Scholar393Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet MicroscopyKeller, Philipp J.; Schmidt, Annette D.; Wittbrodt, Joachim; Stelzer, Ernst H. K.Science (Washington, DC, United States) (2008), 322 (5904), 1065-1069CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A long-standing goal of biol. is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 h of development. Multiview in vivo imaging at 1.5 billion voxels per min provides "digital embryos," i.e., comprehensive databases of cell positions, divisions, and migratory tracks. Our anal. of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
- 394Ritter, J. G.; Veith, R.; Veenendaal, A.; Siebrasse, J. P.; Kubitscheck, U. Light Sheet Microscopy for Single Molecule Tracking in Living Tissue. PLoS One 2010, 5, e11639 DOI: 10.1371/journal.pone.0011639Google ScholarThere is no corresponding record for this reference.
- 395Wan, Y.; McDole, K.; Keller, P. J. Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes. Annu. Rev. Cell Dev. Biol. 2019, 35, 655– 681, DOI: 10.1146/annurev-cellbio-100818-125311Google Scholar395Light-Sheet Microscopy and Its Potential for Understanding Developmental ProcessesWan, Yinan; McDole, Katie; Keller, Philipp J.Annual Review of Cell and Developmental Biology (2019), 35 (), 655-681CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)The ability to visualize and quant. measure dynamic biol. processes in vivo and at high spatiotemporal resoln. is of fundamental importance to exptl. investigations in developmental biol. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resoln. while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biol. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-mol. to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data anal. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technol., and identify exciting opportunities for further advances.
- 396Hillman, E. M. C.; Voleti, V.; Li, W.; Yu, H. Light-Sheet Microscopy in Neuroscience. Annu. Rev. Neurosci. 2019, 42, 295– 313, DOI: 10.1146/annurev-neuro-070918-050357Google Scholar396Light-Sheet Microscopy in NeuroscienceHillman, Elizabeth M. C.; Voleti, Venkatakaushik; Li, Wenze; Yu, HangAnnual Review of Neuroscience (2019), 42 (), 295-313CODEN: ARNSD5; ISSN:0147-006X. (Annual Reviews)Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.
- 397Stelzer, E. H. K. Light-Sheet Fluorescence Microscopy for Quantitative Biology. Nat. Methods 2015, 12, 23– 26, DOI: 10.1038/nmeth.3219Google Scholar397Light-sheet fluorescence microscopy for quantitative biologyStelzer, Ernst H. K.Nature Methods (2015), 12 (1), 23-26CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)In light sheet-based fluorescence microscopy (LSFM), optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects. Because biol. specimens survive long-term three-dimensional imaging at high spatiotemporal resoln., LSFM has become the tool of choice in developmental biol.
- 398Bosse, J. B.; Hogue, I. B.; Feric, M.; Thiberge, S. Y.; Sodeik, B.; Brangwynne, C. P.; Enquist, L. W. Remodeling Nuclear Architecture Allows Efficient Transport of Herpesvirus Capsids by Diffusion. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E5725– E5733, DOI: 10.1073/pnas.1513876112Google Scholar398Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusionBosse, Jens B.; Hogue, Ian B.; Feric, Marina; Thiberge, Stephan Y.; Sodeik, Beate; Brangwynne, Clifford P.; Enquist, Lynn W.Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (42), E5725-E5733CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The nuclear chromatin structure confines the movement of large macromol. complexes to interchromatin corrals. Herpesvirus capsids of approx. 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resoln. Here, the authors use a rotating, oblique light sheet, which they dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resoln. They do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.
- 399Hoyer, P.; de Medeiros, G.; Balazs, B.; Norlin, N.; Besir, C.; Hanne, J.; Krausslich, H. G.; Engelhardt, J.; Sahl, S. J.; Hell, S. W. Breaking the Diffraction Limit of Light-Sheet Fluorescence Microscopy by RESOLFT. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 3442– 3446, DOI: 10.1073/pnas.1522292113Google Scholar399Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFTHoyer, Patrick; de Medeiros, Gustavo; Balazs, Balint; Norlin, Nils; Besir, Christina; Hanne, Janina; Kraeusslich, Hans-Georg; Engelhardt, Johann; Sahl, Steffen J.; Hell, Stefan W.; Hufnagel, LarsProceedings of the National Academy of Sciences of the United States of America (2016), 113 (13), 3442-3446CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent mol. state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biol. specimens with low light dose and axial resoln. far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.
- 400Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793– 795, DOI: 10.1038/nmeth929Google Scholar400Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)Rust, Michael J.; Bates, Mark; Zhuang, XiaoweiNature Methods (2006), 3 (10), 793-796CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors have developed a high-resoln. fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be detd. with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. The authors demonstrated an imaging resoln. of 20 nm. This technique can, in principle, reach mol.-scale resoln.
- 401Malkusch, S.; Muranyi, W.; Muller, B.; Krausslich, H. G.; Heilemann, M. Single-Molecule Coordinate-Based Analysis of the Morphology of HIV-1 Assembly Sites with Near-Molecular Spatial Resolution. Histochem. Cell Biol. 2013, 139, 173– 179, DOI: 10.1007/s00418-012-1014-4Google Scholar401Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolutionMalkusch, Sebastian; Muranyi, Walter; Mueller, Barbara; Kraeusslich, Hans-Georg; Heilemann, MikeHistochemistry and Cell Biology (2013), 139 (1), 173-179CODEN: HCBIFP; ISSN:0948-6143. (Springer)We apply single-mol. super-resoln. microscopy and coordinate-based cluster anal. to ext. information on the distribution and on the morphol. and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly obsd. was in accordance with previous electron microscopy analyses revealing ∼140 nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag mols. in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster anal. and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to ext. information on the properties of the different Gag clusters.
- 402Lehmann, M.; Rocha, S.; Mangeat, B.; Blanchet, F.; Uji, I. H.; Hofkens, J.; Piguet, V. Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction. PLoS Pathog. 2011, 7, e1002456 DOI: 10.1371/journal.ppat.1002456Google Scholar402Quantitative multicolor super-resolution microscopy reveals tetherin HIV-1 interactionLehmann, Martin; Rocha, Susana; Mangeat, Bastien; Blanchet, Fabien; Uji-i, Hiroshi; Hofkens, Johan; Piguet, VincentPLoS Pathogens (2011), 7 (12), e1002456CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resoln. microscopy techniques achieve nanometer resoln. of fluorescently labeled mols. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resoln. anal. of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, resp. Multicolor super-resoln. microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters contg. on av. 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochem. and super-resoln. anal. revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resoln. microscopy anal. of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions.
- 403Inamdar, K.; Floderer, C.; Favard, C.; Muriaux, D. Monitoring HIV-1 Assembly in Living Cells: Insights from Dynamic and Single Molecule Microscopy. Viruses 2019, 11, 72, DOI: 10.3390/v11010072Google Scholar403Monitoring HIV-1 assembly in living cells: insights from dynamic and single molecule microscopyInamdar, Kaushik; Floderer, Charlotte; Favard, Cyril; Muriaux, DelphineViruses (2019), 11 (1), 72CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)The HIV-1 assembly process is a multi-complex mechanism that takes place at the host cell plasma membrane. It requires a spatio-temporal coordination of events to end up with a full mature and infectious virus. The mol. mechanisms of HIV-1 assembly have been extensively studied during the past decades, in order to dissect the resp. roles of the structural and non-structural viral proteins of the viral RNA genome and of some host cell factors. Nevertheless, the time course of HIV-1 assembly was obsd. in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resoln., in addn. to improved fluorescent tags for proteins, now permits study of HIV-1 assembly at the single mol. level within living cells. In this review, after a short description of these new approaches, we will discuss how HIV-1 assembly at the cell plasma membrane has been revisited using advanced super resoln. microscopy techniques and how it can bridge the study of viral assembly from the single mol. to the entire host cell.
- 404Bleck, M.; Itano, M. S.; Johnson, D. S.; Thomas, V. K.; North, A. J.; Bieniasz, P. D.; Simon, S. M. Temporal and Spatial Organization of ESCRT Protein Recruitment During HIV-1 Budding. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 12211– 12216, DOI: 10.1073/pnas.1321655111Google Scholar404Temporal and spatial organization of ESCRT protein recruitment during HIV-1 buddingBleck, Marina; Itano, Michelle S.; Johnson, Daniel S.; Thomas, V. Kaye; North, Alison J.; Bieniasz, Paul D.; Simon, Sanford M.Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (33), 12211-12216CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the host ESCRT (endosomal sorting complex required for transport) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we obsd. that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins like Chmp4b (charged multivesicular body protein 4b) and Vps4A (vacuolar protein sorting-assocd. protein 4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresoln. imaging, we obsd. that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.
- 405Prescher, J.; Baumgartel, V.; Ivanchenko, S.; Torrano, A. A.; Brauchle, C.; Muller, B.; Lamb, D. C. Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites. PLoS Pathog. 2015, 11, e1004677 DOI: 10.1371/journal.ppat.1004677Google Scholar405Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sitesPrescher, Jens; Baumgaertel, Viola; Ivanchenko, Sergey; Torrano, Adriano A.; Braeuchle, Christoph; Mueller, Barbara; Lamb, Don C.PLoS Pathogens (2015), 11 (2), e1004677CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane assocd. lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be obsd., the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resoln. fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an av. size (full-width at half-max.) between 45 and 60 nm or a diam. (detd. using a Ripley's L-function anal.) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual mols. surrounding the central clusters was often obsd., which we attribute to ALIX mols. incorporated into the nascent HIV-1 Gag shell. Expts. performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.
- 406Shao, L.; Kner, P.; Rego, E. H.; Gustafsson, M. G. Super-Resolution 3D Microscopy of Live Whole Cells Using Structured Illumination. Nat. Methods 2011, 8, 1044– 1046, DOI: 10.1038/nmeth.1734Google Scholar406Super-resolution 3D microscopy of live whole cells using structured illuminationShao, Lin; Kner, Peter; Rego, E. Hesper; Gustafsson, Mats G. L.Nature Methods (2011), 8 (12), 1044-1046CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resoln. of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per vol. for >50 time points with 120-nm lateral and 360-nm axial resoln. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.
- 407Jones, S. A.; Shim, S. H.; He, J.; Zhuang, X. Fast, Three-Dimensional Super-Resolution Imaging of Live Cells. Nat. Methods 2011, 8, 499– 508, DOI: 10.1038/nmeth.1605Google Scholar407Fast, three-dimensional super-resolution imaging of live cellsJones, Sara A.; Shim, Sang-Hee; He, Jiang; Zhuang, XiaoweiNature Methods (2011), 8 (6), 499-505CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We report super-resoln. fluorescence imaging of live cells with high spatiotemporal resoln. using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resoln. images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolns. of ∼25 nm and temporal resolns. as fast as 0.5 s. We also demonstrated live-cell 3D super-resoln. imaging. We obtained 3D spatial resoln. of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolns. as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resoln. imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
- 408Nair, D.; Hosy, E.; Petersen, J. D.; Constals, A.; Giannone, G.; Choquet, D.; Sibarita, J. B. Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95. J. Neurosci. 2013, 33, 13204– 13224, DOI: 10.1523/JNEUROSCI.2381-12.2013Google Scholar408Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95Nair, Deepak; Hosy, Eric; Petersen, Jennifer D.; Constals, Audrey; Giannone, Gregory; Choquet, Daniel; Sibarita, Jean-BaptisteJournal of Neuroscience (2013), 33 (32), 13204-13224CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resoln. light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concd. inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them. Nanodomains are dynamic in their shape and position within synapses and can form or disappear within minutes, although they are mostly stable for up to 1 h. AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95, which are generally of larger size than AMPAR nanoclusters. PSD95 expression level regulates AMPAR nanodomain size and compactness in parallel to miniature EPSC amplitude. Monte Carlo simulations further indicate the impact of AMPAR concn. in clusters on the efficacy of synaptic transmission. The observation that AMPARs are highly concd. in nanodomains, instead of diffusively distributed in the PSD as generally thought, has important consequences on our understanding of excitatory neurotransmission. Furthermore, our results indicate that glutamatergic synaptic transmission is controlled by the nanometer-scale regulation of the size of these highly concd. nanodomains.
- 409Izeddin, I.; Recamier, V.; Bosanac, L.; Cisse, I. I.; Boudarene, L.; Dugast-Darzacq, C.; Proux, F.; Benichou, O.; Voituriez, R.; Bensaude, O. Single-Molecule Tracking in Live Cells Reveals Distinct Target-Search Strategies of Transcription Factors in the Nucleus. eLife 2014, 3, e02230 DOI: 10.7554/eLife.02230Google Scholar409Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleusIzeddin, Ignacio; Recamier, Vincent; Bosanac, Lana; Cisse, Ibrahim I.; Boudarene, Lydia; Dugast-Darzacq, Claire; Proux, Florence; Benichou, Olivier; Voituriez, Raphael; Bensaude, Olivier; Dahan, Maxime; Darzacq, XaviereLife (2014), 3 (), e02230/1-e02230/27, 27 pp.CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-mol. tracking assay to det. TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the pos. transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc mol. is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.
- 410Yang, L.; Dun, A. R.; Martin, K. J.; Qiu, Z.; Dunn, A.; Lord, G. J.; Lu, W.; Duncan, R. R.; Rickman, C. Secretory Vesicles Are Preferentially Targeted to Areas of Low Molecular SNARE Density. PLoS One 2012, 7, e49514 DOI: 10.1371/journal.pone.0049514Google Scholar410Secretory vesicles are preferentially targeted to areas of low molecular SNARE densityYang, Lei; Dun, Alison R.; Martin, Kirsty J.; Qiu, Zhen; Dunn, Andrew; Lord, Gabriel J.; Lu, Weiping; Duncan, Rory R.; Rickman, ColinPLoS One (2012), 7 (11), e49514CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (sol. N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters contg. many mols., thus providing a concd. reservoir to promote membrane fusion. However, biophys. expts. suggest that a small no. of SNAREs are sufficient to drive a single fusion event. Here we show, using mol. imaging, that the majority of tSNARE mols. are spatially sepd. from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random mol. distribution and limiting the max. no. of mols. encountered by secretory vesicles. Together our results provide a new model for the mol. mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual mol. machines.
- 411Juette, M. F.; Gould, T. J.; Lessard, M. D.; Mlodzianoski, M. J.; Nagpure, B. S.; Bennett, B. T.; Hess, S. T.; Bewersdorf, J. Three-Dimensional Sub-100 nm Resolution Fluorescence Microscopy of Thick Samples. Nat. Methods 2008, 5, 527– 529, DOI: 10.1038/nmeth.1211Google Scholar411Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samplesJuette, Manuel F.; Gould, Travis J.; Lessard, Mark D.; Mlodzianoski, Michael J.; Nagpure, Bhupendra S.; Bennett, Brian T.; Hess, Samuel T.; Bewersdorf, JoergNature Methods (2008), 5 (6), 527-529CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The ability to image thick vols. with invariant high axial and lateral resoln. is a challenge for existing super-resoln. fluorescence microscopy techniques. The combination of a double-plane detection scheme with fluorescence photoactivation microscopy (FPALM) allows three-dimensional sub-diffraction resoln. imaging of samples as thick as whole cells. Imaging vols. as thick as whole cells at three-dimensional (3D) super-resoln. is required to reveal unknown features of cellular organization. The authors report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resoln. over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resoln. without compromising speed or sensitivity.
- 412Pavani, S. R.; Thompson, M. A.; Biteen, J. S.; Lord, S. J.; Liu, N.; Twieg, R. J.; Piestun, R.; Moerner, W. E. Three-Dimensional, Single-Molecule Fluorescence Imaging Beyond the Diffraction Limit by Using a Double-Helix Point Spread Function. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 2995– 2999, DOI: 10.1073/pnas.0900245106Google Scholar412Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread functionPavani, Sri Rama Prasanna; Thompson, Michael A.; Biteen, Julie S.; Lord, Samuel J.; Liu, Na; Twieg, Robert J.; Piestun, Rafael; Moerner, W. E.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (9), 2995-2999CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We demonstrate single-mol. fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent mols. in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 μm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresoln. imaging of high concns. of mols. in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resoln. beyond the Rayleigh diffraction limit.
- 413Thompson, M. A.; Lew, M. D.; Badieirostami, M.; Moerner, W. E. Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function. Nano Lett. 2010, 10, 211– 218, DOI: 10.1021/nl903295pGoogle Scholar413Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread FunctionThompson, Michael A.; Lew, Matthew D.; Badieirostami, Majid; Moerner, W. E.Nano Letters (2010), 10 (1), 211-218CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a wide-field fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). The authors describe in detail how the localization precision quant. depends upon the no. of photons detected and the z position of the nanoscale emitter, thereby showing a ∼10 nm localization capability along x, y, and z in the limit of weak emitters. Exptl. measurements are compared to Fisher information calcns. of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aq. soln. and a quantum dot-labeled structure inside a living cell in three dimensions.
- 414Yu, B.; Yu, J.; Li, W.; Cao, B.; Li, H.; Chen, D.; Niu, H. Nanoscale Three-Dimensional Single Particle Tracking by Light-Sheet-Based Double-Helix Point Spread Function Microscopy. Appl. Opt. 2016, 55, 449– 453, DOI: 10.1364/AO.55.000449Google Scholar414Nanoscale three-dimensional single particle tracking by light-sheet-based double-helix point spread function microscopyYu, Bin; Yu, Jie; Li, Weihai; Cao, Bo; Li, Heng; Chen, Danni; Niu, HanbenApplied Optics (2016), 55 (3), 449-453CODEN: APOPAI; ISSN:1559-128X. (Optical Society of America)The double-helix point spread function (DH-PSF) microscopy has become an essential tool for nanoscale three-dimensional (3D) localization and tracking of single mols. in living cells. However, its localization precision is limited by fluorescent contrast in thick samples because the signal-to-noise ratio of the system is low due to the inherent low transfer function efficiency and background fluorescence. Here we combine DH-PSF microscopy with light-sheet illumination to eliminate out-of-focus background fluorescence for high-precision 3D single particle tracking. To demonstrate the capability of the method, we obtain the single fluorescent bead image with light-sheet illumination, with three-dimensional localization accuracy better than that of epi-illumination. We also show that the single fluorescent beads in agarose soln. can be tracked, which demonstrates the possibility of our method for the study of dynamic processes in complex biol. specimens.
- 415Holtzer, L.; Meckel, T.; Schmidt, T. Nanometric Three-Dimensional Tracking of Individual Quantum Dots in Cells. Appl. Phys. Lett. 2007, 90, 053902 DOI: 10.1063/1.2437066Google Scholar415Nanometric three-dimensional tracking of individual quantum dots in cellsHoltzer, Laurent; Meckel, Tobias; Schmidt, ThomasApplied Physics Letters (2007), 90 (5), 053902/1-053902/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)Wide-field single-mol. fluorescence microscopy has become an established tool for the study of dynamic biol. processes which occur in the plane of a cellular membrane. In the current study we have extended this technique to the three-dimensional anal. of mol. mobility. Introduction of a cylindrical lens into the emission path of a microscope produced some astigmatism which was used to obtain the full three-dimensional position information. The localization accuracy of fluorescent objects was calcd. theor. and subsequently confirmed by simulations and by expts. For further validation individual quantum dots were followed when passively diffusing and actively transported within life cells.
- 416Huang, B.; Wang, W.; Bates, M.; Zhuang, X. Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy. Science 2008, 319, 810– 813, DOI: 10.1126/science.1153529Google Scholar416Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction MicroscopyHuang, Bo; Wang, Wenqin; Bates, Mark; Zhuang, XiaoweiScience (Washington, DC, United States) (2008), 319 (5864), 810-813CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resoln., achieving a near-mol. resoln. of 20 to 30 nm in the two lateral dimensions. Three-dimensional (3D) nanoscale-resoln. imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to det. both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resoln. of 20 to 30 nm in the lateral dimensions and 50 to 60 nm in the axial dimension. This development allowed us to resolve the 3D morphol. of nanoscopic cellular structures.
- 417Lange, S.; Katayama, Y.; Schmid, M.; Burkacky, O.; Brauchle, C.; Lamb, D. C.; Jansen, R. P. Simultaneous Transport of Different Localized mRNA Species Revealed by Live-Cell Imaging. Traffic 2008, 9, 1256– 1267, DOI: 10.1111/j.1600-0854.2008.00763.xGoogle Scholar417Simultaneous transport of different localized mRNA species revealed by live-cell imagingLange, Susanne; Katayama, Yoshihiko; Schmid, Maria; Burkacky, Ondrej; Braeuchle, Christoph; Lamb, Don C.; Jansen, Ralf-PeterTraffic (Oxford, United Kingdom) (2008), 9 (8), 1256-1267CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)Intracellular mRNA localization is a common mechanism to achieve asym. distributions of proteins. Previous studies have revealed that in a no. of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into sep. or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, the authors analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, the authors have tracked the transport of two different localized mRNA species in real time. Their observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form sep. particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.
- 418Wells, N. P.; Lessard, G. A.; Goodwin, P. M.; Phipps, M. E.; Cutler, P. J.; Lidke, D. S.; Wilson, B. S.; Werner, J. H. Time-Resolved Three-Dimensional Molecular Tracking in Live Cells. Nano Lett. 2010, 10, 4732– 4737, DOI: 10.1021/nl103247vGoogle Scholar418Time-Resolved Three-Dimensional Molecular Tracking in Live CellsWells, Nathan P.; Lessard, Guillaume A.; Goodwin, Peter M.; Phipps, Mary E.; Cutler, Patrick J.; Lidke, Diane S.; Wilson, Bridget S.; Werner, James H.Nano Letters (2010), 10 (11), 4732-4737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses 4 overlapping confocal vol. elements and active feedback once every 5 ms to follow 3-dimensional mol. motion. This method has substantial advantages over three-dimensional mol. tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the mols. being tracked. In particular, the authors show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biol. relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, the authors follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topol. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (∼950 nm/s) vesicular transit through the cell.
- 419Cang, H.; Xu, C. S.; Montiel, D.; Yang, H. Guiding a Confocal Microscope by Single Fluorescent Nanoparticles. Opt. Lett. 2007, 32, 2729– 2731, DOI: 10.1364/OL.32.002729Google Scholar419Guiding a confocal microscope by single fluorescent nanoparticlesCang Hu; Xu C Shan; Montiel Daniel; Yang HawOptics letters (2007), 32 (18), 2729-31 ISSN:0146-9592.Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.
- 420Balzarotti, F.; Eilers, Y.; Gwosch, K. C.; Gynna, A. H.; Westphal, V.; Stefani, F. D.; Elf, J.; Hell, S. W. Nanometer Resolution Imaging and Tracking of Fluorescent Molecules with Minimal Photon Fluxes. Science 2017, 355, 606– 612, DOI: 10.1126/science.aak9913Google Scholar420Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxesBalzarotti, Francisco; Eilers, Yvan; Gwosch, Klaus C.; Gynna, Arvid H.; Westphal, Volker; Stefani, Fernando D.; Elf, Johan; Hell, Stefan W.Science (Washington, DC, United States) (2017), 355 (6325), 606-612CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity min. of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our expts., 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresoln. microscopy, MINFLUX attained ∼1-nm precision, resolving mols. only 6 nm apart. MINFLUX tracking of single fluorescent proteins increased the temporal resoln. and the no. of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromols. in living cells and beyond.
- 421Enderlein, J. Tracking of Fluorescent Molecules Diffusing within Membranes. Appl. Phys. B: Lasers Opt. 2000, 71, 773– 777, DOI: 10.1007/s003400000409Google Scholar421Tracking of fluorescent molecules diffusing within membranesEnderlein, J.Applied Physics B: Lasers and Optics (2000), 71 (5), 773-777CODEN: APBOEM; ISSN:0946-2171. (Springer-Verlag)A new method is proposed for tracking fluorescing single mols. diffusing within a two-dimensional membrane. It is based on a confocal microscopy setup with a constantly rotating laser focus, which follows the position of the mol. The optimization and efficiency of the method are theor. studied for a broad range of exptl. realistic conditions. The proposed method allows for a longtime observation of diffusing mols. while allowing the application of fast spectroscopic techniques such as fluorescence decay time detn. or fluorescence anisotropy measurements.
- 422Kis-Petikova, K.; Gratton, E. Distance Measurement by Circular Scanning of the Excitation Beam in the Two-Photon Microscope. Microsc. Res. Tech. 2004, 63, 34– 49, DOI: 10.1002/jemt.10417Google Scholar422Distance measurement by circular scanning of the excitation beam in the two-photon microscopeKis-Petikova Katarina; Gratton EnricoMicroscopy research and technique (2004), 63 (1), 34-49 ISSN:1059-910X.We developed a method to measure relative distances with nanometer accuracy of fluorescent particles of different color in a two-photon scanning fluorescence microscope, with two-channel photon counting detection. The method can be used in the 10-500 nm range, for distances below the resolution limit of standard far field microscopy. The proposed technique is more efficient than the methods using raster scanning. To achieve maximum sensitivity in the radial direction, the excitation beam is moved periodically in a circular orbit with a radius of the size of the point spread function. The phase and the modulation of the periodic fluorescence signal, calculated by fast Fourier transform, gives the phase and the radial distance of the particle from the center of scanning. The coordinates of particles are recovered simultaneously in the two channels and the relative distance is calculated in real time. Particles can be tracked by moving the center of scanning to the recovered position, while measuring the distance from the second particle. Intensity data are saved and fitted later by a model accounting for light leakage between the channels. The total number of detected photons limited the accuracy of the position and distance measurement. Experiments demonstrating the advantages of the method were performed on fluorescent spheres and single dye molecules immobilized on quartz surface.
- 423Lessard, G. A.; Goodwin, P. M.; Werner, J. H. Three-Dimensional Tracking of Individual Quantum Dots. Appl. Phys. Lett. 2007, 91, 224106, DOI: 10.1063/1.2819074Google Scholar423Three-dimensional tracking of individual quantum dotsLessard, Guillaume A.; Goodwin, Peter M.; Werner, James H.Applied Physics Letters (2007), 91 (22), 224106/1-224106/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)The authors describe an instrument that extends the state of the art in a single-mol. tracking technol., allowing extended observations of single fluorophores and fluorescently labeled proteins as they undergo directed and diffusive transport in three dimensions. The authors demonstrate three-dimensional tracking of individual quantum dots undergoing diffusion for durations of over a second at velocities comparable to those of intracellular signaling processes.
- 424Verdeny-Vilanova, I.; Wehnekamp, F.; Mohan, N.; Alvarez, A. S.; Borbely, J. S.; Otterstrom, J. J.; Lamb, D. C.; Lakadamyali, M. 3D Motion of Vesicles along Microtubules Helps Them to Circumvent Obstacles in Cells. J. Cell Sci. 2017, 130, 1904– 1916, DOI: 10.1242/jcs.201178Google Scholar4243D motion of vesicles along microtubules helps them to circumvent obstacles in cellsVerdeny-Vilanova, Ione; Wehnekamp, Fabian; Mohan, Nitin; Alvarez, AngelSandoval; Borbely, Joseph Steven; Otterstrom, Jason John; Lamb, Don C.; Lakadamyali, MelikeJournal of Cell Science (2017), 130 (11), 1904-1916CODEN: JNCSAI; ISSN:1477-9137. (Company of Biologists Ltd.)Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the sepn. of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them.
- 425Wehnekamp, F.; Plucinska, G.; Thong, R.; Misgeld, T.; Lamb, D. C. Nanoresolution Real-Time 3D Orbital Tracking for Studying Mitochondrial Trafficking in Vertebrate Axons in Vivo. eLife 2019, 8, e46059 DOI: 10.7554/eLife.46059Google Scholar425Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivoWehnekamp, Fabian; Plucinska, Gabriela; Thong, Rachel; Misgeld, Thomas; Lamb, Don C.eLife (2019), 8 (), e46059/1-e46059/22CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resoln. By combining various tech. improvements, we tracked individual mitochondria with unprecedented spatiotemporal resoln. over distances of >100 μm. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resoln. in a living organism offers a new approach to elucidating their function in its complete physiol. context.
- 426Katayama, Y.; Burkacky, O.; Meyer, M.; Brauchle, C.; Gratton, E.; Lamb, D. C. Real-Time Nanomicroscopy via Three-Dimensional Single-Particle Tracking. ChemPhysChem 2009, 10, 2458– 2464, DOI: 10.1002/cphc.200900436Google Scholar426Real-Time Nanomicroscopy via Three-Dimensional Single-Particle TrackingKatayama, Yoshihiko; Burkacky, Ondrej; Meyer, Martin; Braeuchle, Christoph; Gratton, Enrico; Lamb, Don C.ChemPhysChem (2009), 10 (14), 2458-2464CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resoln. in the lateral dimensions and 50 nm in the axial dimension at a temporal resoln. of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resoln.
- 427Hellriegel, C.; Gratton, E. Real-Time Multi-Parameter Spectroscopy and Localization in Three-Dimensional Single-Particle Tracking. J. R. Soc., Interface 2009, 6, S3– S14, DOI: 10.1098/rsif.2008.0313.focusGoogle Scholar427Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle trackingHellriegel, Christian; Gratton, EnricoJournal of the Royal Society, Interface (2009), 6 (Suppl. 1), S3-S14CODEN: JRSICU; ISSN:1742-5689. (Royal Society)Tracking of single particles in optical microscopy has been employed in studies ranging from material sciences to biophysics down to the level of single mols. The technique intrinsically circumvents ensemble averaging and may therefore reveal directly mechanistic details of the involved dynamic processes. Such processes range from translational and rotational motion to spectral dynamics. We distinguish between conventional a posteriori tracking of objects (e.g. from the sequences of images) and the exptl. more refined 'on-the-fly' tracking technique. In this technique, the observation vol. of the microscope is kept centered with respect to the moving object via a feedback algorithm. This approach brings a series of advantages in comparison with the tracking from images, ranging from a superior spatio-temporal resoln. (2-50 nm and 1-32 ms) to the capability of inferring addnl. data (e.g. fluorescence lifetime, emission spectrum, polarization, intensity dynamics) from an object as it moves over several microns in three dimensions. In this contribution, we describe the principle of the tracking technique as implemented on a two-photon laser scanning microscope and illustrate its capabilities with exptl. data, from particles labeled with different dyes moving in a liq. to the characterization of small fluorescently labeled protein assemblies in living cells.
- 428Liu, Z.; Lavis, L. D.; Betzig, E. Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level. Mol. Cell 2015, 58, 644– 659, DOI: 10.1016/j.molcel.2015.02.033Google Scholar428Imaging Live-Cell Dynamics and Structure at the Single-Molecule LevelLiu, Zhe; Lavis, Luke D.; Betzig, EricMolecular Cell (2015), 58 (4), 644-659CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Observation of mol. processes inside living cells is fundamental to a quant. understanding of how biol. systems function. Specifically, decoding the complex behavior of single mols. enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble expts. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new expts. to investigate the robustness and stochasticity of diverse mol. mechanisms with high spatiotemporal resoln. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-mol. level with minimal perturbations to the specimen.
- 429Saxton, M. J. Single-Particle Tracking: Connecting the Dots. Nat. Methods 2008, 5, 671– 672, DOI: 10.1038/nmeth0808-671Google Scholar429Single-particle tracking: connecting the dotsSaxton, Michael J.Nature Methods (2008), 5 (8), 671-672CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review commentary on the accompanying articles by (ibid. A. Serge et al., 687-694 and K. Jaqaman et al., 695-702). Algorithms for analyzing single-particle tracking images to obtain the paths of individual particles are challenged by high-d. data. Improvements in algorithms help to overcome these limitations.
- 430Carter, B. C.; Shubeita, G. T.; Gross, S. P. Tracking Single Particles: A User-Friendly Quantitative Evaluation. Phys. Biol. 2005, 2, 60– 72, DOI: 10.1088/1478-3967/2/1/008Google Scholar430Tracking single particles: a user-friendly quantitative evaluationCarter Brian C; Shubeita George T; Gross Steven PPhysical biology (2005), 2 (1), 60-72 ISSN:.As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.
- 431Thompson, R. E.; Larson, D. R.; Webb, W. W. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophys. J. 2002, 82, 2775– 2783, DOI: 10.1016/S0006-3495(02)75618-XGoogle Scholar431Precise nanometer localization analysis for individual fluorescent probesThompson, Russell E.; Larson, Daniel R.; Webb, Watt W.Biophysical Journal (2002), 82 (5), 2775-2783CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Calcn. of the centroid of the images of individual fluorescent particles and mols. allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resoln. The factors that limit the precision of these techniques are examd. and a simple equation derived that describes the precision of localization over a wide range of conditions. In addn., a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an exptl. app. and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the no. of photons in the spot for the shot noise limited case and as the inverse of the no. of photons for the background noise limited case. The optimal image magnification depends on the expected no. of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the std. deviation of the point spread function.
- 432Small, A.; Stahlheber, S. Fluorophore Localization Algorithms for Super-Resolution Microscopy. Nat. Methods 2014, 11, 267– 279, DOI: 10.1038/nmeth.2844Google Scholar432Fluorophore localization algorithms for super-resolution microscopySmall, Alex; Stahlheber, ShaneNature Methods (2014), 11 (3), 267-279CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Super-resoln. localization microscopy methods provide powerful new capabilities for probing biol. at the nanometer scale via fluorescence. These methods rely on two key innovations: switchable fluorophores (which blink on and off and can be sequentially imaged) and powerful localization algorithms (which est. the positions of the fluorophores in the images). These techniques have spurred a flurry of innovation in algorithm development over the last several years. In this Review, we survey the fundamental issues for single-fluorophore fitting routines, localization algorithms based on principles other than fitting, three-dimensional imaging, dipole imaging and techniques for estg. fluorophore positions from images of multiple activated fluorophores. We offer practical advice for users and adopters of algorithms, and we identify areas for further development.
- 433Ernst, D.; Kohler, J. How the Number of Fitting Points for the Slope of the Mean-Square Displacement Influences the Experimentally Determined Particle Size Distribution from Single-Particle Tracking. Phys. Chem. Chem. Phys. 2013, 15, 3429– 3432, DOI: 10.1039/c3cp44391dGoogle Scholar433How the number of fitting points for the slope of the mean-square displacement influences the experimentally determined particle size distribution from single-particle trackingErnst, Dominique; Koehler, JuergenPhysical Chemistry Chemical Physics (2013), 15 (10), 3429-3432CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The size distribution of nanoparticles can be detd. by single-particle tracking. This yields the mean-squared displacement (MSD) as a function of the lag time, and for normal diffusion the slope of this curve is directly related to the diffusion coeff. or via the Stokes-Einstein relation to the particle size. Here we demonstrate how the exptl. detd. size distributions are affected by the no. of fitting points used to det. the slope of the MSD curve.
- 434Born, M.; Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light; Elsevier: 2013.Google ScholarThere is no corresponding record for this reference.
- 435Liu, S. L.; Li, J.; Zhang, Z. L.; Wang, Z. G.; Tian, Z. Q.; Wang, G. P.; Pang, D. W. Fast and High-Accuracy Localization for Three-Dimensional Single-Particle Tracking. Sci. Rep. 2013, 3, 2462, DOI: 10.1038/srep02462Google Scholar435Fast and high-accuracy localization for three-dimensional single-particle trackingLiu Shu-Lin; Li Jicun; Zhang Zhi-Ling; Wang Zhi-Gang; Tian Zhi-Quan; Wang Guo-Ping; Pang Dai-WenScientific reports (2013), 3 (), 2462 ISSN:.We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization. Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. The precision is almost the same as that of the iterative nonlinear least-squares 3D Gaussian fitting method, but with two orders of magnitude higher computation speed. This approach can reduce considerably the time and costs for processing the large volume data of 3D images for 3D single-particle tracking, which is especially suited for 3D high-precision single-particle tracking, 3D single-molecule imaging and even new microscopy techniques.
- 436Qu, X. H.; Wu, D.; Mets, L.; Scherer, N. F. Nanometer-Localized Multiple Single-Molecule Fluorescence Microscopy. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11298– 11303, DOI: 10.1073/pnas.0402155101Google Scholar436Nanometer-localized multiple single-molecule fluorescence microscopyQu, Xiaohui; Wu, David; Mets, Laurens; Scherer, Norbert F.Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (31), 11298-11303CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Fitting the image of a single mol. to the point spread function of an optical system greatly improves the precision with which single mols. can be located. Centroid localization with nanometer precision has been achieved when a sufficient no. of photons are collected. However, if multiple single mols. reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-mol. (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single mols. within a diffraction-limited area. NALMS microscopy will greatly facilitate single-mol. study of biol. systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resoln. is demonstrated.
- 437Hess, S. T.; Girirajan, T. P.; Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 2006, 91, 4258– 4272, DOI: 10.1529/biophysj.106.091116Google Scholar437Ultra-high resolution imaging by fluorescence photoactivation localization microscopyHess, Samuel T.; Girirajan, Thanu P. K.; Mason, Michael D.Biophysical Journal (2006), 91 (11), 4258-4272CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Biol. structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resoln. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large nos. of fluorescent mols. on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small nos. of them at a time, at low excitation intensity. To control the no. of visible fluorophores in the field of view and ensure that optically active mols. are sepd. by much more than the width of the point spread function, photoactivatable fluorescent mols. are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable mols., the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive mols. are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the mols. are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small no. of mols. are visible at a given time, their positions can be detd. precisely; with only ∼100 detected photons per mol., the localization precision can be as much as 10-fold better than the resoln., depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are obsd. by FPALM of PA-GFP on glass. FPALM images are compared with images of the same mols. by wide-field fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with at. force microscopy and show that the full width at half-max. of features ∼86±4 nm is significantly better than the expected diffraction-limited optical resoln. The no. of fluorescent mols. and their brightness distribution have also been detd. using FPALM. This new method suggests a means to address a significant no. of biol. questions that had previously been limited by microscope resoln.
- 438Parthasarathy, R. Rapid, Accurate Particle Tracking by Calculation of Radial Symmetry Centers. Nat. Methods 2012, 9, 724– 726, DOI: 10.1038/nmeth.2071Google Scholar438Rapid, accurate particle tracking by calculation of radial symmetry centersParthasarathy, RaghuveerNature Methods (2012), 9 (7_part3), 724-726CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)I introduce an algorithm for subpixel localization of imaged objects based on an analytic, non-iterative calcn. of the best-fit radial symmetry center. This approach yields tracking accuracies that are near theor. limits, similarly to Gaussian fitting, but with orders-of-magnitude faster execution time, lower sensitivity to nearby particles and applicability to any radially sym. intensity distribution. I demonstrate the method with several types of data, including super-resoln. microscopy images.
- 439Sage, D.; Neumann, F. R.; Hediger, F.; Gasser, S. M.; Unser, M. Automatic Tracking of Individual Fluorescence Particles: Application to the Study of Chromosome Dynamics. IEEE Trans. Image Process. 2005, 14, 1372– 1383, DOI: 10.1109/TIP.2005.852787Google Scholar439Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamicsSage Daniel; Neumann Franck R; Hediger Florence; Gasser Susan M; Unser MichaelIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2005), 14 (9), 1372-83 ISSN:1057-7149.We present a new, robust, computational procedure for tracking fluorescent markers in time-lapse microscopy. The algorithm is optimized for finding the time-trajectory of single particles in very noisy dynamic (two- or three-dimensional) image sequences. It proceeds in three steps. First, the images are aligned to compensate for the movement of the biological structure under investigation. Second, the particle's signature is enhanced by applying a Mexican hat filter, which we show to be the optimal detector of a Gaussian-like spot in 1/omega2 noise. Finally, the optimal trajectory of the particle is extracted by applying a dynamic programming optimization procedure. We have used this software, which is implemented as a Java plug-in for the public-domain ImageJ software, to track the movement of chromosomal loci within nuclei of budding yeast cells. Besides reducing trajectory analysis time by several 100-fold, we achieve high reproducibility and accuracy of tracking. The application of the method to yeast chromatin dynamics reveals different classes of constraints on mobility of telomeres, reflecting differences in nuclear envelope association. The generic nature of the software allows application to a variety of similar biological imaging tasks that require the extraction and quantitation of a moving particle's trajectory.
- 440Godinez, W. J.; Lampe, M.; Worz, S.; Muller, B.; Eils, R.; Rohr, K. Deterministic and Probabilistic Approaches for Tracking Virus Particles in Time-Lapse Fluorescence Microscopy Image Sequences. Med. Image Anal. 2009, 13, 325– 342, DOI: 10.1016/j.media.2008.12.004Google Scholar440Deterministic and probabilistic approaches for tracking virus particles in time-lapse fluorescence microscopy image sequencesGodinez W J; Lampe M; Worz S; Muller B; Eils R; Rohr KMedical image analysis (2009), 13 (2), 325-42 ISSN:.Modern developments in time-lapse fluorescence microscopy enable the observation of a variety of processes exhibited by viruses. The dynamic nature of these processes requires the tracking of viruses over time to explore spatial-temporal relationships. In this work, we developed deterministic and probabilistic approaches for multiple virus tracking in multi-channel fluorescence microscopy images. The deterministic approaches follow a traditional two-step paradigm comprising particle localization based on either the spot-enhancing filter or 2D Gaussian fitting, as well as motion correspondence based on a global nearest neighbor scheme. Our probabilistic approaches are based on particle filters. We describe approaches based on a mixture of particle filters and based on independent particle filters. For the latter, we have developed a penalization strategy that prevents the problem of filter coalescence (merging) in cases where objects lie in close proximity. A quantitative comparison based on synthetic image sequences is carried out to evaluate the performance of our approaches. In total, eight different tracking approaches have been evaluated. We have also applied these approaches to real microscopy images of HIV-1 particles and have compared the tracking results with ground truth obtained from manual tracking. It turns out that the probabilistic approaches based on independent particle filters are superior to the deterministic schemes as well as to the approaches based on a mixture of particle filters.
- 441Chenouard, N.; Smal, I.; de Chaumont, F.; Maska, M.; Sbalzarini, I. F.; Gong, Y.; Cardinale, J.; Carthel, C.; Coraluppi, S.; Winter, M. Objective Comparison of Particle Tracking Methods. Nat. Methods 2014, 11, 281– 289, DOI: 10.1038/nmeth.2808Google Scholar441Objective comparison of particle tracking methodsChenouard, Nicolas; Smal, Ihor; de Chaumont, Fabrice; Maska, Martin; Sbalzarini, Ivo F.; Gong, Yuanhao; Cardinale, Janick; Carthel, Craig; Coraluppi, Stefano; Winter, Mark; Cohen, Andrew R.; Godinez, William J.; Rohr, Karl; Kalaidzidis, Yannis; Liang, Liang; Duncan, James; Shen, Hongying; Xu, Yingke; Magnusson, Klas E. G.; Jalden, Joakim; Blau, Helen M.; Paul-Gilloteaux, Perrine; Roudot, Philippe; Kervrann, Charles; Waharte, Francois; Tinevez, Jean-Yves; Shorte, Spencer L.; Willemse, Joost; Celler, Katherine; van Wezel, Gilles P.; Dan, Han-Wei; Tsai, Yuh-Show; Ortiz de Solorzano, Carlos; Olivo-Marin, Jean-Christophe; Meijering, ErikNature Methods (2014), 11 (3), 281-289CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Particle tracking is of key importance for quant. anal. of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large nos. of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.
- 442Reid, D. An Algorithm for Tracking Multiple Targets. IEEE Trans. Autom. Control 1979, 24, 843– 854, DOI: 10.1109/TAC.1979.1102177Google ScholarThere is no corresponding record for this reference.
- 443Veenman, C. J.; Reinders, M. J.; Backer, E. Resolving Motion Correspondence for Densely Moving Points. IEEE T. Pattern Anal. 2001, 23, 54– 72, DOI: 10.1109/34.899946Google ScholarThere is no corresponding record for this reference.
- 444Jiang, S.; Zhou, X.; Kirchhausen, T.; Wong, S. T. Tracking Molecular Particles in Live Cells Using Fuzzy Rule-Based System. Cytometry, Part A 2007, 71, 576– 584, DOI: 10.1002/cyto.a.20411Google Scholar444Tracking molecular particles in live cells using fuzzy rule-based systemJiang Shan; Zhou Xiaobo; Kirchhausen Tom; Wong Stephen T CCytometry. Part A : the journal of the International Society for Analytical Cytology (2007), 71 (8), 576-84 ISSN:1552-4922.Recent development of detection techniques of molecular particles in live cells has stimulated interest in developing the new powerful techniques to track the molecular particles in live cells. One special type of cellular microscopy images is about the formation and transportation of clathrin-coated pits and vesicles. Clathrin-coated pits are very important in studying the behavior of proteins and lipids in live cells. To answer the question, whether there exist "hot spots" for the formation of Clathrin-coated pits or the pits and arrays formed randomly on the plasma membrane, it is necessary to track many hundreds of individual pits dynamically in live-cell microscope movies to capture and monitor how pits and vesicles were formed. Therefore, a motion correspondence algorithm based on fuzzy rule-based system is proposed to resolve the problem of ambiguous association encountered in these dynamic, live-cell images of clathrin assemblies. Results show that this method can accurately track most of the particles in the high volume images.
- 445Shafique, K.; Shah, M. A Noniterative Greedy Algorithm for Multiframe Point Correspondence. IEEE Trans. Pattern Anal. Mach. Intell. 2005, 27, 51– 65, DOI: 10.1109/TPAMI.2005.1Google Scholar445A noniterative greedy algorithm for multiframe point correspondenceShafique Khurram; Shah MubarakIEEE transactions on pattern analysis and machine intelligence (2005), 27 (1), 51-65 ISSN:0162-8828.This paper presents a framework for finding point correspondences in monocular image sequences over multiple frames. The general problem of multiframe point correspondence is NP-hard for three or more frames. A polynomial time algorithm for a restriction of this problem is presented and is used as the basis of the proposed greedy algorithm for the general problem. The greedy nature of the proposed algorithm allows it to be used in real-time systems for tracking and surveillance, etc. In addition, the proposed algorithm deals with the problems of occlusion, missed detections, and false positives by using a single noniterative greedy optimization scheme and, hence, reduces the complexity of the overall algorithm as compared to most existing approaches where multiple heuristics are used for the same purpose. While most greedy algorithms for point tracking do not allow for entry and exit of the points from the scene, this is not a limitation for the proposed algorithm. Experiments with real and synthetic data over a wide range of scenarios and system parameters are presented to validate the claims about the performance of the proposed algorithm.
- 446Jaqaman, K.; Loerke, D.; Mettlen, M.; Kuwata, H.; Grinstein, S.; Schmid, S. L.; Danuser, G. Robust Single-Particle Tracking in Live-Cell Time-Lapse Sequences. Nat. Methods 2008, 5, 695– 702, DOI: 10.1038/nmeth.1237Google Scholar446Robust single-particle tracking in live-cell time-lapse sequencesJaqaman, Khuloud; Loerke, Dinah; Mettlen, Marcel; Kuwata, Hirotaka; Grinstein, Sergio; Schmid, Sandra L.; Danuser, GaudenzNature Methods (2008), 5 (8), 695-702CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here the authors present a tracking algorithm that addresses the principal challenges of SPT, namely high particle d., particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose soln. identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, the authors show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.
- 447Saxton, M. J. Single-Particle Tracking: The Distribution of Diffusion Coefficients. Biophys. J. 1997, 72, 1744– 1753, DOI: 10.1016/S0006-3495(97)78820-9Google Scholar447Single-particle tracking: the distribution of diffusion coefficientsSaxton, Michael J.Biophysical Journal (1997), 72 (4), 1744-1753CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)In single-particle tracking expts., the diffusion coeff. D may be measured from the trajectory of an individual particle in the cell membrane. The statistical distribution of single-trajectory diffusion coeffs. is examd. by Monte Carlo calcns. The width of this distribution may be useful as a measure of the heterogeneity of the membrane and as a test of models of hindered diffusion in the membrane. For some models, the distribution of the short-range diffusion coeff. is much narrower than the obsd. distribution for proteins diffusing in cell membranes. To aid in the anal. of single-particle tracking measurements, the distribution of D is examd. for various definitions of D and for various trajectory lengths.
- 448Dahan, M. From Analog to Digital: Exploring Cell Dynamics with Single Quantum Dots. Histochem. Cell Biol. 2006, 125, 451– 456, DOI: 10.1007/s00418-005-0105-xGoogle Scholar448From analog to digital: exploring cell dynamics with single quantum dotsDahan, MaximeHistochemistry and Cell Biology (2006), 125 (5), 451-456CODEN: HCBIFP; ISSN:0948-6143. (Springer)Semiconductor quantum dots (QDs) have emerged as new fluorescent probes for biol. When combined with ultrasensitive optical techniques, they allow motions of individual biomols. to be tracked in live cells with high signal-to-noise and over unprecedented durations. Single QD imaging readily offers a powerful tool to investigate the organization in cell membranes. Altogether QDs will contribute to more advanced biol. imaging and enable new studies on the dynamics of cellular processes.
- 449Dupont, A.; Gorelashvili, M.; Schuller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D. Three-Dimensional Single-Particle Tracking in Live Cells: News from the Third Dimension. New J. Phys. 2013, 15, 075008 DOI: 10.1088/1367-2630/15/7/075008Google Scholar449Three-dimensional single-particle tracking in live cells: news from the third dimensionDupont, A.; Gorelashvili, M.; Schueller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D.New Journal of Physics (2013), 15 (July), 075008CODEN: NJOPFM; ISSN:1367-2630. (IOP Publishing Ltd.)Single-particle tracking (SPT) is of growing importance in the biophys. community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its tech. simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort. Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) anal. introduced previously. The 3D trajectories and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells' cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coeff. values derived from the 2D anal. were therefore biased.
- 450Bannai, H.; Lévi, S.; Schweizer, C.; Dahan, M.; Triller, A. Imaging the Lateral Diffusion of Membrane Molecules with Quantum Dots. Nat. Protoc. 2006, 1, 2628– 2634, DOI: 10.1038/nprot.2006.429Google Scholar450Imaging the lateral diffusion of membrane molecules with quantum dotsBannai, Hiroko; Levi, Sabine; Schweizer, Claude; Dahan, Maxime; Triller, AntoineNature Protocols (2006), 1 (6), 2628-2634CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a sensitive approach to tracking the motion of membrane mols. such as lipids and proteins with mol. resoln. in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane mols. of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and org. dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The exptl. procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.
- 451Saxton, M. J. Single-Particle Tracking: Effects of Corrals. Biophys. J. 1995, 69, 389– 398, DOI: 10.1016/S0006-3495(95)79911-8Google Scholar451Single-particle tracking: effects of corralsSaxton, Michael J.Biophysical Journal (1995), 69 (2), 389-95CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Structural proteins of the membrane skeleton are thought to form "corrals" at the membrane surface, and these corrals may restrict lateral diffusion of membrane proteins. Recent exptl. developments in single-particle tracking and laser trapping make it possible to examine the corral model in detail. Techniques to interpret these expts. are presented. First, escape times for a diffusing particle in a corral are obtained from Monte Carlo calcns. and anal. solns. for various corral sizes, shapes, and escape probabilities, and reduced to a common curve. Second, the identification of corrals in tracking expts. is considered. The simplest way to identify corrals is by sight. If the walls are impermeable enough, a trajectory fills the corral before the diffusing particle escapes. The fraction of distinct sites visited before escape is calcd. for corrals of various sizes, shapes, and escape probabilities, and reduced to a common curve. This fraction is also a measure of the probability that the diffusing species will react with another species in the corral before escaping. Finally, the effect of the sampling interval on the measurement of the short-range diffusion coeff. is examd.
- 452Rock, R. S. Myosin VI Is a Processive Motor with a Large Step Size. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 13655– 13659, DOI: 10.1073/pnas.191512398Google Scholar452Myosin VI is a processive motor with a large step sizeRock, Ronald S.; Rice, Sarah E.; Wells, Amber L.; Purcell, Thomas J.; Spudich, James A.; Sweeney, H. LeeProceedings of the National Academy of Sciences of the United States of America (2001), 98 (24), 13655-13659CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Myosin VI is a mol. motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mech. advances before it releases from actin. Here we show that myosin VI is also processive by using single mol. motility and optical trapping expts. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.
- 453Ma, S.; Chisholm, R. L. Cytoplasmic Dynein-Associated Structures Move Bidirectionally in Vivo. J. Cell Sci. 2002, 115, 1453– 1460Google Scholar453Cytoplasmic dynein-associated structures move bidirectionally in vivoMa, Shuo; Chisholm, Rex L.Journal of Cell Science (2002), 115 (7), 1453-1460CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubule-based motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively, different motors could assoc. with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-assocd. structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addn., dynein puncta often rapidly reverse their direction. Dynein stably assocs. with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo assocn. but rather by switching activity of motors assocd. with the cargo. These observations suggest that both plus- and minus-end-directed motors assoc. with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.
- 454Sieczkarski, S. B.; Whittaker, G. R. Dissecting Virus Entry via Endocytosis. J. Gen. Virol. 2002, 83, 1535– 1545, DOI: 10.1099/0022-1317-83-7-1535Google Scholar454Dissecting virus entry via endocytosisSieczkarski, Sara B.; Whittaker, Gary R.Journal of General Virology (2002), 83 (7), 1535-1545CODEN: JGVIAY; ISSN:0022-1317. (Society for General Microbiology)A review. Numerous virus families utilize endocytosis to infect host cells, mediating virus internalization as well as trafficking to the site of replication. Recent research has demonstrated that viruses employ the full endocytic capabilities of the cell. The endocytic pathways utilized include clathrin-mediated endocytosis, caveolae, macropinocytosis and novel non-clathrin, non-caveolae pathways. The tools to study endocytosis and, consequently, virus entry are becoming more effective and specific as the amt. of information on endocytic component structure and function increases. The use of inhibitory drugs, although still quite common, often leads to non-specific disruptions in the cell. Mol. inhibitors in the form of dominant-neg. proteins have surpassed the use of chem. inhibitors in terms of specificity to individual pathways. Dominant-neg. mols. are derived from both structural proteins of endocytosis, such as dynamin and caveolin, and regulatory proteins, primarily small GTPases and kinases. This review focuses on the exptl. approaches taken to examine virus entry and provides both classic examples and recent research on a variety of virus families.
- 455Boulant, S.; Stanifer, M.; Lozach, P. Y. Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis. Viruses 2015, 7, 2794– 2815, DOI: 10.3390/v7062747Google Scholar455Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosisBoulant, Steeve; Stanifer, Megan; Lozach, Pierre-YvesViruses (2015), 7 (6), 2794-2815CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization.
- 456Mudhakir, D.; Harashima, H. Learning from the Viral Journey: How to Enter Cells and How to Overcome Intracellular Barriers to Reach the Nucleus. AAPS J. 2009, 11, 65– 77, DOI: 10.1208/s12248-009-9080-9Google Scholar456Learning from the viral journey: how to enter cells and how to overcome intracellular barriers to reach the nucleusMudhakir Diky; Harashima HideyoshiThe AAPS journal (2009), 11 (1), 65-77 ISSN:.Viruses deliver their genome into host cells where they subsequently replicate and multiply. A variety of relevant strategies have evolved by which viruses gain intracellular access and utilize cellular machinery for the synthesis of their genome. Therefore, the viral journey provides insight into the cell's trafficking machinery and how it can be best exploited to improve nonviral gene delivery systems. This review summarizes viral internalization pathways and intracellular trafficking of viruses, with an emphasis on the endosomal escape processes of nonenveloped viruses. Intracellular events from viral entry through nuclear delivery of the viral complementary DNA are also discussed.
- 457Damm, E. M.; Pelkmans, L. Systems Biology of Virus Entry in Mammalian Cells. Cell. Microbiol. 2006, 8, 1219– 1227, DOI: 10.1111/j.1462-5822.2006.00745.xGoogle Scholar457Systems biology of virus entry in mammalian cellsDamm, Eva-Maria; Pelkmans, LucasCellular Microbiology (2006), 8 (8), 1219-1227CODEN: CEMIF5; ISSN:1462-5814. (Blackwell Publishing Ltd.)A review. In this article, the authors define systems biol. of virus entry in mammalian cells as the discipline that combines several approaches to comprehensively understand the collective phys. behavior of virus entry routes, and to understand the coordinated operation of the functional modules and mol. machineries that lead to this phys. behavior. Clearly, these are extremely ambitious aims, but recent developments in different life science disciplines slowly allow us to set them as realistic, although very distant, goals. Besides classical approaches to obtain high-resoln. information of the mols., particles and machines involved, the authors require approaches that can monitor collective behavior of many mols., particles and machines simultaneously, to reveal design principles of the systems as a whole. Here the authors will discuss approaches that fall in the latter category, namely time-lapse imaging and single-particle tracking (SPT) combined with computational anal. and modeling, and genome-wide RNA interference approaches to reveal the host components required for virus entry. These techniques should in the future allow us to assign host genes to the systems' functions and characteristics, and allow emergence-driven, in silico assembly of networks that include interactions with increasing hierarchy (mols.-multiprotein complexes-vesicles and organelles), and kinetics and subcellular spatiality, to allow realistic simulations of virus entry in real time.
- 458Chang, K.; Baginski, J.; Hassan, S. F.; Volin, M.; Shukla, D.; Tiwari, V. Filopodia and Vruses: An Analysis of Membrane Processes in Entry Mechanisms. Front. Microbiol. 2016, 7, 300, DOI: 10.3389/fmicb.2016.00300Google Scholar458Filopodia and Viruses: An Analysis of Membrane Processes in Entry MechanismsChang Kenneth; Baginski John; Volin Michael; Tiwari Vaibhav; Hassan Samer F; Shukla DeepakFrontiers in microbiology (2016), 7 (), 300 ISSN:1664-302X.Filopodia are thin, actin rich bundles protruding from cell plasma membranes, serving physiological purposes, such as probing the environment and facilitating cell-to-cell adhesion. Recent studies have highlighted that actively polymerized filopodial-protrusions are exploited during virus entry, trafficking, spread, and the development of clinical pathology of viral diseases. These observations have caused a surge in investigation of the key determinants of filopodial induction and their influence on cell topography including receptor expression for viral entry. It is now very clear that filopodia can provide unique opportunities for many viruses to invade host cells vertically during primary infection, or horizontally during virus spread from cell-to-cell. These emerging concepts can explain the unprecedented ability of viruses to invade both nearby and long-distant host cells, a feature that may directly contribute to viral tropism. In this review, we summarize the significance of filopodia in viral diseases and discuss future therapeutic possibilities to precisely target filopodial-flyovers to prevent or control infectious diseases.
- 459Shinya, K.; Ebina, M.; Yamada, S.; Ono, M.; Kasai, N.; Kawaoka, Y. Avian Flu: Influenza Virus Receptors in the Human Airway. Nature 2006, 440, 435– 436, DOI: 10.1038/440435aGoogle Scholar459Influenza virus receptors in the human airway: avian and human flu viruses seem to target different regions of a patient's respiratory tract.Shinya, Kyoko; Ebina, Masahito; Yamada, Shinya; Ono, Masao; Kasai, Noriyuki; Kawaoka, YoshihiroNature (London, United Kingdom) (2006), 440 (7083), 435-436CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)H5N1 influenza viruses derived from humans and birds target different regions of a patient's respiratory tract. Although the viruses preferentially recognizing SAα2,3Gal can be transmitted from birds to humans, they can replicate only efficiently in the lower parts of respiratory tract. The may explain the phenomenon that H5N1 viruses rarely infect and spread between humans although they can replicate efficiently in the lungs.
- 460Ibricevic, A.; Pekosz, A.; Walter, M. J.; Newby, C.; Battaile, J. T.; Brown, E. G.; Holtzman, M. J.; Brody, S. L. Influenza Virus Receptor Specificity and Cell Tropism in Mouse and Human Airway Epithelial Cells. J. Virol. 2006, 80, 7469– 7480, DOI: 10.1128/JVI.02677-05Google Scholar460Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cellsIbricevic, Aida; Pekosz, Andrew; Walter, Michael J.; Newby, Celeste; Battaile, John T.; Brown, Earl G.; Holtzman, Michael J.; Brody, Steven L.Journal of Virology (2006), 80 (15), 7469-7480CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an α2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an α2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the α2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The α2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, α2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the α2,3-linked SA receptor.
- 461Lakadamyali, M.; Rust, M. J.; Zhuang, X. Endocytosis of Influenza Viruses. Microbes Infect. 2004, 6, 929– 936, DOI: 10.1016/j.micinf.2004.05.002Google Scholar461Endocytosis of influenza virusesLakadamyali, Melike; Rust, Michael J.; Zhuang, XiaoweiMicrobes and Infection (2004), 6 (10), 929-936CODEN: MCINFS; ISSN:1286-4579. (Elsevier B.V.)A review. Receptor-mediated endocytosis is known to play an important role in the entry of many viruses into host cells. However, the exact internalization mechanism has, until recently, remained poorly understood for many medically important viruses, including influenza. Developments in real-time imaging of single viruses as well as the use of dominant-neg. mutants to selectively block specific endocytic pathways have improved our understanding of the influenza infection process.
- 462Mercer, J.; Schelhaas, M.; Helenius, A. Virus Entry by Endocytosis. Annu. Rev. Biochem. 2010, 79, 803– 833, DOI: 10.1146/annurev-biochem-060208-104626Google Scholar462Virus entry by endocytosisMercer, Jason; Schelhaas, Mario; Helenius, AriAnnual Review of Biochemistry (2010), 79 (), 803-833CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Although viruses are simple in structure and compn., their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biol. of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
- 463Nisole, S.; Saib, A. Early Steps of Retrovirus Replicative Cycle. Retrovirology 2004, 1, 9, DOI: 10.1186/1742-4690-1-9Google Scholar463Early steps of retrovirus replicative cycleNisole Sebastien; Saib AliRetrovirology (2004), 1 (), 9 ISSN:.During the last two decades, the profusion of HIV research due to the urge to identify new therapeutic targets has led to a wealth of information on the retroviral replication cycle. However, while the late stages of the retrovirus life cycle, consisting of virus replication and egress, have been partly unraveled, the early steps remain largely enigmatic. These early steps consist of a long and perilous journey from the cell surface to the nucleus where the proviral DNA integrates into the host genome. Retroviral particles must bind specifically to their target cells, cross the plasma membrane, reverse-transcribe their RNA genome, while uncoating the cores, find their way to the nuclear membrane and penetrate into the nucleus to finally dock and integrate into the cellular genome. Along this journey, retroviruses hijack the cellular machinery, while at the same time counteracting cellular defenses. Elucidating these mechanisms and identifying which cellular factors are exploited by the retroviruses and which hinder their life cycle, will certainly lead to the discovery of new ways to inhibit viral replication and to improve retroviral vectors for gene transfer. Finally, as proven by many examples in the past, progresses in retrovirology will undoubtedly also provide some priceless insights into cell biology.
- 464Spear, P. G.; Eisenberg, R. J.; Cohen, G. H. Three Classes of Cell Surface Receptors for Alphaherpesvirus Entry. Virology 2000, 275, 1– 8, DOI: 10.1006/viro.2000.0529Google Scholar464Three classes of cell surface receptors for alphaherpesvirus entrySpear, Patricia G.; Eisenberg, Roselyn J.; Cohen, Gary H.Virology (2000), 275 (1), 1-8CODEN: VIRLAX; ISSN:0042-6822. (Academic Press)A review with 66 refs. (c) 2000 Academic Press.
- 465Bailey, C. J.; Crystal, R. G.; Leopold, P. L. Association of Adenovirus with the Microtubule Organizing Center. J. Virol. 2003, 77, 13275– 13287, DOI: 10.1128/JVI.77.24.13275-13287.2003Google Scholar465Association of adenovirus with the microtubule organizing centerBailey, Christopher J.; Crystal, Ronald G.; Leopold, Philip L.Journal of Virology (2003), 77 (24), 13275-13287CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an assocn. with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and assocs. with the microtubule organizing center (MTOC). To test this hypothesis, we established an exptl. system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymn. of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-assocd. Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymg. agent nocodazole, but not to the microfilament-depolymg. agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissocn. of Ad from microtubules likely requires nuclear factors.
- 466Kielian, M.; Rey, F. A. Virus Membrane-Fusion Proteins: More Than One Way to Make a Hairpin. Nat. Rev. Microbiol. 2006, 4, 67– 76, DOI: 10.1038/nrmicro1326Google Scholar466Virus membrane-fusion proteins: more than one way to make a hairpinKielian, Margaret; Rey, Felix A.Nature Reviews Microbiology (2006), 4 (1), 67-76CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Structure-function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. In both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. This review will focus in particular on the properties of the more recently described class II proteins.
- 467Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D. C. Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation. Biophys. J. 2013, 104, 2373– 2382, DOI: 10.1016/j.bpj.2013.04.005Google Scholar467Tracking Image Correlation: Combining Single-Particle Tracking and Image CorrelationDupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D. C.Biophysical Journal (2013), 104 (11), 2373-2382CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The interactions and coordination of biomols. are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quant. global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomols., usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization anal. along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle's position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addn., the cross-correlation anal. shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.
- 468Alenquer, M.; Vale-Costa, S.; Etibor, T. A.; Ferreira, F.; Sousa, A. L.; Amorim, M. J. Influenza A Virus Ribonucleoproteins Form Liquid Organelles at Endoplasmic Reticulum Exit Sites. Nat. Commun. 2019, 10, 1629, DOI: 10.1038/s41467-019-09549-4Google Scholar468Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sitesAlenquer Marta; Vale-Costa Silvia; Etibor Temitope Akhigbe; Ferreira Filipe; Sousa Ana Laura; Amorim Maria Joao; Sousa Ana LauraNature communications (2019), 10 (1), 1629 ISSN:.Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.
- 469Jo, S.; Kawaguchi, A.; Takizawa, N.; Morikawa, Y.; Momose, F.; Nagata, K. Involvement of Vesicular Trafficking System in Membrane Targeting of the Progeny Influenza Virus Genome. Microbes Infect. 2010, 12, 1079– 1084, DOI: 10.1016/j.micinf.2010.06.011Google Scholar469Involvement of vesicular trafficking system in membrane targeting of the progeny influenza virus genomeJo, Shuichi; Kawaguchi, Atsushi; Takizawa, Naoki; Morikawa, Yuko; Momose, Fumitaka; Nagata, KyosukeMicrobes and Infection (2010), 12 (12-13), 1079-1084CODEN: MCINFS; ISSN:1286-4579. (Elsevier Masson SAS)The genome of influenza type A virus consists of single-stranded RNAs of neg. polarity. Progeny viral RNA (vRNA) replicated in the nucleus is nuclear-exported, and finally transported to the budding site beneath the plasma membrane. However, the precise process of the membrane targeting of vRNA is unclear, although viral proteins and cytoskeleton are thought to play roles. Here, we have visualized the translocation process of progeny vRNA using fluorescence in situ hybridization method. Our results provide an evidence of the involvement of vesicular trafficking in membrane targeting of progeny vRNA independent of that of viral membrane proteins.
- 470Avilov, S. V.; Moisy, D.; Naffakh, N.; Cusack, S. Influenza A Virus Progeny vRNP Trafficking in Live Infected Cells Studied with the Virus-Encoded Fluorescently Tagged PB2 Protein. Vaccine 2012, 30, 7411– 7417, DOI: 10.1016/j.vaccine.2012.09.077Google Scholar470Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 proteinAvilov, Sergiy V.; Moisy, Dorothee; Naffakh, Nadia; Cusack, StephenVaccine (2012), 30 (51), 7411-7417CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, the authors have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells. Here, using this virus, the authors studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection. In agreement with recent reports, upon nuclear export, progeny vRNPs accumulate in the particles contg. Rab11, a multifunctional protein involved in vesicle trafficking which resides at recycling endosomes. Fluorescence resonance energy transfer microscopy indicated a distance <10 nm between PB2 and Rab11, suggesting that a direct interaction occurs. Single particle tracking anal. showed that most of the motions of vRNP-pos. particles in infected cells are slow, while rapid directional motions intermittently occur. Anal. focused on these intermittent motions indicated that depolymn. of either microtubules or actin filaments moderately reduced their occurrence, while disruption of both cytoskeleton components in combination suppressed the rapid motions entirely. Thus, the split-GFP based virus enabled the authors to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of vRNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.
- 471Amorim, M. J.; Bruce, E. A.; Read, E. K. C.; Foeglein, A.; Mahen, R.; Stuart, A. D.; Digard, P. A Rab11-and Microtubule-Dependent Mechanism for Cytoplasmic Transport of Influenza A Virus Viral RNA. J. Virol. 2011, 85, 4143– 4156, DOI: 10.1128/JVI.02606-10Google Scholar471A Rab11- and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNAAmorim, Maria Joao; Bruce, Emily A.; Read, Eliot K. C.; Foeglein, Agnes; Mahen, Robert; Stuart, Amanda D.; Digard, PaulJournal of Virology (2011), 85 (9), 4143-4156CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Copptn. expts. showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.
- 472Hendrix, J.; Baumgartel, V.; Schrimpf, W.; Ivanchenko, S.; Digman, M. A.; Gratton, E.; Krausslich, H. G.; Muller, B.; Lamb, D. C. Live-Cell Observation of Cytosolic HIV-1 Assembly onset Reveals RNA-Interacting Gag Oligomers. J. Cell Biol. 2015, 210, 629– 646, DOI: 10.1083/jcb.201504006Google Scholar472Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomersHendrix, Jelle; Baumgaertel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kraeusslich, Hans-Georg; Mueller, Barbara; Lamb, Don C.Journal of Cell Biology (2015), 210 (4), 629-646CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Redn. of mobility is assocd. with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was obsd. on the seconds timescale that oligomerized in a concn.-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.
- 473Jose, J.; Tang, J.; Taylor, A. B.; Baker, T. S.; Kuhn, R. J. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells. Viruses 2015, 7, 6182– 6199, DOI: 10.3390/v7122926Google Scholar473Fluorescent protein-tagged sindbis virus E2 glycoprotein allows single particle analysis of virus budding from live cellsJose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.Viruses (2015), 7 (12), 6182-6199CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examd. the virions by transmission electron cryo-microscopy and detd. the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, resp. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry anal., contributed to a 10-fold redn. in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking expts. We used the FP-tagged virus for high-resoln. live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close assocn. with filopodial extensions.
- 474Baumgartel, V.; Muller, B.; Lamb, D. C. Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly. Viruses 2012, 4, 777– 799, DOI: 10.3390/v4050777Google Scholar474Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assemblyBaumgartel Viola; Muller Barbara; Lamb Don CViruses (2012), 4 (5), 777-99 ISSN:.Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.
- 475Perlman, M.; Resh, M. D. Identification of an Intracellular Trafficking and Assembly Pathway for HIV-1 Gag. Traffic 2006, 7, 731– 745, DOI: 10.1111/j.1398-9219.2006.00428.xGoogle Scholar475Identification of an intracellular trafficking and assembly pathway for HIV-1 GagPerlman, Mira; Resh, Marilyn D.Traffic (Oxford, United Kingdom) (2006), 7 (6), 731-745CODEN: TRAFFA; ISSN:1398-9219. (Blackwell Publishing Ltd.)Retroviral Gag proteins are membrane-bound poly-proteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle prodn. is detd. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amts. of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag assocn. with MVB-like compartments regulates the site of HIV-1 budding and particle formation.
- 476Gunzenhauser, J.; Olivier, N.; Pengo, T.; Manley, S. Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions. Nano Lett. 2012, 12, 4705– 4710, DOI: 10.1021/nl3021076Google Scholar476Quantitative super-resolution imaging reveals protein stoichiometry and nanoscale morphology of assembling HIV-Gag virionsGunzenhauser, Julia; Olivier, Nicolas; Pengo, Thomas; Manley, SulianaNano Letters (2012), 12 (9), 4705-4710CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The HIV structural protein Gag assembles to form spherical particles of radius ∼70 nm. During the assembly process, the no. of Gag proteins increases over several orders of magnitude from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as std. fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, the authors demonstrate an approach using super-resoln. fluorescence imaging that permits quant. morphol. and mol. counting anal. over a wide range of protein cluster sizes. The authors applied this technique to the anal. of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions.
- 477Fogarty, K. H.; Chen, Y.; Grigsby, I. F.; Macdonald, P. J.; Smith, E. M.; Johnson, J. L.; Rawson, J. M.; Mansky, L. M.; Mueller, J. D. Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color z-Scan Fluorescence Fluctuation Spectroscopy. Biophys. J. 2011, 100, 1587– 1595, DOI: 10.1016/j.bpj.2011.02.008Google Scholar477Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation SpectroscopyFogarty, Keir H.; Chen, Yan; Grigsby, Iwen F.; MacDonald, Patrick J.; Smith, Elizabeth M.; Johnson, Jolene L.; Rawson, Jonathan M.; Mansky, Louis M.; Mueller, Joachim D.Biophysical Journal (2011), 100 (6), 1587-1595CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness anal. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation vol., cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addn. of a distinctly colored ref. protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concn.-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.
- 478Chen, Y.; Wu, B.; Musier-Forsyth, K.; Mansky, L. M.; Mueller, J. D. Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry. Biophys. J. 2009, 96, 1961– 1969, DOI: 10.1016/j.bpj.2008.10.067Google Scholar478Fluorescence fluctuation spectroscopy on viral-like particles reveals variable Gag stoichiometryChen, Yan; Wu, Bin; Musier-Forsyth, Karin; Mansky, Louis M.; Mueller, Joachim D.Biophysical Journal (2009), 96 (5), 1961-1969CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence fluctuation spectroscopy dets. the brightness, size, and concn. of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation vol. Brightness provides a measure of the no. of fluorescently labeled proteins within a complex and has been used previously to det. the stoichiometry of small oligomers in cells. We extend brightness anal. to large macromol. protein complexes contg. thousands of proteins and det. their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to det. the stoichiometry of HIV-1 Gag within the particles. Control expts. establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The expts. further show that the brightness scales linearly with the amt. of labeled Gag within the particle. Brightness anal. shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not const., but varies with the amt. of transfected DNA plasmid. We obsd. HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP prodn. This study shows that brightness anal. has the potential to become an important tool for investigating large mol. complexes by providing quant. information about their size and compn.
- 479Sardo, L.; Hatch, S. C.; Chen, J.; Nikolaitchik, O.; Burdick, R. C.; Chen, D.; Westlake, C. J.; Lockett, S.; Pathak, V. K.; Hu, W. S. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly. J. Virol. 2015, 89, 10832– 10840, DOI: 10.1128/JVI.01146-15Google Scholar479Dynamics of HIV-1 RNA near the plasma membrane during virus assemblySardo, Luca; Hatch, Steven C.; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C.; Chen, De; Westlake, Christopher J.; Lockett, Stephen; Pathak, Vinay K.; Hu, Wei-ShauJournal of Virology (2015), 89 (21), 10832-10840CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)To increase our understanding of the events that lead to HIV-1 genome packaging, we examd. the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we obsd. that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions.
- 480Rahman, S. A.; Koch, P.; Weichsel, J.; Godinez, W. J.; Schwarz, U.; Rohr, K.; Lamb, D. C.; Krausslich, H. G.; Muller, B. Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy. J. Virol. 2014, 88, 7904– 7914, DOI: 10.1128/JVI.00431-14Google Scholar480Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopyRahman Sheikh Abdul; Koch Peter; Krausslich Hans-Georg; Muller Barbara; Weichsel Julian; Schwarz Ulrich; Godinez William J; Rohr Karl; Lamb Don CJournal of virology (2014), 88 (14), 7904-14 ISSN:.Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.
- 481Baumgartel, V.; Ivanchenko, S.; Dupont, A.; Sergeev, M.; Wiseman, P. W.; Krausslich, H. G.; Brauchle, C.; Muller, B.; Lamb, D. C. Live-Cell Visualization of Dynamics of HIV Budding Site Interactions with an ESCRT Component. Nat. Cell Biol. 2011, 13, 469– 474, DOI: 10.1038/ncb2215Google Scholar481Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT componentBaumgartel Viola; Ivanchenko Sergey; Dupont Aurelie; Sergeev Mikhail; Wiseman Paul W; Krausslich Hans-Georg; Brauchle Christoph; Muller Barbara; Lamb Don CNature cell biology (2011), 13 (4), 469-74 ISSN:.HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.
- 482Dimitrov, D. S.; Willey, R. L.; Sato, H.; Chang, L. J.; Blumenthal, R.; Martin, M. A. Quantitation of Human Immunodeficiency Virus Type 1 Infection Kinetics. J. Virol. 1993, 67, 2182– 2190, DOI: 10.1128/JVI.67.4.2182-2190.1993Google Scholar482Quantitation of human immunodeficiency virus type 1 infection kineticsDimitrov, Dimiter S.; Willey, Ronald L.; Sato, Hironori; Chang, Lung Ji; Blumenthal, Robert; Martin, Malcolm A.Journal of Virology (1993), 67 (4), 2182-90CODEN: JOVIAM; ISSN:0022-538X.Tissue culture infections of CD4-pos. human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in 3 stages: (1) a period following the initiation of an infection during which no detectable virus is produced; (2) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (3) a final period when virus prodn. declines. Equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection were derived. The analyses indicated that the crit. parameter affecting the kinetics of HIV-1 infection is the infection rate const. k = lnn/ti, where n is the no. of infectious virions produced by one cell (about 102) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was the finding that the infectivity of HIV-1 during cell-to-cell transmission is 102 to 103 times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. It was also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small no. of infectious particles produced per cycle.
- 483Johnson, D. C.; Huber, M. T. Directed Egress of Animal Viruses Promotes Cell-to-Cell Spread. J. Virol. 2002, 76, 1– 8, DOI: 10.1128/JVI.76.1.1-8.2002Google Scholar483Directed egress of animal viruses promotes cell-to-cell spreadJohnson, David C.; Huber, Mary T.Journal of Virology (2002), 76 (1), 1-8CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A review focuses on direct cell-to-cell spread of animal viruses in solid tissues. Three interesting examples of how animal viruses have organized their egress strategies to promote cell-to-cell spread are described. These examples include alpha herpesviruses, HIV, and poxviruses. The alpha herpesviruses provide fascinating examples of viruses that replicate in polarized cells, epithelial cells, and neurons and imitate intracellular sorting pathways to direct nascent virions to cell junctions, promoting infection of adjacent epithelial cells and directed spread within the nervous system. HIV normally replicates in lymphocytes and macrophages, cells that are not usually considered polarized. Poxviruses can induce the formation of actin tails that launch virus particles from the cell surface on the tips of microvilli toward neighboring cells.
- 484Sattentau, Q. Avoiding the Void: Cell-to-Cell Spread of Human Viruses. Nat. Rev. Microbiol. 2008, 6, 815– 826, DOI: 10.1038/nrmicro1972Google Scholar484Avoiding the void: cell-to-cell spread of human virusesSattentau, QuentinNature Reviews Microbiology (2008), 6 (11), 815-826CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. The initial stages of animal virus infection are generally described as the binding of free virions to permissive target cells followed by entry and replication. Although this route of infection is undoubtedly important, many viruses that are pathogenic for humans, including HIV-1, herpes simplex virus and measles, can also move between cells without diffusing through the extracellular environment. Cell-to-cell spread not only facilitates rapid viral dissemination, but may also promote immune evasion and influence disease. The author discusses the various mechanisms by which viruses move directly between cells and the implications of this for viral dissemination and pathogenesis.
- 485Sherer, N. M.; Lehmann, M. J.; Jimenez-Soto, L. F.; Horensavitz, C.; Pypaert, M.; Mothes, W. Retroviruses Can Establish Filopodial Bridges for Efficient Cell-to-Cell Transmission. Nat. Cell Biol. 2007, 9, 310– 315, DOI: 10.1038/ncb1544Google Scholar485Retroviruses can establish filopodial bridges for efficient cell-to-cell transmissionSherer, Nathan M.; Lehmann, Maik J.; Jimenez-Soto, Luisa F.; Horensavitz, Christina; Pypaert, Marc; Mothes, WaltherNature Cell Biology (2007), 9 (3), 310-315CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)The spread of retroviruses between cells is estd. to be 2-3 orders of magnitude more efficient when cells can phys. interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virol. or infectious synapses. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong assocn. of the viral envelope glycoprotein (Env) in an infected cell with the receptor mols. in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.
- 486Mothes, W.; Sherer, N. M.; Jin, J.; Zhong, P. Virus Cell-to-Cell Transmission. J. Virol. 2010, 84, 8360– 8368, DOI: 10.1128/JVI.00443-10Google Scholar486Virus cell-to-cell transmissionMothes, Walther; Sherer, Nathan M.; Jin, Jing; Zhong, PengJournal of Virology (2010), 84 (17), 8360-8368CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A review. Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion mols., and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections.
- 487Eugenin, E. A.; Gaskill, P. J.; Berman, J. W. Tunneling Nanotubes (TNT) Are Induced by HIV-Infection of Macrophages: A Potential Mechanism for Intercellular HIV Trafficking. Cell. Immunol. 2009, 254, 142– 148, DOI: 10.1016/j.cellimm.2008.08.005Google Scholar487Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: A potential mechanism for intercellular HIV traffickingEugenin, E. A.; Gaskill, P. J.; Berman, J. W.Cellular Immunology (2009), 254 (2), 142-148CODEN: CLIMB8; ISSN:0008-8749. (Elsevier B.V.)Cell to cell communication is essential for the organization/coordination of multicellular systems and cellular development. Cellular communication is mediated by sol. factors, including growth factors, neurotransmitters, cytokines/chemokines, gap junctions, and the recently described tunneling nanotubes (TNT). TNT are long cytoplasmatic bridges that enable long range directed communication between cells. The proposed function for TNT is the cell-to-cell transfer of large cellular structures such as vesicles and organelles. We demonstrate that HIV-infection of human macrophages results in an increased no. of TNT, and show HIV particles within these structures. We propose that HIV "highjacks" TNT communication to spread HIV through an intercellular route between communicated cells, contributing to the pathogenesis of AIDS.
- 488Gousset, K.; Schiff, E.; Langevin, C.; Marijanovic, Z.; Caputo, A.; Browman, D. T.; Chenouard, N.; de Chaumont, F.; Martino, A.; Enninga, J. Prions Hijack Tunnelling Nanotubes for Intercellular Spread. Nat. Cell Biol. 2009, 11, 328– 336, DOI: 10.1038/ncb1841Google Scholar488Prions hijack tunnelling nanotubes for intercellular spreadGousset, Karine; Schiff, Edwin; Langevin, Christelle; Marijanovic, Zrinka; Caputo, Anna; Browman, Duncan T.; Chenouard, Nicolas; de Chaumont, Fabrice; Martino, Angelo; Enninga, Jost; Olivo-Marin, Jean-Christophe; Maennel, Daniela; Zurzolo, ChiaraNature Cell Biology (2009), 11 (3), 328-336CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)In variant Creutzfeldt-Jakob disease, prions (PrPSc) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunneling nanotubes (TNTs) have been identified between cells, both in vitro and in vivo, and may represent a conserved means of cell-to-cell communication. Here we show that TNTs allow transfer of exogenous and endogenous PrPSc between infected and naive neuronal CAD cells. Significantly, transfer of endogenous PrPSc aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrPSc spreading in neuronal cells. In addn., we detected the transfer of labeled PrPSc from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS. We therefore propose that TNTs are involved in the spreading of PrPSc within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.
- 489Sowinski, S.; Jolly, C.; Berninghausen, O.; Purbhoo, M. A.; Chauveau, A.; Köhler, K.; Oddos, S.; Eissmann, P.; Brodsky, F. M.; Hopkins, C. Membrane Nanotubes Physically Connect T Cells over Long Distances Presenting a Novel Route for HIV-1 Transmission. Nat. Cell Biol. 2008, 10, 211– 219, DOI: 10.1038/ncb1682Google Scholar489Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmissionSowinski, Stefanie; Jolly, Clare; Berninghausen, Otto; Purbhoo, Marco A.; Chauveau, Anne; Koehler, Karsten; Oddos, Stephane; Eissmann, Philipp; Brodsky, Frances M.; Hopkins, Colin; Oenfelt, Bjoern; Sattentau, Quentin; Davis, Daniel M.Nature Cell Biology (2008), 10 (2), 211-219CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Transmission of HIV-1 via intercellular connections has been estd. as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virol. synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.
- 490Kumar, A.; Kim, J. H.; Ranjan, P.; Metcalfe, M. G.; Cao, W.; Mishina, M.; Gangappa, S.; Guo, Z.; Boyden, E. S.; Zaki, S. Influenza Virus Exploits Tunneling Nanotubes for Cell-to-Cell Spread. Sci. Rep. 2017, 7, 40360, DOI: 10.1038/srep40360Google Scholar490Influenza virus exploits tunneling nanotubes for cell-to-cell spreadKumar, Amrita; Kim, Jin Hyang; Ranjan, Priya; Metcalfe, Maureen G.; Cao, Weiping; Mishina, Margarita; Gangappa, Shivaprakash; Guo, Zhu; Boyden, Edward S.; Zaki, Sherif; York, Ian; Garcia-Sastre, Adolfo; Shaw, Michael; Sambhara, SuryaprakashScientific Reports (2017), 7 (), 40360CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naive cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naive cell, resulting in productive viral replication in the naive cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.
- 491Nzounza, P.; Chazal, M.; Guedj, C.; Schmitt, A.; Masse, J. M.; Randriamampita, C.; Pique, C.; Ramirez, B. C. The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells. PLoS One 2012, 7, e30130 DOI: 10.1371/journal.pone.0030130Google Scholar491The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cellsNzounza, Patrycja; Chazal, Maxime; Guedj, Chloe; Schmitt, Alain; Masse, Jean-Marc; Randriamampita, Clotilde; Pique, Claudine; Ramirez, Bertha CeciliaPLoS One (2012), 7 (1), e30130CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Background: Cell-to-cell virus transmission of human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virol. synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunol. synapse (IS). We have previously identified the human homolog of the Drosophila Disks Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a neg. regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodol./Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quant. cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. Conclusion: Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.
- 492Igakura, T.; Stinchcombe, J. C.; Goon, P. K.; Taylor, G. P.; Weber, J. N.; Griffiths, G. M.; Tanaka, Y.; Osame, M.; Bangham, C. R. Spread of HTLV-I between Lymphocytes by Virus-Induced Polarization of the Cytoskeleton. Science 2003, 299, 1713– 1716, DOI: 10.1126/science.1080115Google Scholar492Spread of HTLV-I Between Lymphocytes by Virus-Induced Polarization of the CytoskeletonIgakura, Tadahiko; Stinchcombe, Jane C.; Goon, Peter K. C.; Taylor, Graham P.; Weber, Jonathan N.; Griffiths, Gillian M.; Tanaka, Yuetsu; Osame, Mitsuhiro; Bangham, Charles R. M.Science (Washington, DC, United States) (2003), 299 (5613), 1713-1716CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell contact is required for efficient transmission of human T cell leukemia virus-type 1 (HTLV-I) between cells and between individuals, because naturally infected lymphocytes produce virtually no cell-free infectious HTLV-I particles. However, the mechanism of cell-to-cell spread of HTLV-I is not understood. We show here that cell contact rapidly induces polarization of the cytoskeleton of the infected cell to the cell-cell junction. HTLV-I core (Gag protein) complexes and the HTLV-I genome accumulate at the cell-cell junction and are then transferred to the uninfected cell. Other lymphotropic viruses, such as HIV-1, may similarly subvert normal T cell physiol. to allow efficient propagation between cells.
- 493Barnard, A. L.; Igakura, T.; Tanaka, Y.; Taylor, G. P.; Bangham, C. R. Engagement of Specific T-Cell Surface Molecules Regulates Cytoskeletal Polarization in HTLV-1–Infected Lymphocytes. Blood 2005, 106, 988– 995, DOI: 10.1182/blood-2004-07-2850Google Scholar493Engagement of specific T-cell surface molecules regulates cytoskeletal polarization in HTLV-1-infected lymphocytesBarnard, Amanda L.; Igakura, Tadahiko; Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.Blood (2005), 106 (3), 988-995CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Cell-cell contact is required for efficient transmission of human T-lymphotropic virus type 1 (HTLV-1). An HTLV-1-infected cell polarizes its microtubule-organizing center (MTOC) toward the cell-cell junction; HTLV-1 core (Gag) complexes and the HTLV-1 genome accumulate at the point of contact and are then transferred to the uninfected cell. However, the mechanisms involved in this cytoskeletal polarization and transport of HTLV-1 complexes are unknown. Here, we tested the hypothesis that engagement of a specific T-cell surface ligand is synergistic with HTLV-1 infection in causing polarization of the MTOC to the cell contact region. We show that antibodies to intercellular adhesion mol.-1 (ICAM-1; CD54) caused MTOC polarization at a higher frequency in HTLV-1-infected cells. ICAM-1 is upregulated on HTLV-1-infected cells, and, in turn, ICAM-1 on the cell surface upregulates HTLV-1 gene expression. We propose that a pos. feedback loop involving ICAM-1 and HTLV-1 Tax protein facilitates the formation of the virol. synapse and contributes to the T-cell tropism of HTLV-1. In contrast, MTOC polarization induced in T cells by antibodies to CD3 or CD28 was significantly inhibited by HTLV-1 infection.
- 494Johnson, D. C.; Webb, M.; Wisner, T. W.; Brunetti, C. Herpes Simplex Virus gE/gI Sorts Nascent Virions to Epithelial Cell Junctions, Promoting Virus Spread. J. Virol. 2001, 75, 821– 833, DOI: 10.1128/JVI.75.2.821-833.2001Google Scholar494Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spreadJohnson, David C.; Webb, Mike; Wisner, Todd W.; Brunetti, CraigJournal of Virology (2001), 75 (2), 821-833CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. GE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, μ1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles. Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.
- 495Rustom, A.; Saffrich, R.; Markovic, I.; Walther, P.; Gerdes, H. H. Nanotubular Highways for Intercellular Organelle Transport. Science 2004, 303, 1007– 1010, DOI: 10.1126/science.1093133Google Scholar495Nanotubular Highways for Intercellular Organelle TransportRustom, Amin; Saffrich, Rainer; Markovic, Ivanka; Walther, Paul; Gerdes, Hans-HermannScience (Washington, DC, United States) (2004), 303 (5660), 1007-1010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chem. synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small mols. Accordingly, we propose a novel biol. principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.
- 496Davis, D. M.; Sowinski, S. Membrane Nanotubes: Dynamic Long-Distance Connections between Animal Cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 431– 436, DOI: 10.1038/nrm2399Google Scholar496Membrane nanotubes: dynamic long-distance connections between animal cellsDavis, Daniel M.; Sowinski, StefanieNature Reviews Molecular Cell Biology (2008), 9 (6), 431-436CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)Membrane nanotubes are thin extensions of the plasma membrane that connect cells transiently and might facilitate intercellular communication. Recent studies have revealed considerable heterogeneity in their structure, formation, mode of cargo transport and functional properties, depending on the cell types involved. Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.
- 497Efros, A. L.; Nesbitt, D. J. Origin and Control of Blinking in Quantum Dots. Nat. Nanotechnol. 2016, 11, 661– 671, DOI: 10.1038/nnano.2016.140Google Scholar497Origin and control of blinking in quantum dotsEfros, Alexander L.; Nesbitt, David J.Nature Nanotechnology (2016), 11 (8), 661-671CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)A review. Semiconductor nanocrystals offer an enormous diversity of potential device applications, based on their size-tunable luminescence, high optical stability and bottom-up chem. approaches to self-assembly. The promise of such applications can be seriously limited by luminescence intermittency in nanocrystal emission, i.e., blinking, arising from the escape of either 1 or both of the photoexcited carriers to the nanocrystal surface. In the 1st scenario, the remaining nanocrystal charge quenches luminescence via nonradiative Auger recombination, whereas for the other, the exciton probably is intercepted before thermalization and does not contribute to the luminescence. This summarizes the current understanding of the mechanisms responsible for nanocrystal blinking kinetics as well as core-shell engineering efforts to control such phenomena. Softening of the core-shell confinement potential strongly suppresses nonradiative Auger processes in charged nanocrystals, with successful nonblinking implementations demonstrated in CdSe-CdS core-thick-shell nanocrystals and their modifications.
- 498Marchuk, K.; Guo, Y.; Sun, W.; Vela, J.; Fang, N. High-Precision Tracking with Non-Blinking Quantum Dots Resolves Nanoscale Vertical Displacement. J. Am. Chem. Soc. 2012, 134, 6108– 6111, DOI: 10.1021/ja301332tGoogle Scholar498High-Precision Tracking with Non-blinking Quantum Dots Resolves Nanoscale Vertical DisplacementMarchuk, Kyle; Guo, Yijun; Sun, Wei; Vela, Javier; Fang, NingJournal of the American Chemical Society (2012), 134 (14), 6108-6111CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Novel nonblinking quantum dots (NBQDs) were utilized in three-dimensional super-localization, high-precision tracking applications under an automated scanning-angle total internal reflection fluorescence microscope (SA-TIRFM). NBQDs were randomly attached to stationary microtubules along the radial axis under gliding assay conditions. By automatically scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves were obtained. Fit with theor. decay functions, the abs. vertical positions were detd. with sub-10-nm localization precision. The emission intensity profile of the NBQDs attached to kinesin-propelled microtubules was used to resolve the self-rotation of gliding microtubules within a small vertical distance of ∼50 nm. The authors demonstrate the applicability of NBQDs in high-precision fluorescence imaging expts.
- 499Keller, A. M.; Ghosh, Y.; DeVore, M. S.; Phipps, M. E.; Stewart, M. H.; Wilson, B. S.; Lidke, D. S.; Hollingsworth, J. A.; Werner, J. H. 3-Dimensional Tracking of Non-blinking ’Giant’ Quantum Dots in Live Cells. Adv. Funct. Mater. 2014, 24, 4796– 4803, DOI: 10.1002/adfm.201400349Google Scholar4993-Dimensional Tracking of Non-blinking 'Giant' Quantum Dots in Live CellsKeller, Aaron M.; Ghosh, Yagnaseni; DeVore, Matthew S.; Phipps, Mary E.; Stewart, Michael H.; Wilson, Bridget S.; Lidke, Diane S.; Hollingsworth, Jennifer A.; Werner, James H.Advanced Functional Materials (2014), 24 (30), 4796-4803CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or 'blinking,' which causes ambiguity in particle trajectory anal. and limits tracking duration. Here, non-blinking 'giant' quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to com. available QDs. A time-gated photon-pair correlation anal. is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coeff. and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, esp. in cases where changes in cellular dynamics are occurring on the time scale of several minutes.
- 500Patterson, G.; Davidson, M.; Manley, S.; Lippincott-Schwartz, J. Superresolution Imaging Using Single-Molecule Localization. Annu. Rev. Phys. Chem. 2010, 61, 345– 367, DOI: 10.1146/annurev.physchem.012809.103444Google Scholar500Superresolution imaging using single-molecule localizationPatterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, JenniferAnnual Review of Physical Chemistry (2010), 61 (), 345-368CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. Superresoln. imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resoln. of light microscopy by over an order of magnitude (∼10-20-nm resoln.), allowing biol. processes to be described at the mol. scale. Here, we discuss a form of superresoln. microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent mols. In this single-mol.-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresoln. imaging: from three-dimensional, multicolor mol. localization to tracking of nanometric structures and mols. in living cells. Single-mol.-based superresoln. imaging thus offers exciting possibilities for obtaining mol.-scale information on biol. events occurring at variable timescales.
- 501Lee, A.; Tsekouras, K.; Calderon, C.; Bustamante, C.; Presse, S. Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis. Chem. Rev. 2017, 117, 7276– 7330, DOI: 10.1021/acs.chemrev.6b00729Google Scholar501Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data AnalysisLee, Antony; Tsekouras, Konstantinos; Calderon, Christopher; Bustamante, Carlos; Presse, SteveChemical Reviews (Washington, DC, United States) (2017), 117 (11), 7276-7330CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Super-resoln. microscopy provides direct insight into fundamental biol. processes occurring at length scales smaller than light's diffraction limit. The anal. of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data anal. methods starting from an overview of basic statistical techniques underlying the anal. of super-resoln. and, more broadly, imaging data. We subsequently break down the anal. of super-resoln. data into four problems: the localization problem, the counting problem, the linking problem, and what we've termed the interpretation problem.
- 502Huang, B.; Bates, M.; Zhuang, X. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem. 2009, 78, 993– 1016, DOI: 10.1146/annurev.biochem.77.061906.092014Google Scholar502Super-resolution fluorescence microscopyHuang, Bo; Bates, Mark; Zhuang, XiaoweiAnnual Review of Biochemistry (2009), 78 (), 993-1016CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Achieving a spatial resoln. that is not limited by the diffraction of light, recent developments of super-resoln. fluorescence microscopy techniques allow the observation of many biol. structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resoln. fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biol. structures and processes.
- 503Pan, W.; Dong, Z.; Li, F.; Meng, W.; Feng, L.; Niu, X.; Li, C.; Luo, Q.; Li, Z.; Sun, C. Visualizing Influenza Virus Infection in Living Mice. Nat. Commun. 2013, 4, 2369, DOI: 10.1038/ncomms3369Google Scholar503Visualizing influenza virus infection in living micePan Weiqi; Dong Zhenyuan; Li Feng; Meng Weixu; Feng Liqiang; Niu Xuefeng; Li Chufang; Luo Qinfang; Li Zhengfeng; Sun Caijun; Chen LingNature communications (2013), 4 (), 2369 ISSN:.Preventing and treating influenza virus infection remain a challenge because of incomplete understanding of the host-pathogen interactions, limited therapeutics and lack of a universal vaccine. So far, methods for monitoring the course of infection with influenza virus in real time in living animals are lacking. Here we report the visualization of influenza viral infection in living mice using an engineered replication-competent influenza A virus carrying luciferase reporter gene. After intranasal inoculation, bioluminescence can be detected in the chest and nasopharyngeal passage of living mice. The intensity of bioluminescence in the chest correlates with the dosage of infection and the viral load in the lung. Bioluminescence in the chest of infected mice diminishes on antiviral treatment. This work provides a novel approach that enables real-time study of influenza virus infection and effects of antiviral therapeutics in living animals.
- 504Pan, H.; Zhang, P.; Gao, D.; Zhang, Y.; Li, P.; Liu, L.; Wang, C.; Wang, H.; Ma, Y.; Cai, L. Noninvasive Visualization of Respiratory Viral Infection Using Bioorthogonal Conjugated Near-Infrared-Emitting Quantum Dots. ACS Nano 2014, 8, 5468– 5477, DOI: 10.1021/nn501028bGoogle Scholar504Noninvasive Visualization of Respiratory Viral Infection Using Bioorthogonal Conjugated Near-Infrared-Emitting Quantum DotsPan, Hong; Zhang, Pengfei; Gao, Duyang; Zhang, Yijuan; Li, Ping; Liu, Lanlan; Wang, Ce; Wang, Hanzhong; Ma, Yifan; Cai, LintaoACS Nano (2014), 8 (6), 5468-5477CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Highly pathogenic avian influenza A viruses are emerging pandemic threats in human beings. Monitoring the in vivo dynamics of avian influenza viruses is extremely important for understanding viral pathogenesis and developing antiviral drugs. Although a no. of technologies have been applied for tracking viral infection in vivo, most of them are laborious with unsatisfactory detection sensitivity. Herein the authors labeled avian influenza H5N1 pseudotype virus (H5N1p) with near-IR (NIR)-emitting QDs by bioorthogonal chem. The conjugation of QDs onto H5N1p was highly efficient with superior stability both in vitro and in vivo. Furthermore, QD-labeled H5N1p (QD-H5N1p) demonstrated bright and sustained fluorescent signals in mouse lung tissues, allowing the authors to visualize respiratory viral infection in a noninvasive and real-time manner. The fluorescence signals of QD-H5N1p in lung were correlated with the severity of virus infection and significantly attenuated by antiviral agents, such as oseltamivir carboxylate and mouse antiserum against H5N1p. The biodistribution of QD-H5N1p in lungs and other organs could be easily quantified by measuring fluorescent signals and cadmium concn. of virus-conjugated QDs in tissues. Hence, virus labeling with NIR QDs provides a simple, reliable, and quant. strategy for tracking respiratory viral infection and for antiviral drug screening.
- 505Zong, W.; Wu, R.; Li, M.; Hu, Y.; Li, Y.; Li, J.; Rong, H.; Wu, H.; Xu, Y.; Lu, Y. Fast High-Resolution Miniature Two-Photon Microscopy for Brain Imaging in Freely Behaving Mice. Nat. Methods 2017, 14, 713– 719, DOI: 10.1038/nmeth.4305Google Scholar505Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving miceZong, Weijian; Wu, Runlong; Li, Mingli; Hu, Yanhui; Li, Yijun; Li, Jinghang; Rong, Hao; Wu, Haitao; Xu, Yangyang; Lu, Yang; Jia, Hongbo; Fan, Ming; Zhou, Zhuan; Zhang, Yunfeng; Wang, Aimin; Chen, Liangyi; Cheng, HepingNature Methods (2017), 14 (7), 713-719CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-detd. behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resoln., miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resoln. (0.64 microm laterally and 3.35 microm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.
- 506Wang, K.; Sun, W.; Richie, C. T.; Harvey, B. K.; Betzig, E.; Ji, N. Direct Wavefront Sensing for High-Resolution in Vivo Imaging in Scattering Tissue. Nat. Commun. 2015, 6, 7276, DOI: 10.1038/ncomms8276Google Scholar506Direct wavefront sensing for high-resolution in vivo imaging in scattering tissueWang, Kai; Sun, Wenzhi; Richie, Christopher T.; Harvey, Brandon K.; Betzig, Eric; Ji, NaNature Communications (2015), 6 (), 7276CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-IR guide stars. The method enables in vivo two-photon morphol. and functional imaging down to 700 μm inside the mouse brain.
- 507Hong, G.; Antaris, A. L.; Dai, H. Near-Infrared Fluorophores for Biomedical Imaging. Nat. Biomed. Eng. 2017, 1, 0010 DOI: 10.1038/s41551-016-0010Google Scholar507Near-infrared fluorophores for biomedical imagingHong, Guosong; Antaris, Alexander L.; Dai, HongjieNature Biomedical Engineering (2017), 1 (1), 0010CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)A review. In this Review, we cover recent progress made on NIR fluorescence imaging in both the 700-900 nm NIR-I and the 1,000-1,700 nm NIR-II windows by highlighting an increasingly developing palette of biocompatible NIR fluorophores that span the entire NIR window and include inorg. nanoparticles, org. macromols. and small mols. with tunable emission wavelengths. Together with advances in imaging instrumentation allowing for the efficient detection of long-wavelength NIR photons, recently developed NIR fluorophores have fuelled biomedical imaging from contrast-enhanced imaging of anatomical structures and mol. imaging of specific biomarkers to functional imaging of physiol. activities, both for preclin. animal studies and clin. diagnostics and interventions.
- 508Ding, F.; Zhan, Y. B.; Lu, X. J.; Sun, Y. Recent Advances in Near-Infrared II Fluorophores for Multifunctional Biomedical Imaging. Chem. Sci. 2018, 9, 4370– 4380, DOI: 10.1039/C8SC01153BGoogle Scholar508Recent advances in near-infrared II fluorophores for multifunctional biomedical imagingDing, Feng; Zhan, Yibei; Lu, Xiaoju; Sun, YaoChemical Science (2018), 9 (19), 4370-4380CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. In recent years, owing to unsatisfactory clin. imaging clarity and depths in the living body for early diagnosis and prognosis, novel imaging modalities with high bioimaging performance have been actively explored. The remarkable headway made in the second near-IR region (NIR-II, 1000-1700 nm) has promoted the development of biomedical imaging significantly. NIR-II fluorescence imaging possesses a no. of merits which prevail over the traditional and NIR-I (400-900 nm) imaging modalities in fundamental research, such as reduced photon scattering, as well as auto-fluorescence and improved penetration depth. Functional probes for instant and precise feedback of in vivo information are at the core of this modality for superb imaging. Herein, we review the recently developed fluorophores including carbon nanotubes, org. small mols., quantum dots, conjugated polymers and rare-earth-doped materials to present superior and multifunctionality of biomedical imaging in the NIR-II regions (1000-1700 nm).
- 509Smith, A. M.; Mancini, M. C.; Nie, S. Bioimaging: Second Window for in Vivo Imaging. Nat. Nanotechnol. 2009, 4, 710, DOI: 10.1038/nnano.2009.326Google Scholar509Second window for in vivo imagingSmith, Andrew M.; Mancini, Michael C.; Nie, ShumingNature Nanotechnology (2009), 4 (11), 710-711CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Enhanced fluorescence from carbon nanotubes and advances in near-IR cameras have opened up a new wavelength window for small animal imaging.
- 510Zhang, J. J.; Lin, Y.; Zhou, H.; He, H.; Ma, J. J.; Luo, M. Y.; Zhang, Z. L.; Pang, D. W. Cell Membrane-Camouflaged NIR II Fluorescent Ag2Te Quantum Dots-Based Nanobioprobes for Enhanced in Vivo Homotypic Tumor Imaging. Adv. Healthcare Mater. 2019, 8, e1900341 DOI: 10.1002/adhm.201900341Google ScholarThere is no corresponding record for this reference.
- 511Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared Region. Angew. Chem., Int. Ed. 2012, 51, 9818– 9821, DOI: 10.1002/anie.201206059Google Scholar511In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared RegionHong, Guosong; Robinson, Joshua T.; Zhang, Yejun; Diao, Shuo; Antaris, Alexander L.; Wang, Qiangbin; Dai, HongjieAngewandte Chemie, International Edition (2012), 51 (39), 9818-9821, S9818/1-S9818/11CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors have developed biocompatible, heavy-metal free 6PEG-Ag2S QDs as an imaging contrast agent that are brightly fluorescent in the NIR-II window. Imaging with these NIR-II QDs afforded deep inner organ registration, dynamic tumor contrast, and fast tumor detection. The in vivo pharmacokinetics of the QDs was studied, suggesting an unprecedented degree of accumulation of 6PEG-Ag2S QDs in the tumor(> 10% ID/g) through the EPR effect. The short-term excretion profile of the 6PEG-Ag2S QDs suggested biliary clearance as the main clearance pathway. Further studies of genotoxicity and reproductive toxicity will be used to evaluate the potential of this new type of NIR-II fluorophores for pre-clin. use.
- 512Jiang, P.; Zhu, C. N.; Zhang, Z. L.; Tian, Z. Q.; Pang, D. W. Water-Soluble Ag2S Quantum Dots for Near-Infrared Fluorescence Imaging in Vivo. Biomaterials 2012, 33, 5130– 5135, DOI: 10.1016/j.biomaterials.2012.03.059Google Scholar512Water-soluble Ag2S quantum dots for near-infrared fluorescence imaging in vivoJiang, Peng; Zhu, Chun-Nan; Zhang, Zhi-Ling; Tian, Zhi-Quan; Pang, Dai-WenBiomaterials (2012), 33 (20), 5130-5135CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)A one-step method for synthesizing water-sol. Ag2S quantum dots terminated with carboxylic acid group has been reported. The crystal structure and surface of the prepd. Ag2S quantum dots were characterized. The prepd. Ag2S quantum dots exhibited bright photoluminescence and excellent photostabilities. The photoluminescence emissions could be tuned from visible region to near-IR (NIR) region (from 510 nm to 1221 nm). Ultra-small sized Ag2S nanoclusters were synthesized with high initial monomer concn. in the current system. The in vivo imaging expts. of nude mice showed that the NIR photoluminescence of the prepd. Ag2S quantum dots could penetrate the body of mice. Compared to the conventional NIR quantum dots, the Ag2S quantum dots don't contain toxic elements to body (such as Cd and Pb), thus, the prepd. Ag2S quantum dots could serve as excellent NIR optical imaging probes and would open the opportunity to study nanodiagnostics and imaging in vivo.
Cited By
Click to copy section linkSection link copied!
Citation Statements
beta
Smart citations by scite.ai include citation statements extracted from the full text of the citing article. The number of the statements may be higher than the number of citations provided by ACS Publications if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
Explore this article's citation statements on scite.ai
This article is cited by 160 publications.
- Jing Li, Jiao Hu, An-An Liu, Cui Liu, Dai-Wen Pang. Quantum Dots for Chemical Metrology. Analytical Chemistry 2025, 97
(13)
, 6891-6910. https://doi.org/10.1021/acs.analchem.4c06794
- Yusuke Sato, Yusaku Hatanaka, Yoshitaka Sato, Kota Matsumoto, Shion Osana, Ryoichi Nagatomi, Seiichi Nishizawa. Viral Membrane-Targeting Amphipathic Helical Peptide-Based Fluorogenic Probes for the Analysis of Infectious Titers of Enveloped Viruses. Analytical Chemistry 2025, 97
(10)
, 5480-5487. https://doi.org/10.1021/acs.analchem.4c04852
- Meng-Die Zhu, Xue-Hui Shi, Hui-Ping Wen, Li-Ming Chen, Dan-Dan Fu, Lei Du, Jing Li, Qian-Qian Wan, Zhi-Gang Wang, Chuanming Yu, Dai-Wen Pang, Shu-Lin Liu. Rapid Deployment of Antiviral Drugs Using Single-Virus Tracking and Machine Learning. ACS Nano 2024, 18
(52)
, 35256-35268. https://doi.org/10.1021/acsnano.4c10136
- Nathan J. Brooks, Chih-Chen Liu, Yan-Hsien Chen, Chia-Lung Hsieh. Point Spread Function Engineering for Spiral Phase Interferometric Scattering Microscopy Enables Robust 3D Single-Particle Tracking and Characterization. ACS Photonics 2024, 11
(12)
, 5239-5250. https://doi.org/10.1021/acsphotonics.4c01481
- Nicolas Mateos, Enric Gutierrez-Martinez, Jessica Angulo-Capel, Irene Carlon-Andres, Sergi Padilla-Parra, Maria F. Garcia-Parajo, Juan A. Torreno-Pina. Early Steps of Individual Multireceptor Viral Interactions Dissected by High-Density, Multicolor Quantum Dot Mapping in Living Cells. ACS Nano 2024, 18
(42)
, 28881-28893. https://doi.org/10.1021/acsnano.4c09085
- Dan-Dan Fu, Li-Juan Zhang, Bo Tang, Lei Du, Jing Li, Jian Ao, Zhi-Ling Zhang, Zhi-Gang Wang, Shu-Lin Liu, Dai-Wen Pang. Quantitatively Dissecting Triple Roles of Dynactin in Dynein-Driven Transport of Influenza Virus by Quantum Dot-Based Single-Virus Tracking. ACS Nano 2024, 18
(37)
, 25893-25905. https://doi.org/10.1021/acsnano.4c10564
- Lei Du, Yi-Ning Hou, Dan-Dan Fu, Jing Li, Jian Ao, Ai-Xin Ma, Qian-Qian Wan, Zhi-Gang Wang, Shu-Lin Liu, Li-Juan Zhang, Dai-Wen Pang. Revealing Different Pathways for Influenza A Virus To Reach Microtubules after Endocytosis by Quantum Dot-Based Single-Virus Tracking. ACS Nano 2024, 18
(34)
, 23090-23103. https://doi.org/10.1021/acsnano.4c05261
- Shannan-Leigh Macleod, Elana H. Super, Lauren J. Batt, Eleanor Yates, Samuel T. Jones. Plate-Based High-Throughput Fluorescence Assay for Assessing Enveloped Virus Integrity. Biomacromolecules 2024, 25
(8)
, 4925-4933. https://doi.org/10.1021/acs.biomac.4c00358
- Leping Lin, An-An Liu, Wei Zhao, Yin Yang, Dong-Liang Zhu, Bo-Ran Dong, Fei Ding, Di Ning, Xiaobo Zhu, Dingbin Liu, Dai-Wen Pang. Multihierarchical Regulation To Achieve Quantum Dot Nanospheres with a Photoluminescence Quantum Yield Close to 100%. Journal of the American Chemical Society 2024, 146
(31)
, 21348-21356. https://doi.org/10.1021/jacs.4c03308
- Yuxin Lin, Jack Exell, Haoting Lin, Chen Zhang, Kevin D. Welsher. Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions. The Journal of Physical Chemistry B 2024, 128
(23)
, 5590-5600. https://doi.org/10.1021/acs.jpcb.4c01710
- Yibin Zhu, Lei Ji, Chenying Li, Chunhuan Zhang, Jiatao Zhang. Fluorescence Enhancement of CdS:Ag Quantum Dots Co-Assembled with Au Nanoparticles in a Hollow Nanosphere Form. Langmuir 2024, 40
(22)
, 11642-11649. https://doi.org/10.1021/acs.langmuir.4c00918
- Zhi-Gang Wang, Yusi Hu, Hao-Yang Liu, Hai-Yan Wen, Bao-Ping Qi, Shu-Lin Liu. Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells. Analytical Chemistry 2024, 96
(18)
, 7231-7239. https://doi.org/10.1021/acs.analchem.4c00805
- Ai-Xin Ma, Cong Yu, Ming-Yu Zhang, Jian Ao, Hao-Yang Liu, Meng-Qian Zhang, Qian-Qian Sun, Dan-Dan Fu, Lei Du, Jing Li, Shu-Lin Liu, Zhi-Gang Wang, Dai-Wen Pang. One-Step Dual-Color Labeling of Viral Envelope and Intraviral Genome with Quantum Dots Harnessing Virus Infection. Nano Letters 2024, 24
(8)
, 2544-2552. https://doi.org/10.1021/acs.nanolett.3c04600
- Jian Ao, Ai-Xin Ma, Jing Li, Chun-Yu Wang, Dan-Dan Fu, Lei Du, Cong Yu, Shu-Lin Liu, Zhi-Gang Wang, Dai-Wen Pang. Real-Time Dissection of the Exosome Pathway for Influenza Virus Infection. ACS Nano 2024, 18
(5)
, 4507-4519. https://doi.org/10.1021/acsnano.3c11309
- Yan Zhang, Ning Xu, Chunyu Yan, Xuelian Zhou, Qinglong Qiao, Lu Miao, Zhaochao Xu. Live-Cell Imaging to Resolve Salt-Induced Liquid–Liquid Phase Separation of FUS Protein by Dye Self-Labeling. Chemical & Biomedical Imaging 2024, 2
(1)
, 70-80. https://doi.org/10.1021/cbmi.3c00094
- Marvin Jungblut, Simone Backes, Marcel Streit, Georg Gasteiger, Sören Doose, Markus Sauer, Gerti Beliu. Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection. ACS Nano 2023, 17
(21)
, 21822-21828. https://doi.org/10.1021/acsnano.3c07767
- Yi-Fan Tian, Yu-Peng Zhang, Qiu-Mei Wu, Dai-Wen Pang, Shu-Lin Liu, Zhi-Gang Wang. Real-Time Imaging of Single Viral mRNA Translation in Live Cells Using CRISPR/dCas13. Analytical Chemistry 2023, 95
(44)
, 16298-16304. https://doi.org/10.1021/acs.analchem.3c03365
- Florian Semmer, Marie-Charlotte Emperauger, Colin Lopez, Christine Bogicevic, François Treussart, Karen Perronet, François Marquier. 3D Real-Time Two-Photon Microscopy Device for Single-Particle Holographic Tracking (3D-Red Shot). ACS Photonics 2023, 10
(9)
, 3426-3434. https://doi.org/10.1021/acsphotonics.3c00949
- Lulu Huang, Xiuhai Mao, Jie Li, Qian Li, Jianlei Shen, Mengmeng Liu, Chunhai Fan, Yang Tian. Nanoparticle Spikes Enhance Cellular Uptake via Regulating Myosin IIA Recruitment. ACS Nano 2023, 17
(10)
, 9155-9166. https://doi.org/10.1021/acsnano.2c12660
- Weiqing Yang, Zhihong Wei, Yan Nie, Yuxi Tian. Optical Detection and Imaging of Nonfluorescent Matter at the Single-Molecule/Particle Level. The Journal of Physical Chemistry Letters 2022, 13
(41)
, 9618-9631. https://doi.org/10.1021/acs.jpclett.2c02228
- José A. Epstein, Guy Z. Ramon. Connecting the Non-Brownian Dots: Increasing Near-Neighbor Particle-Tracking Efficiency by Coordinate System Manipulation. Langmuir 2022, 38
(35)
, 10729-10735. https://doi.org/10.1021/acs.langmuir.2c00584
- Meng-Qian Zhang, Zhi-Gang Wang, Dan-Dan Fu, Ju-Mei Zhang, Hao-Yang Liu, Shu-Lin Liu, Dai-Wen Pang. Quantum Dots Tracking Endocytosis and Transport of Proteins Displayed by Mammalian Cells. Analytical Chemistry 2022, 94
(21)
, 7567-7575. https://doi.org/10.1021/acs.analchem.2c00411
- Zhi-Gang Wang, Shu-Lin Liu, Dai-Wen Pang. Quantum Dots: A Promising Fluorescent Label for Probing Virus Trafficking. Accounts of Chemical Research 2021, 54
(14)
, 2991-3002. https://doi.org/10.1021/acs.accounts.1c00276
- Li Xia, Li-Juan Zhang, Hong-Wu Tang, Dai-Wen Pang. Revealing Microtubule-Dependent Slow-Directed Motility by Single-Particle Tracking. Analytical Chemistry 2021, 93
(12)
, 5211-5217. https://doi.org/10.1021/acs.analchem.0c05377
- Meng-Ni Bao, Li-Juan Zhang, Bo Tang, Dan-Dan Fu, Jing Li, Lei Du, Yi-Ning Hou, Zhi-Ling Zhang, Hong-Wu Tang, Dai-Wen Pang. Influenza A Viruses Enter Host Cells via Extracellular Ca2+ Influx-Involved Clathrin-Mediated Endocytosis. ACS Applied Bio Materials 2021, 4
(3)
, 2044-2051. https://doi.org/10.1021/acsabm.0c00968
- Zhi-Gang Wang, Lei Wang, Don C. Lamb, Hua-Jie Chen, Yusi Hu, Hong-Peng Wang, Dai-Wen Pang, Shu-Lin Liu. Real-Time Dissecting the Dynamics of Drug Transportation in the Live Brain. Nano Letters 2021, 21
(1)
, 642-650. https://doi.org/10.1021/acs.nanolett.0c04216
- Ji Liu, Zongqiang Cui. Fluorescent Labeling of Proteins of Interest in Live Cells: Beyond Fluorescent Proteins. Bioconjugate Chemistry 2020, 31
(6)
, 1587-1595. https://doi.org/10.1021/acs.bioconjchem.0c00181
- Aditya Bansal, Shalini Sharma, Manasa Kethamreddy, Mukesh K. Pandey. PET imaging of AAV9 and AAVBR1 trafficking in normal mice. Scientific Reports 2025, 15
(1)
https://doi.org/10.1038/s41598-025-86815-0
- Mingzhi Li, Li Pan, Caoyuan Ma, Hongxia Wu, Guangtao Xiang, Lian-Feng Li, Tao Wang, Rui Luo, Yongfeng Li, Di Liu, Huanjie Zhai, Moon Assad, Xin Song, Yanjin Wang, Franck Gallardo, Hua-Ji Qiu, Yuan Sun. Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells. Veterinary Microbiology 2025, 304 , 110503. https://doi.org/10.1016/j.vetmic.2025.110503
- Anjana Singh, Yasheshwar, Naveen K. Kaushik, Deepak Kala, Rupak Nagraik, Shagun Gupta, Ankur Kaushal, Yashika Walia, Sunny Dhir, Md Salik Noorani. Conventional and cutting-edge advances in plant virus detection: emerging trends and techniques. 3 Biotech 2025, 15
(4)
https://doi.org/10.1007/s13205-025-04253-1
- An-An Liu, Ran Cui, Xia Zong, Jianhong Jia, Yusi Hu, Jing-Ya Zhao, Dai-Wen Pang. Live-cell synthesis of biocompatible quantum dots. Nature Protocols 2025, 120 https://doi.org/10.1038/s41596-024-01133-5
- Yiqing Lu, Parinaz Jabbari, Anton Mukhamedshin, Andrei V. Zvyagin. Fluorescence lifetime imaging in drug delivery research. Advanced Drug Delivery Reviews 2025, 218 , 115521. https://doi.org/10.1016/j.addr.2025.115521
- Hana M. Dobrovolny. How do viruses get around? A review of mathematical modeling of in-host viral transmission. Virology 2025, 604 , 110444. https://doi.org/10.1016/j.virol.2025.110444
- Shujun Liu, Wenfeng Xu, Pengfei Jin, Li-Li Huang. Real-time monitoring of virus infection dynamics in established infection models for mechanism analysis. Chinese Chemical Letters 2025, 33 , 111076. https://doi.org/10.1016/j.cclet.2025.111076
- Fayu Yang, Saba Aliyari, Zixiang Zhu, Haixue Zheng, Genhong Cheng, Shilei Zhang. CRISPR-Cas: a game-changer in vaccine development and the fight against viral infections. Trends in Microbiology 2025, 22 https://doi.org/10.1016/j.tim.2025.02.006
- Prashant Hitaishi, Akash Anandrao Parab, Rohit Verma, Parul Khurana, Sheenam Thatai. Spectroscopic Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2). Coronaviruses 2025, 6
(2)
https://doi.org/10.2174/0126667975284619240222051806
- Ping Ping Hu, Lin Ling Zheng, Lei Zhan, Cheng Zhi Huang. Imaging of Viral Genomic RNA Replication with Nanoprobes. 2025, 145-153. https://doi.org/10.1007/978-1-0716-4248-1_12
- Asuka Nanbo, Miako Sakaguchi, Wakako Furuyama. Visualizing the Internalization of Marburg Viruslike Particles into Living Cells. 2025, 75-90. https://doi.org/10.1007/978-1-0716-4256-6_6
- Jiaxin Liu, Liqun Wang, Xinyue Zhang, Shaowen Wang, Qiwei Qin. Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process. Virulence 2024, 15
(1)
https://doi.org/10.1080/21505594.2023.2301244
- Yi-Ning Hou, Li-Juan Zhang, Lei Du, Dan-Dan Fu, Jing Li, Liu Liu, Peng-Fei Xu, Ya-Wen Zheng, Dai-Wen Pang, Hong-Wu Tang. Analyzing the factors affecting virus invasion by quantitative single-particle analysis. Virulence 2024, 15
(1)
https://doi.org/10.1080/21505594.2024.2367671
- Cong Yu, Hua-Jie Chen, Hao-Yang Liu, Di Ning, Lei Wang, Xue-Hui Shi, Zhi-Gang Wang, Dai-Wen Pang, Shu-Lin Liu. Dual-lock-and-key virus-mimicking nanoprobes for ultra-high accurate and sensitive imaging of viral infections in vivo. Nano Today 2024, 59 , 102527. https://doi.org/10.1016/j.nantod.2024.102527
- Enbo Zhu, Yan‐Ruide Li, Samuel Margolis, Jing Wang, Kaidong Wang, Yaran Zhang, Shaolei Wang, Jongchan Park, Charlie Zheng, Lili Yang, Alison Chu, Yuhua Zhang, Liang Gao, Tzung K. Hsiai. Frontiers in artificial intelligence‐directed light‐sheet microscopy for uncovering biological phenomena and multiorgan imaging. VIEW 2024, 5
(5)
https://doi.org/10.1002/VIW.20230087
- Theodhora Ziu, Ezgi Sambur, Zsolt Ruzsics, Hartmut Hengel, Reingard Grabherr, Siegfried Höfinger, Hanna Harant. In Vitro Profiling of the Antiviral Peptide TAT-I24. International Journal of Molecular Sciences 2024, 25
(19)
, 10463. https://doi.org/10.3390/ijms251910463
- Sobhana A. Sripada, Mahshid Hosseini, Srivatsan Ramesh, Junhyeong Wang, Kimberly Ritola, Stefano Menegatti, Michael A. Daniele. Advances and opportunities in process analytical technologies for viral vector manufacturing. Biotechnology Advances 2024, 74 , 108391. https://doi.org/10.1016/j.biotechadv.2024.108391
- Kaito Takanami, Daisuke Taniguchi, Masafumi Kuroda, Sawako Enoki, Yasushi Okada, Yoshiyuki Kabashima. Detection of diffusion anisotropy from an individual short particle trajectory. Physical Review Research 2024, 6
(3)
https://doi.org/10.1103/PhysRevResearch.6.033272
- Jiarong Cai, Wei Zhao, Wenhui Yan, Xinyu Wang, Yuning Zhang, Dai-Wen Pang. Circularly polarized luminescence inorganic quantum dots: synthesis strategies and luminescence mechanism. SCIENTIA SINICA Chimica 2024, 54
(8)
, 1165-1177. https://doi.org/10.1360/SSC-2024-0050
- Razvan Bocu. Dynamic Monitoring of Time-Dependent Evolution of Biomolecules Using Quantum Dots-Based Biosensors Assemblies. Biosensors 2024, 14
(8)
, 380. https://doi.org/10.3390/bios14080380
- Yu-Peng Zhang, Jin-Wei Bu, Ru-Xin Shu, Shu-Lin Liu. Advances in rapid point-of-care virus testing. The Analyst 2024, 149
(9)
, 2507-2525. https://doi.org/10.1039/D4AN00238E
- Judith A. Müller, Nathalie Schäffler, Thomas Kellerer, Gerlinde Schwake, Thomas S. Ligon, Joachim O. Rädler. Kinetics of RNA-LNP delivery and protein expression. European Journal of Pharmaceutics and Biopharmaceutics 2024, 197 , 114222. https://doi.org/10.1016/j.ejpb.2024.114222
- Yu Gu, Pierre‐Yves Lozach. Illuminating bunyavirus entry into host cells with fluorescence. Molecular Microbiology 2024, 121
(4)
, 671-678. https://doi.org/10.1111/mmi.15165
- Xiao Luo, Jie Zhang, Handong Tan, Jiahao Jiang, Junda Li, Weijia Wen. Real-Time 3D Tracking of Multi-Particle in the Wide-Field Illumination Based on Deep Learning. Sensors 2024, 24
(8)
, 2583. https://doi.org/10.3390/s24082583
- Cornelia Holler, Richard William Taylor, Alexandra Schambony, Leonhard Möckl, Vahid Sandoghdar. A paintbrush for delivery of nanoparticles and molecules to live cells with precise spatiotemporal control. Nature Methods 2024, 21
(3)
, 512-520. https://doi.org/10.1038/s41592-024-02177-x
- YingQi LIANG, GuoBin MAO, JunBiao DAI, YingXin MA. Single virus tracking. SCIENTIA SINICA Vitae 2024, 54
(3)
, 396-408. https://doi.org/10.1360/SSV-2023-0070
- Hao-Yang Liu, Yusi Hu, Cong Yu, Zhi-Gang Wang, Shu-Lin Liu, Dai-Wen Pang. Quantitative single-virus tracking for revealing the dynamics of SARS-CoV-2 fusion with plasma membrane. Science Bulletin 2024, 69
(4)
, 502-511. https://doi.org/10.1016/j.scib.2023.11.020
- B. Megala Devi, Samyuktha Guruprasath, Pooraniammal Balu, Anirudha Chattopadhyay, Siva Sudha Thilagar, Kanaga Vijayan Dhanabalan, Manoj Choudhary, Swarnalatha Moparthi, A. Abdul Kader Jailani. Dissecting Diagnostic and Management Strategies for Plant Viral Diseases: What Next?. Agriculture 2024, 14
(2)
, 284. https://doi.org/10.3390/agriculture14020284
- Lulu Jin, Zhengwei Mao. Living virus‐based nanohybrids for biomedical applications. WIREs Nanomedicine and Nanobiotechnology 2024, 16
(1)
https://doi.org/10.1002/wnan.1923
- Yu Gu, Jana Koch, Céline Garnier, Alexandra Erny, Pierre-Yves Lozach. Making Rift Valley Fever Viral Particles Fluorescent. 2024, 165-188. https://doi.org/10.1007/978-1-0716-3926-9_12
- Yongge Li, Kheder Suleiman, Yong Xu. Anomalous diffusion, non-Gaussianity, nonergodicity, and confinement in stochastic-scaled Brownian motion with diffusing diffusivity dynamics. Physical Review E 2024, 109
(1)
https://doi.org/10.1103/PhysRevE.109.014139
- Iestyn Pope, Hugh Tanner, Francesco Masia, Lukas Payne, Kenton Paul Arkill, Judith Mantell, Wolfgang Langbein, Paola Borri, Paul Verkade. Correlative light-electron microscopy using small gold nanoparticles as single probes. Light: Science & Applications 2023, 12
(1)
https://doi.org/10.1038/s41377-023-01115-4
- Long Chen, Chao He. Single probes and resonant four-wave-mixing enabling novel correlative light electron microscopy workflow. Light: Science & Applications 2023, 12
(1)
https://doi.org/10.1038/s41377-023-01154-x
- Hiroto Furukawa, Yuuna Kimura, Hiroshi Inaba, Kazunori Matsuura. A supramolecular system mimicking the infection process of an enveloped virus through membrane fusion. Scientific Reports 2023, 13
(1)
https://doi.org/10.1038/s41598-023-47347-7
- Jian-Fei Lu, Sheng Luo, Hao Tang, Jia-Hui Liang, Yi-Fan Zhao, Yang Hu, Guan-Jun Yang, Jiong Chen, . Micropterus salmoides
rhabdovirus enters cells via clathrin-mediated endocytosis pathway in a pH-, dynamin-, microtubule-, rab5-, and rab7-dependent manner. Journal of Virology 2023, 97
(10)
https://doi.org/10.1128/jvi.00714-23
- S. V. Von Gratowski, V.V. Koledov, S. Balashov. Towards Micro Nanorobotic Platform for Single Virusonics. 2023, 1-6. https://doi.org/10.1109/MARSS58567.2023.10294116
- Xianbin Ma, Mingchuan Mao, Jiaqi He, Chao Liang, Hai-Yan Xie. Nanoprobe-based molecular imaging for tumor stratification. Chemical Society Reviews 2023, 52
(18)
, 6447-6496. https://doi.org/10.1039/D3CS00063J
- Anthony Petkidis, Vardan Andriasyan, Urs F. Greber. Machine learning for cross-scale microscopy of viruses. Cell Reports Methods 2023, 3
(9)
, 100557. https://doi.org/10.1016/j.crmeth.2023.100557
- A.A. El-Barbary, I.F.M. AL-Dosari. New insights into electronic and adsorption energies of metallofullerenes C19M. Results in Materials 2023, 19 , 100428. https://doi.org/10.1016/j.rinma.2023.100428
- Ming-Yu Zhang, An-An Liu, Luyang Jiao, Shuhan Zhang, Lin-Han Jiang, Xianglei Kong, Dai-Wen Pang. Capture of small clusters by ligand–solvent interaction. The Journal of Chemical Physics 2023, 159
(6)
https://doi.org/10.1063/5.0155373
- Ju-Mei Zhang, Zhi-Gang Wang, Lei Du, Dan-Dan Fu, Meng-Qian Zhang, Jing Li, Shu-Lin Liu, Dai-Wen Pang. Quantitative analysis of the effects of vimentin intermediate filaments on the early stages of influenza A virus infection. New Journal of Chemistry 2023, 47
(30)
, 14344-14354. https://doi.org/10.1039/D3NJ00952A
- Jonas Skeivalas, Eimuntas Paršeliūnas, Audrius Paršeliūnas, Dominykas Šlikas. Analysis of Results of Digital Electroencephalography and Digital Vectors of Coronavirus Images upon Applying the Theory of Covariance Functions. Symmetry 2023, 15
(7)
, 1330. https://doi.org/10.3390/sym15071330
- Anthony Petkidis, Vardan Andriasyan, Urs F Greber. Label-free microscopy for virus infections. Microscopy 2023, 72
(3)
, 204-212. https://doi.org/10.1093/jmicro/dfad024
- Di Liu, Li Pan, Huanjie Zhai, Hua-Ji Qiu, Yuan Sun. Virus tracking technologies and their applications in viral life cycle: research advances and future perspectives. Frontiers in Immunology 2023, 14 https://doi.org/10.3389/fimmu.2023.1204730
- Lei Wang, Zhi-Gang Wang, Di Ning, Yusi Hu, Shu-Lin Liu, Dai-Wen Pang. Real-time monitoring of biomolecular dynamics on cell membranes by quantum dot-based multicolor electrochemiluminescence. Nano Today 2023, 50 , 101855. https://doi.org/10.1016/j.nantod.2023.101855
- Suparna Paul, Subhajit Mukherjee, Priyabrata Banerjee. Recent advancement in nanomaterial-encapsulated drug delivery vehicles for combating cancer, COVID-19, and HIV-like chronic diseases. Materials Advances 2023, 4
(9)
, 2042-2061. https://doi.org/10.1039/D2MA01075E
- Yongchao Du, Yaxi Li, Yunliang Liu, Naiyun Liu, Yuanyuan Cheng, Qiuzhong Shi, Xiang Liu, Zhimin Tao, Yumeng Guo, Jianguo Zhang, Najmeh Askaria, Haitao Li. Stalk-derived carbon dots as nanosensors for Fe3+ ions detection and biological cell imaging. Frontiers in Bioengineering and Biotechnology 2023, 11 https://doi.org/10.3389/fbioe.2023.1187632
- Ana Ortiz-Perez, Cristina Izquierdo-Lozano, Rens Meijers, Francesca Grisoni, Lorenzo Albertazzi. Identification of fluorescently-barcoded nanoparticles using machine learning. Nanoscale Advances 2023, 5
(8)
, 2307-2317. https://doi.org/10.1039/D2NA00648K
- Lu Miao, Chunyu Yan, Yingzhu Chen, Wei Zhou, Xuelian Zhou, Qinglong Qiao, Zhaochao Xu. SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells. Cell Chemical Biology 2023, 30
(3)
, 248-260.e4. https://doi.org/10.1016/j.chembiol.2023.02.001
- Yingying Cao, Seungah Lee, Kyungsoo Kim, Jong-Young Kwak, Seong Ho Kang. Real-time six-dimensional spatiotemporal tracking of single anisotropic nanoparticles in live cells by integrated multifunctional light-sheet nanoscopy. Microchimica Acta 2023, 190
(2)
https://doi.org/10.1007/s00604-023-05633-1
- Hao-Yang Liu, Zhi-Gang Wang, Shu-Lin Liu, Dai-Wen Pang. Single-virus tracking with quantum dots in live cells. Nature Protocols 2023, 18
(2)
, 458-489. https://doi.org/10.1038/s41596-022-00775-7
- Alexander P. Demchenko. Detection of Harmful Microbes. 2023, 453-491. https://doi.org/10.1007/978-3-031-19089-6_13
- Xiaowei Zhang, Wei Li, Zongqiang Cui. Single-Particle Tracking of Virus Entry in Live Cells. 2023, 153-168. https://doi.org/10.1007/978-3-031-40086-5_5
- Xianxun Sun, Zongqiang Cui. Microbiological Nanotechnology. 2023, 525-553. https://doi.org/10.1007/978-981-16-8984-0_16
- 余永建 Yu Yongjian, 王越 Wang Yue, 李寰 Li Huan, 周文超 Zhou Wenchao, 舒风风 Shu Fengfeng, 高明 Gao Ming, 吴一辉 Wu Yihui. 融合通道层注意力机制的UNet的衍射极限荧光点检测和定位. Laser & Optoelectronics Progress 2023, 60
(14)
, 1412004. https://doi.org/10.3788/LOP230718
- Fatemeh Sabzian‐Molaei, Seyedrafi Hosseini, Azam Bolhassani, Vahid Eskandari, Saeed Norouzi, Amin Hadi. Antiviral Effect of Saffron Compounds on the GP120 of HIV‐1: an In Silico Study. ChemistrySelect 2022, 7
(47)
https://doi.org/10.1002/slct.202203471
- Shaun M. Christie, Takuya Tada, Yandong Yin, Amit Bhardwaj, Nathaniel R. Landau, Eli Rothenberg. Single-virus tracking reveals variant SARS-CoV-2 spike proteins induce ACE2-independent membrane interactions. Science Advances 2022, 8
(49)
https://doi.org/10.1126/sciadv.abo3977
- Courtney Johnson, Jack Exell, Yuxin Lin, Jonathan Aguilar, Kevin D. Welsher. Capturing the start point of the virus–cell interaction with high-speed 3D single-virus tracking. Nature Methods 2022, 19
(12)
, 1642-1652. https://doi.org/10.1038/s41592-022-01672-3
- Nicolas Moreno, Daniela Moreno-Chaparro, Florencio Balboa Usabiaga, Marco Ellero. Hydrodynamics of spike proteins dictate a transport-affinity competition for SARS-CoV-2 and other enveloped viruses. Scientific Reports 2022, 12
(1)
https://doi.org/10.1038/s41598-022-14884-6
- Xianbin Ma, Meng‐Jie Zhang, Jingting Wang, Tian Zhang, Peng Xue, Yuejun Kang, Zhi‐Jun Sun, Zhigang Xu. Emerging Biomaterials Imaging Antitumor Immune Response. Advanced Materials 2022, 34
(42)
https://doi.org/10.1002/adma.202204034
- Danni Li, Liyan Xiong, Guixia Pan, Tingfang Wang, Rou Li, Lizhi Zhu, Qianqian Tong, Qinqin Yang, Ye Peng, Changjing Zuo, Cong Wang, Xiao Li. Molecular imaging on ACE2‐dependent transocular infection of coronavirus. Journal of Medical Virology 2022, 94
(10)
, 4878-4889. https://doi.org/10.1002/jmv.27958
- Geetanjali Negi, Anurag Sharma, Manorama Dey, Garvita Dhanawat, Nagma Parveen. Membrane attachment and fusion of HIV-1, influenza A, and SARS-CoV-2: resolving the mechanisms with biophysical methods. Biophysical Reviews 2022, 14
(5)
, 1109-1140. https://doi.org/10.1007/s12551-022-00999-7
- Muqsit Pirzada, Zeynep Altintas. Nanomaterials for virus sensing and tracking. Chemical Society Reviews 2022, 51
(14)
, 5805-5841. https://doi.org/10.1039/D1CS01150B
- Bertus van Heerden, Nicholas A. Vickers, Tjaart P. J. Krüger, Sean B. Andersson. Real‐Time Feedback‐Driven Single‐Particle Tracking: A Survey and Perspective. Small 2022, 18
(29)
https://doi.org/10.1002/smll.202107024
- Deepti Mittal, Syed Azmal Ali. Use of Nanomaterials for Diagnosis and Treatment: The Advancement of Next-Generation Antiviral Therapy. Microbial Drug Resistance 2022, 28
(6)
, 670-697. https://doi.org/10.1089/mdr.2021.0281
- Hamza Abu Owida, Jamal I. Al-Nabulsi, Nidal M. Turab, Nawal Louzi, . Nanotechnology Role Development for COVID-19 Pandemic Management. Journal of Nanotechnology 2022, 2022 , 1-12. https://doi.org/10.1155/2022/1872933
- Jung Min Kim, Young-Mi Kang. Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group. International Journal of Molecular Sciences 2022, 23
(10)
, 5841. https://doi.org/10.3390/ijms23105841
- Shimin Li, Dianbing Wang, Abbas Ghulam, Xia Li, Min Li, Qin Li, Yingxin Ma, Lei Wang, Hangshen Wu, Zongqiang Cui, Xian-En Zhang, . Tracking the Replication-Competent Zika Virus with Tetracysteine-Tagged Capsid Protein in Living Cells. Journal of Virology 2022, 96
(7)
https://doi.org/10.1128/jvi.01846-21
- Junfang Liu, Minhong Su, Xin Chen, Zhongli Li, Zekui Fang, Li Yi. Lipid-mediated biosynthetic labeling strategy for in vivo dynamic tracing of avian influenza virus infection. Journal of Biomaterials Applications 2022, 36
(9)
, 1689-1699. https://doi.org/10.1177/08853282211063298
- Fan Yang, Huiting Lu, Xiangdan Meng, Haifeng Dong, Xueji Zhang. Shedding Light on DNA‐Based Nanoprobes for Live‐Cell MicroRNA Imaging. Small 2022, 18
(10)
https://doi.org/10.1002/smll.202106281
- Anuradha Ramoji, Susanne Pahlow, Aikaterini Pistiki, Jan Rueger, Tanveer Ahmed Shaik, Haodong Shen, Christina Wichmann, Christoph Krafft, Juergen Popp. Understanding viruses and viral infections by biophotonic methods. Translational Biophotonics 2022, 4
(1-2)
https://doi.org/10.1002/tbio.202100008
- Gihan S. Gunaratne, Jonathan S. Marchant. The ins and outs of virus trafficking through acidic Ca2+ stores. Cell Calcium 2022, 102 , 102528. https://doi.org/10.1016/j.ceca.2022.102528
- Jing-Ya Zhao, Zhi-Gang Wang, Hui Hu, Zhi-Ling Zhang, Bo Tang, Meng-Yao Luo, Ling-Ling Yang, Baoshan Wang, Dai-Wen Pang. How different are the surfaces of semiconductor Ag2Se quantum dots with various sizes?. Science Bulletin 2022, 67
(6)
, 619-625. https://doi.org/10.1016/j.scib.2021.12.004
Learn about these metrics
Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.
Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.
The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.
Recommended Articles
Abstract
Figure 1
Figure 1. Timeline of the key developments of the single-virus tracking technique.
Figure 2
Figure 2. Comparison of the size scales of fluorescent labels and the spatial resolutions of biological imaging techniques.
Figure 3
Figure 3. Uptake of Cy5-labeled adeno-associated virus (AAV) by a live HeLa cell. (a) Representative trajectories of AAV particles in cells at different stages of the infection. (b) Zoom in of trajectory 2 showing several membrane interactions of the AAV at the cell surface. (c) Mean number of consecutive cell interactions derived for viruses that did not dock. (d–e) Distribution of adsorption times for (d) 137 nondocking and (e) 42 membrane penetrating trajectories. Adapted with permission from ref (69). Copyright 2001 The American Association for the Advancement of Science.
Figure 4
Figure 4. Clathrin structures capture vesicular stomatitis viruses (VSVs) and defective interfering particles (DI-T) with similar kinetics. (a) Schematic of the clathrin-dependent virus internalization pathway. (b) VSVs and DI-T particles captured by clathrin structures in the same cell. BSC1 cells stably expressing s2-eGFP (green) were inoculated with Alexa Fluor 647-labeled DI-T (blue, blue arrowheads) and Alexa Fluor 568-labeled VSV (red, red arrowheads). Adapted with permission from ref (140). Copyright 2010 Public Library of Science.
Figure 5
Figure 5. Tracking the transport and fusion of individual influenza viruses. (a) Trajectory of a DiD-labeled virus inside a cell. (b) Time trajectories of the velocity (black) and the DiD fluorescence intensity (blue) of a virus. (c–e) Histogram of the viral velocity in each stage. (Inset) Shown is the measured average mean square displacement (⟨Δr2⟩) vs time (Δt) for a virus. Adapted with permission from ref (68). Copyright 2003 National Academy of Sciences, U.S.A.
Figure 6
Figure 6. Characterization of near-infrared FPs. (a–c) Normalized excitation (a), emission (b), and full absorption spectra of different iRFPs. (d) Schematic representation of directed molecular evolution that led to iRFPs with distinct spectral properties. (e) Brightness of HeLa cells transiently transfected with iRFPs, normalized to the value for iRFP713-expressing cells. Adapted with permission from ref (181). Copyright 2013 Springer Nature.
Figure 7
Figure 7. Generation of recombinant influenza viruses carrying a GFP reporter. (a) Schematic representation of the NS segment of WT PR8 virus and NS1-GFP virus. (b) A549 cells were infected with recombinant PR8 virus carrying NS1-GFP. At 10 h postinfection, cells were fixed and stained for NP. NP staining is shown in red and NS1-GFP is shown in green. (c) Fluorescent micrographs of NS1-GFP virus plaques taken at 20× magnification. Adapted with permission from ref (217). Copyright 2010 National Academy of Sciences, U.S.A.
Figure 8
Figure 8. Single ASLV-A entry into acidic endosomes and virus-endosome fusion. (a) Schematic diagram illustrating virus labeling and how the endosomal pH drops and subsequent ASLV-A fusion is visualized. (b) ASLV-A (yellow) fusion with TVA950 cells transiently expressing mKO-Rab5 (blue). Pseudoviruses were labeled with EcpH-ICAM (green) and Gag-mKate2 (red). The right top image panels show consecutive snapshots of the boxed region showing the virus prior to internalization (left), immediately after entry into acidic Rab5-positive endosomes (middle), and after fusion with early endosomes (right). The graph in panel (c) shows the fluorescence intensities of mKO-Rab5 and the viral EcpH-ICAM (green) and Gag-mKate2 (red) signals as a function of time. Adapted with permission from ref (215). Copyright 2014 BioMed Central Ltd.
Figure 9
Figure 9. Use of PtFPs for investigating focal adhesions. (a) Protocol for dual-label super resolution imaging by PALM. (b) Dual-color PALM super resolution image overlay of paxillin (green) and zyxin (red). (c) Diffraction-limited, summed molecule, dual-color TIRF image. (d) DIC image. Adapted with permission from ref (228). Copyright 2007 National Academy of Sciences, U.S.A.
Figure 10
Figure 10. Properties of QDs. (a) Schematic drawing of a core–shell QD and fluorescent images of QDs of different sizes under UV light. (b) Absorption (left) and emission (right) spectra of CdSe/ZnS QDs. All QD samples and data were obtained in the group of Pang.
Figure 11
Figure 11. Gold nanocluster labeling of enteroviruses. (a) Synthesis of the maleimide functionalized Au102(pMBA)44 clusters and their site-specific conjugation to enteroviruses. (b–c) TEM images of CVB3 viruses treated with functionalized gold clusters. (c) Control TEM image with conventional negative staining of a virus sample incubated with nonfunctionalized clusters. Adapted with permission from ref (272). Copyright 2014 National Academy of Sciences, U.S.A.
Figure 12
Figure 12. Labeling strategies and labeling sites for fluorescent labels in SVT.
Figure 13
Figure 13. Cross-linking reactions for conjugating fluorescent labels to viruses.
Figure 14
Figure 14. Covalent attachment of QDs to AAV and characterization of the QD-AAV conjugates. (a) QD-AAV networks are generated by an amide bond formation between the carboxylic source on QDs and the primary amines from lysine residues on the AAV capsid via carbodiimide chemistry. (b) Transmission electron microscope (TEM) images of (left) unconjugated QD525, (middle) AAV only, and (right) QD525-labeled AAV. Adapted with permission from ref (136). Copyright 2011 American Chemical Society.
Figure 15
Figure 15. Engineering the hepatitis D virus (HDV) assembly process for site-specific incorporation of unnatural amino acids (UAAs) into its surface envelope proteins. (a) Two-step procedure for the assembly of intact HDV carrying site-specifically incorporated UAA-recognized non-natural amino acids in human hepatocytes Huh-7 cells. (b) Structures of five Pyl analogues used in this study: PenK (Ne-pent-4-ynyloxycarbonyl-l-lysine), ACPK (Ne-((1R,2R)-2-azido-cyclopentyl oxy-carbonyl)-l-lysine), BCN (bicyclo[6.1.0]non-4-yn-9-ylmethanol), DiZPK (3-(3-methyl-3H-diazirine-3-yl)-propaminocarbonyl-Ne-l-lysine), and ONBK (o-nitrobenzyloxy carbonyl-Ne-lysine). Adapted with permission from ref (296). Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Figure 16
Figure 16. Schematic of a two-step labeling strategy of labeling virus with QDs via the biotin–streptavidin interaction. Adapted with permission from ref (108). Copyright 2012 American Chemical Society.
Figure 17
Figure 17. A schematic diagram for the generation of Halo tag-labeled pseudorabies viruses. Adapted with permission from ref (324). Copyright 2016 American Chemical Society.
Figure 18
Figure 18. Scheme of the general strategy for in situ virus labeling during progeny virus assembly. The labeling procedure includes (1) infection of the host cells with the virus, (2) after 2 days’ cultivation, the proteins on the host cell surface were conjugated with polypeptides containing his-tags and carboxyl groups, (3) progeny viruses assemble and are released from the cell surface, incorporating the his-tags in the process, and (4) the progeny viruses are further tagged with Ni2+-NTA modified QDs. Adapted with permission from ref (335). Copyright 2016 The Royal Society of Chemistry.
Figure 19
Figure 19. Labeling the HIV-1 proviral loci. Within the cytosol of a live cell, the TALEs fused with a short LplA acceptor peptide (LAP) are decorated with trans-cyclooctene and subsequently labeled with tetrazine-conjugated red QDs via Diels–Alder cycloaddition chemistry. The TALEs fused with an AP tag are biotinylated and labeled with streptavidin-conjugated green QDs. The two QD-TALEs bind to the target HIV-1 proviral DNA sequences, and their fluorescence colocalization demonstrates a single-copy HIV-1 provirus loci in the human chromosomes. Adapted with permission from ref (116). Copyright 2017 Springer Nature.
Figure 20
Figure 20. Working principle of encapsulating SA-QDs into HIV-based lentivirus in living cells. Adapted with permission from ref (355). Copyright 2013 American Chemical Society.
Figure 21
Figure 21. Schematic drawing of an objective-based TIRF and a prism-based TIRF.
Figure 22
Figure 22. Live cell imaging of individual HIV-1 assembly sites. HeLa cell transfected with a mixture of pCHIV and pCHIVeGFP imaged at 25 h post transfection (a) in WF and (b) in TIRF mode. The scale bar represents 5 μm. Adapted with permission from ref (370). Copyright 2009 Public Library of Science.
Figure 23
Figure 23. Schematic drawing of (a) a laser scanning confocal microscope and (b) a spinning disk confocal microscope.
Figure 24
Figure 24. SPT-PALM imaging of VSVG in COS7cells. (left) PALM image of VSVG tagged with EosFP. (middle) All SPT-PALM trajectories of localized VSVG molecules. (right) Diffusion map of individual EosFP-VSVG molecules in the cell. Adapted with permission from ref (235). Copyright 2008 Springer Nature.
Figure 25
Figure 25. 3D orbital tracking. (a) Light microscopy transmission image of a zebrafish embryo and a zoom in on the tail with a typical Rohon–Beard neuron labeled by a membrane-targeted fluorescent protein (shown in yellow). (scale bar, 200 μm). (b) Schematic of the custom-built 3D real-time orbital tracking microscope consisting of a laser scanning confocal modality for tracking and a wide-field modality for simultaneous environmental observation. (c) (Upper image) Confocal reconstruction of a sensory neuron where both the membrane and the individual mitochondria are fluorescently labeled (scale bar, 100 μm). (Lower Image) Photoactivation of a single PA-GFP-labeled axonal mitochondrion (in yellow) (scale bar, 5 mm). (d) Schematic representation of the 3D orbital tracking approach. Different particle locations are indicated through spheres of varying color. Depending on the location of the particle, the phase and modulation of the signal vary. (e) Trajectory of an anterograde moving mitochondrion (100 Hz, 20,000 data points). (f) Autocorrelation carpet (top) of the angle between consecutive orbits and segregation of the trajectory into regions of directed transport (green) and stationary phases (red). Adapted with permission from ref (425). Copyright 2019 ELife Sciences Publications.
Figure 26
Figure 26. Schematic representation of single-virus tracking. (a) Time-series of images acquired using fluorescence microscopy. The optical spatial resolution is about 250 nm in the lateral direction and about 500 nm in the axial direction, although the localization of the particle can be determined with much higher precision. (b) Steps in SVT analysis. From the collected images, the location of the different particles is first determined, and then the same particle needs to be linked through the different frames. Once the trajectories have been determined, various analyses can be performed such as a mean-squared-displacement analysis.
Figure 27
Figure 27. Detection of the accurate position of particles with subdiffraction resolution. (a) Image generated by sampling the point spread function (PSF) of wide-field microscopy on a 3D grid with a lattice size of 20 nm. (b) 3D CCD image simulated from the PSF image (a) with a signal-to-noise ratio of 20. The blue crosses indicate the true center of the 3D particle. (c) 3D scatter plots of the errors illustrating the error ranges of the centroid (green), Gaussian fitting (blue), and radial symmetry algorithm (red), respectively. Adapted with permission from ref (435). Copyright 2013 Springer Nature.
Figure 28
Figure 28. CD36 receptor aggregation activity depending on motion type. (a) Epifluorescence image of a macrophage where CD36 has been immunolabeled using a primary Fab fragment followed by a Cy3-conjugated secondary Fab fragment. (b) CD36 tracks in a control macrophage. (c and d) CD36 tracks in (c) a blebbistatin-treated and (d) a nocodazole-treated macrophage. Scale bars, 1 μm. (e) Bar plot showing the fraction of particles undergoing linear motion. (f) x coordinate, y coordinate, and amplitude of two sample trajectories as a function of time where merging events (green ovals), splitting events (purple ovals), and closed gaps (orange ovals) have been highlighted. Adapted with permission from ref (446). Copyright 2008 Springer Nature.
Figure 29
Figure 29. Virus cell surfing along filopodia. (a) Individual murine leukemia virus (MLV) labeled with YFP (red) surfing along the filopodium of a HEK 293 cell transduced with mCAT-1-CFP (green). The time in the upper righthand corner is given in seconds, and the motion from two particles is summarized as white arrows in the right-most panel. (b) Image summarizing the overall movement of selected particles on filopodia of a single HEK 293 cell. (c) Thirty-one frames from a recorded movie superimposed to show the transport and photobleaching of MLVs during the SVT experiment (moving particles are highlighted in white). (d) To quantify virus cell surfing, the motility of 85 individual MLV particles was plotted over time where time point 0 represents the moment the virus attaches to a filopodium. Adapted with permission from ref (208). Copyright 2005 The Rockefeller University Press.
Figure 30
Figure 30. Real-time imaging of clathrin-dependent CPV internalization. (a) Example of clathrin-dependent CPV endocytosis. Left panels, CRFK σ2-eGFP cells (green, AP-2) were inoculated with fluorescent capsids (red) and imaged as before. Right panel, diffusion path of the capsid shown at left. A color-coded line trace of the capsid diffusion path is overlaid onto the t = 51-s image. (b) Plot of the background-corrected AP-2 (green) and capsid (red) fluorescence intensities with respect to time for the event in panel a. For frames prior to pit initiation, the AP-2 fluorescence intensity was quantified at the eventual site of pit initiation. (c) Efficiency of clathrin-dependent CPV entry. (d) Examples of CPV dissociation from the cell surface. Time-lapse images showing the attachment (downward-facing arrows) of two capsids (red; no. 1, no. 2) and subsequent capsid dissociation (upward-facing arrows). (e) Residence time of CPV capsids that dissociated from CRFK cells. Adapted with permission from ref (142). Copyright 2012 American Society for Microbiology.
Figure 31
Figure 31. Identification of HIV-1 fusion sites by single-virus imaging. (a) Schematic presentation of redistribution of viral lipid and content markers upon fusion with a plasma membrane (left) and with an endosome (right). Viruses colabeled with membrane (red) and content (green) markers are pseudocolored yellow. (b and c) Partial fusion of JRFL with the plasma membrane of TZM-bl cells. The time from the beginning of imaging is shown. The two-dimensional projection of the particle’s trajectory (cyan) is overlaid on the last image. Changes in fluorescence intensities (in arbitrary units) of membrane (red) and content (green) markers, as well as the instantaneous velocity (blue trace) of the particle, are shown. Adapted with permission from ref (83). Copyright 2009 Elsevier.
Figure 32
Figure 32. Fusion of PFV. (a) Bright field image of a cell overlaid with the trajectory of a dual-color PFV virus during fusion at or near the plasma membrane. The dual-color signal is shown in yellow and the single-labeled capsid in green. (b) Tracking image correlation (TrIC) analysis of the fusion event observed in panel (a). (upper panel) The fluorescence intensity of the Gag-eGFP signal (green) and mCherry-Env (red) signal, (second panel) instantaneous velocity, (third panel) cross-correlation amplitude (blue) and randomized cross-correlation signal (black), and (lower panel) relative distance between the Gag-eGFP and mCherry-Env signals plotted as a function of time. (c) 3D relative trajectory of the Env-labeled envelop with respect to the Gag-capsid showing movement in the order of hundreds of nanometers between the two labels before the completion of the fusion process. Adapted with permission from ref (467). Copyright 2013 Elsevier.
Figure 33
Figure 33. Real-time imaging of vRNP of influenza A viruses (IAV) release from a Rab7-positive endosome. (a) A fluorescence movie of a cell containing QD625-labeled influenza viruses (red) and Rab7-ECFP labeled endosomes (cyan) was recorded and SVT was performed. (b) Trajectories of the QD-labeled influenza virus colocalizing with the fluorescently labeled endosome from panel a. (c) Fluorescence image of an infected cell treated with NH4Cl. (d) Trajectories of the QD625 and ECFP fluorescent signals in NH4Cl-treated cells. (e) Model for IAV uncoating and vRNP dynamics. An IAV virion enters the host cell via endocytosis. Individual vRNPs finally undergo a three-stage active transport process to arrive at the cell nucleus and display two diffusion patterns within the nucleus. Adapted with permission from ref (113). Copyright 2019 National Academy of Sciences, U.S.A.
Figure 34
Figure 34. Recruitment of VPS4A to HIV assembly sites. (a) Wide-field image and time projections (5,926 s) from a TIRFM image series exemplifying frequent colocalization of eGFP-VPS4A bursts (green) with nascent HIV particles (magenta). (b) TIRFM images of the assembly and release of a HIV particle (top panel, arrows) and the corresponding eGFP-VPS4A channel (bottom panel, arrows). (c) Number of VPS4A bursts detected within 528 s (200 frames) in the presence of the indicated HIV derivatives (left) and number of HIV budding sites detected (right). (d) Wide-field image and time-projected TIRFM image of cells coexpressing eGFP-VPS4A (green) and the nonbudding HIV late minus mutant (magenta). All scale bars, 800 nm. Adapted with permission from ref (481). Copyright 2011 Springer Nature.
Figure 35
Figure 35. Membrane nanotubes present a novel route for HIV-1 to spread between T cells. (a) Membrane nanotubes were formed after intercellular contact between an infected Jurkat T cell (red) and an uninfected Jurkat T cell. (b) The frequency of membrane nanotubes formed between uninfected and infected Jurkat T cells or between two populations of uninfected Jurkat T cells. (c) Time-lapse imaging of Gag-GFP (green), expressed in the context of the fully infectious virus, along a membrane nanotube connecting infected with uninfected Jurkat T cells (red). (d) The arrow indicates Gag-GFP within the cytoplasm of the initially uninfected T cell. (e) The position of Gag-GFP is plotted against time showing generally directed movement from uninfected to infected cells. Adapted with permission from ref (489). Copyright 2008 Springer Nature.
References
This article references 512 other publications.
- 1Smith, A. E.; Helenius, A. How Viruses Enter Animal Cells. Science 2004, 304, 237– 242, DOI: 10.1126/science.10948231How Viruses Enter Animal CellsSmith, Alicia E.; Helenius, AriScience (Washington, DC, United States) (2004), 304 (5668), 237-242CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Viruses replicate within living cells and use the cellular machinery for the synthesis of their genome and other components. To gain access, they have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into the host cell. Many animal viruses take advantage of endocytic pathways and rely on the cell to guide them through a complex entry and uncoating program. In the dialogue between the cell and the intruder, the cell provides crit. cues that allow the virus to undergo mol. transformations that lead to successful internalization, intracellular transport, and uncoating.
- 2Greber, U. F.; Way, M. A Superhighway to Virus Infection. Cell 2006, 124, 741– 754, DOI: 10.1016/j.cell.2006.02.0182A superhighway to virus infectionGreber, Urs F.; Way, MichaelCell (Cambridge, MA, United States) (2006), 124 (4), 741-754CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Microtubule-mediated transport of macromols. and organelles (also known as "cargo") is essential for cells to function. Deficiencies in cytoplasmic transport are frequently assocd. with severe diseases and syndromes. Cytoplasmic transport also provides viruses with the means to reach their site of replication and is the route for newly assembled progeny to leave the infected cell. This parasitic relationship of viruses with the host cytoskeleton provides an excellent basis for cell biologists to unlock the secrets of cytoplasmic transport and unravel mechanisms of disease. Recent advances in live cell imaging and computational tracking of fluorescently labeled viruses are now revealing how complex the movements of single viruses are in infected cells. This review focuses on microtubule-based motility of viruses and highlights the mechanisms regulating cytoplasmic transport.
- 3Marsh, M.; Helenius, A. Virus Entry: Open Sesame. Cell 2006, 124, 729– 740, DOI: 10.1016/j.cell.2006.02.0073Virus entry: open sesameMarsh, Mark; Helenius, AriCell (Cambridge, MA, United States) (2006), 124 (4), 729-740CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biol. studies, live-cell imaging, and systems biol. have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addn., such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.
- 4Gruenberg, J. Viruses and Endosome Membrane Dynamics. Curr. Opin. Cell Biol. 2009, 21, 582– 588, DOI: 10.1016/j.ceb.2009.03.0084Viruses and endosome membrane dynamicsGruenberg, JeanCurrent Opinion in Cell Biology (2009), 21 (4), 582-588CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)A review. Cell surface mols., ligands, and solutes can be endocytosed into animal cells via several pathways in addn. to clathrin-mediated endocytosis, which all seem to lead to canonical endosomes. It seems that viruses can enter and infect cells through most of, if not all, endocytic routes, having evolved different, sometimes elaborate, strategies to (mis)use cellular machineries to their own benefit during infection. Here, the author discusses recent progress in understanding the pathways followed by animal viruses into cells, and how these studies are also providing novel insights into the understanding of some mol. mechanisms that control endocytic membrane transport.
- 5Sun, E.; He, J.; Zhuang, X. Live Cell Imaging of Viral Entry. Curr. Opin. Virol. 2013, 3, 34– 43, DOI: 10.1016/j.coviro.2013.01.0055Live cell imaging of viral entrySun, Eileen; He, Jiang; Zhuang, XiaoweiCurrent Opinion in Virology (2013), 3 (1), 34-43CODEN: COVUAF; ISSN:1879-6257. (Elsevier B. V.)A review. Viral entry encompasses the initial steps of infection starting from virion host cell attachment to viral genome release. Given the dynamic interactions between the virus and the host, many questions related to viral entry can be directly addressed by live cell imaging. Recent advances in fluorescent labeling of viral and cellular components, fluorescence microscopy with high sensitivity and spatiotemporal resoln., and image anal. enabled studies of a broad spectrum across many viral entry steps, including virus-receptor interactions, internalization, intracellular transport, genomic release, nuclear transport, and cell-to-cell transmission. Collectively, these live cell imaging studies have not only enriched our understandings of the viral entry mechanisms, but also provided novel insights into basic cellular biol. processes.
- 6Pelkmans, L.; Helenius, A. Insider Information: What Viruses Tell Us about Endocytosis. Curr. Opin. Cell Biol. 2003, 15, 414– 422, DOI: 10.1016/S0955-0674(03)00081-46Insider information: what viruses tell us about endocytosisPelkmans, Lucas; Helenius, AriCurrent Opinion in Cell Biology (2003), 15 (4), 414-422CODEN: COCBE3; ISSN:0955-0674. (Elsevier Science Ltd.)A review. Viruses have long served as tools in mol. and cellular biol. to study a variety of complex cellular processes. Currently, there is a revived interest in virus entry into animal cells because it is evident that incoming viruses make use of numerous endocytic pathways that are otherwise difficult to study. Besides the classical clathrin-mediated uptake route, viruses use caveolae-mediated endocytosis, lipid-raft-mediated endocytic pathways, and macropinocytosis. Some of these are subject to regulation, involve novel endocytic organelles, and some of them connect organelles that were previously not known to communicate by membrane traffic.
- 7Conner, S. D.; Schmid, S. L. Regulated Portals of Entry into the Cell. Nature 2003, 422, 37– 44, DOI: 10.1038/nature014517Regulated portals of entry into the cellConner, Sean D.; Schmid, Sandra L.Nature (London, United Kingdom) (2003), 422 (6927), 37-44CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)A review. The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. "Endocytosis" encompasses several diverse mechanisms by which cells internalize macromols. and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of mol. interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiol. and, thus, must be viewed in a broader context than simple vesicular trafficking.
- 8Horan, P. K.; Melnicoff, M. J.; Jensen, B. D.; Slezak, S. E. Fluorescent Cell Labeling for in Vivo and in Vitro Cell Tracking. Methods Cell Biol. 1990, 33, 469– 490, DOI: 10.1016/S0091-679X(08)60547-68Fluorescent cell labeling for in vivo and in vitro cell trackingHoran P K; Melnicoff M J; Jensen B D; Slezak S EMethods in cell biology (1990), 33 (), 469-90 ISSN:0091-679X.There is no expanded citation for this reference.
- 9Giepmans, B. N. G. The Fluorescent Toolbox for Assessing Protein Location and Function. Science 2006, 312, 217– 224, DOI: 10.1126/science.11246189The Fluorescent Toolbox for Assessing Protein Location and FunctionGiepmans, Ben N. G.; Adams, Stephen R.; Ellisman, Mark H.; Tsien, Roger Y.Science (Washington, DC, United States) (2006), 312 (5771), 217-224CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Advances in mol. biol., org. chem., and materials science have recently created several new classes of fluorescent probes for imaging in cell biol. Here the authors review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live vs. fixed cells: detn. of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small org. fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
- 10Alcor, D.; Gouzer, G.; Triller, A. Single-Particle Tracking Methods for the Study of Membrane Receptors Dynamics. Eur. J. Neurosci. 2009, 30, 987– 997, DOI: 10.1111/j.1460-9568.2009.06927.x10Single-particle tracking methods for the study of membrane receptors dynamicsAlcor Damien; Gouzer Geraldine; Triller AntoineThe European journal of neuroscience (2009), 30 (6), 987-97 ISSN:.Single-particle tracking (SPT) applications have been growing rapidly in the field of cell biology, and in particular in neurobiology, as a means of unravelling the involvement of diffusion dynamics of neurotransmitter receptors and other synaptic proteins in the regulation of neuronal activity. Suitable probes and technological improvements make SPT more accessible than it used to be and open up broad applications in cellular biology. In this technical highlight, we give an overview of the experimental approach in SPT. The concepts and results in neurobiology have already been the object of detailed reviews. Here, we focus on a qualitative description of the implementation of SPT, from molecule labelling to acquisition, data treatment and analysis of protein diffusion properties. Constraints, limitations and future developments are discussed.
- 11Cang, H.; Shan Xu, C.; Yang, H. Progress in Single-Molecule Tracking Spectroscopy. Chem. Phys. Lett. 2008, 457, 285– 291, DOI: 10.1016/j.cplett.2008.03.09811Progress in single-molecule tracking spectroscopyCang, Hu; Shan Xu, C.; Yang, HawChemical Physics Letters (2008), 457 (4-6), 285-291CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)A review; a topic of much current interest is the chem. reactivity in complex environments, involving dynamical processes spanning multiple scales in both time and space. While challenging to study using std. ensemble-averaged methods, it is amenable to investigations using single-mol. approaches. Ultimately, one would like to monitor the dynamics of individual mols. as they undergo chem. transformations through time-dependent spectroscopic studies, and to correlate various mol. properties with their immediate phys. and chem. environments. This article provides an overview of the recent advances toward this goal and describes future developments and potential applications.
- 12Levi, V.; Gratton, E. Exploring Dynamics in Living Cells by Tracking Single Particles. Cell Biochem. Biophys. 2007, 48, 1– 15, DOI: 10.1007/s12013-007-0010-012Exploring dynamics in living cells by tracking single particlesLevi, Valeria; Gratton, EnricoCell Biochemistry and Biophysics (2007), 48 (1), 1-15CODEN: CBBIFV; ISSN:1085-9195. (Humana Press Inc.)A review. In the last years, significant advances in microscopy techniques and the introduction of a novel technol. to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biol. The authors' understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to the actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein mols. by following their way through the cell. In this review, the authors introduce the single particle tracking technol. to new users. The instrumentation is briefly described and some of the algorithms commonly used to locate and track particles explained. Also, the authors present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells.
- 13Shen, H.; Tauzin, L. J.; Baiyasi, R.; Wang, W.; Moringo, N.; Shuang, B.; Landes, C. F. Single Particle Tracking: From Theory to Biophysical Applications. Chem. Rev. 2017, 117, 7331– 7376, DOI: 10.1021/acs.chemrev.6b0081513Single Particle Tracking: From Theory to Biophysical ApplicationsShen, Hao; Tauzin, Lawrence J.; Baiyasi, Rashad; Wang, Wenxiao; Moringo, Nicholas; Shuang, Bo; Landes, Christy F.Chemical Reviews (Washington, DC, United States) (2017), 117 (11), 7331-7376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theor. background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biol. and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.
- 14Xia, T.; Li, N.; Fang, X. H. Single-Molecule Fluorescence Imaging in Living Cells. Annu. Rev. Phys. Chem. 2013, 64, 459– 480, DOI: 10.1146/annurev-physchem-040412-11012714Single-molecule fluorescence imaging in living cellsXia, Tie; Li, Nan; Fang, XiaohongAnnual Review of Physical Chemistry (2013), 64 (), 459-480CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. The transition of single-mol. fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomols. in their native environments and their roles in cellular processes. Here we review recent advances of single-mol. biophys. approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-mol. fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochem. parameters that can be obtained by imaging individually labeled biomols. in cells and what can be learned from such characterizations.
- 15Manzo, C.; Garcia-Parajo, M. F. A Review of Progress in Single Particle Tracking: from Methods to Biophysical Insights. Rep. Prog. Phys. 2015, 78, 124601, DOI: 10.1088/0034-4885/78/12/12460115A review of progress in single particle tracking: from methods to biophysical insightsManzo, Carlo; Garcia-Parajo, Maria F.Reports on Progress in Physics (2015), 78 (12), 124601/1-124601/29CODEN: RPPHAG; ISSN:1361-6633. (IOP Publishing Ltd.)Optical microscopy has for centuries been a key tool to study living cells with min. invasiveness. The advent of single mol. techniques over the past two decades has revolutionized the field of cell biol. by providing a more quant. picture of the complex and highly dynamic organization of living systems. Amongst these techniques, single particle tracking (SPT) has emerged as a powerful approach to study a variety of dynamic processes in life sciences. SPT provides access to single mol. behavior in the natural context of living cells, thereby allowing a complete statistical characterization of the system under study. In this review we describe the foundations of SPT together with novel optical implementations that nowadays allow the investigation of single mol. dynamic events with increasingly high spatiotemporal resoln. using mol. densities closer to physiol. expression levels. We outline some of the algorithms for the faithful reconstruction of SPT trajectories as well as data anal., and highlight biol. examples where the technique has provided novel insights into the role of diffusion regulating cellular function. The last part of the review concs. on different theor. models that describe anomalous transport behavior and ergodicity breaking obsd. from SPT studies in living cells.
- 16Miller, H.; Zhou, Z.; Shepherd, J.; Wollman, A. J. M.; Leake, M. C. Single-Molecule Techniques in Biophysics: A Review of the Progress in Methods and Applications. Rep. Prog. Phys. 2018, 81, 024601 DOI: 10.1088/1361-6633/aa8a0216Single-molecule techniques in biophysics: a review of the progress in methods and applicationsMiller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J. M.; Leake, Mark C.Reports on Progress in Physics (2018), 81 (2), 024601/1-024601/47CODEN: RPPHAG; ISSN:1361-6633. (IOP Publishing Ltd.)A review. Single-mol. biophysics has transformed our understanding of biol., but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equil., covering multiple lengths from the nanoscale of single mols. to up to several orders of magnitude higher in cells, tissues and organisms. Biomols. are often characterized by underlying instability: multiple metastable free energy states exist, sepd. by levels of just a few multiples of the thermal energy scale kBT, where kB is the Boltzmann const. and T abs. temp., implying complex inter-conversion kinetics in the relatively hot, wet environment of active biol. matter. A key benefit of single-mol. biophysics techniques is their ability to probe heterogeneity of free energy states across a mol. population, too challenging in general for conventional ensemble av. approaches. Parallel developments in exptl. and computational techniques have catalyzed the birth of multiplexed, correlative techniques to tackle previously intractable biol. questions. Exptl., progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent expts., including the underpinning mathematics. These methods are broadly divided into tools which detect mols. and those which manipulate them. For the former we discuss the progress of super-resoln. microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mech. perturb mols. We also consider in silico progress of single-mol. computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative at. force microscopy and fluorescence imaging, to probe questions closer to native physiol. behavior. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.
- 17Wieser, S.; Schutz, G. J. Tracking Single Molecules in the Live Cell Plasma Membrane-Do’s and Don’t’s. Methods 2008, 46, 131– 140, DOI: 10.1016/j.ymeth.2008.06.01017Tracking single molecules in the live cell plasma membrane-Do's and Don't'sWieser, Stefan; Schuetz, Gerhard J.Methods (Amsterdam, Netherlands) (2008), 46 (2), 131-140CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)A review. In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomol. within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomols. labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resoln. of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single mols. is state-of-the-art. The advances in the fields of microscopy brought new cell biol. questions into reach. The investigation of a single fluorescent mol.-or simultaneously of an ensemble of individual mols.-provides principally new information, which is generally hidden in ensemble-averaged signals of mols. In this paper we describe strategies how to make use of single mol. trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single mol. tracking. A diffusing membrane constituent-e.g. a protein or a lipid-experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently obsd. as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available anal. models and point out potential pitfalls, which may result in quant. or three even qual. misinterpretations.
- 18Kusumi, A.; Tsunoyama, T. A.; Hirosawa, K. M.; Kasai, R. S.; Fujiwara, T. K. Tracking Single Molecules at Work in Living Cells. Nat. Chem. Biol. 2014, 10, 524– 532, DOI: 10.1038/nchembio.155818Tracking single molecules at work in living cellsKusumi, Akihiro; Tsunoyama, Taka A.; Hirosawa, Kohichiro M.; Kasai, Rinshi S.; Fujiwara, Takahiro K.Nature Chemical Biology (2014), 10 (7), 524-532CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Methods for imaging and tracking single mols. conjugated with fluorescent probes, called single-mol. tracking (SMT), are now providing researchers with the unprecedented ability to directly observe mol. behaviors and interactions in living cells. Current SMT methods are achieving almost the ultimate spatial precision and time resoln. for tracking single mols., detd. by the currently available dyes. In cells, various mol. interactions and reactions occur as stochastic and probabilistic processes. SMT provides an ideal way to directly track these processes by observing individual mols. at work in living cells, leading to totally new views of the biochem. and mol. processes used by cells whether in signal transduction, gene regulation or formation and disintegration of macromol. complexes. Here we review SMT methods, summarize the recent results obtained by SMT, including related superresoln. microscopy data, and describe the special concerns when SMT applications are shifted from the in vitro paradigms to living cells.
- 19Kusumi, A.; Shirai, Y. M.; Koyama-Honda, I.; Suzuki, K. G. N.; Fujiwara, T. K. Hierarchical Organization of the Plasma Membrane: Investigations by Single-Molecule Tracking vs. Fluorescence Correlation Spectroscopy. FEBS Lett. 2010, 584, 1814– 1823, DOI: 10.1016/j.febslet.2010.02.04719Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopyKusumi, Akihiro; Shirai, Yuki M.; Koyama-Honda, Ikuko; Suzuki, Kenichi G. N.; Fujiwara, Takahiro K.FEBS Letters (2010), 584 (9), 1814-1823CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Single-mol. tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a no. of unprecedented observations, thus fostering a new basic understanding of mol. diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, contg. diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane mols. stay together for limited durations. Mol. interactions occur in the time-dependent inhomogeneous two-dimensional liq. of the plasma membrane, which might be a key for plasma membrane functions.
- 20Kusumi, A.; Fujiwara, T. K.; Chadda, R.; Xie, M.; Tsunoyama, T. A.; Kalay, Z.; Kasai, R. S.; Suzuki, K. G. Dynamic Organizing Principles of the Plasma Membrane That Regulate Signal Transduction: Commemorating the Fortieth Anniversary of Singer and Nicolson’s Fluid-Mosaic Model. Annu. Rev. Cell Dev. Biol. 2012, 28, 215– 250, DOI: 10.1146/annurev-cellbio-100809-15173620Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic modelKusumi, Akihiro; Fujiwara, Takahiro K.; Chadda, Rahul; Xie, Min; Tsunoyama, Taka A.; Kalay, Ziya; Kasai, Rinshi S.; Suzuki, Kenichi G. N.Annual Review of Cell and Developmental Biology (2012), 28 (), 215-250CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)A review. The recent rapid accumulation of knowledge on the dynamics and structure of the plasma membrane has prompted major modifications of the textbook fluid-mosaic model. However, because the new data have been obtained in a variety of research contexts using various biol. paradigms, the impact of the crit. conceptual modifications on biomedical research and development has been limited. In this review, we try to synthesize our current biol., chem., and phys. knowledge about the plasma membrane to provide new fundamental organizing principles of this structure that underlie every mol. mechanism that realizes its functions. Special attention is paid to signal transduction function and the dynamic aspect of the organizing principles. We propose that the cooperative action of the hierarchical three-tiered mesoscale (2-300 nm) domains-actin-membrane-skeleton induced compartments (40-300 nm), raft domains (2-20 nm), and dynamic protein complex domains (3-10 nm)-is crit. for membrane function and distinguishes the plasma membrane from a classical Singer-Nicolson-type model.
- 21Suzuki, K. G.; Kasai, R. S.; Hirosawa, K. M.; Nemoto, Y. L.; Ishibashi, M.; Miwa, Y.; Fujiwara, T. K.; Kusumi, A. Transient GPI-Anchored Protein Homodimers Are Units for Raft Organization and Function. Nat. Chem. Biol. 2012, 8, 774– 783, DOI: 10.1038/nchembio.102821Transient GPI-anchored protein homodimers are units for raft organization and functionSuzuki, Kenichi G. N.; Kasai, Rinshi S.; Hirosawa, Koichiro M.; Nemoto, Yuri L.; Ishibashi, Munenori; Miwa, Yoshihiro; Fujiwara, Takahiro K.; Kusumi, AkihiroNature Chemical Biology (2012), 8 (9), 774-783CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Advanced single-mol. fluorescent imaging was applied to study the dynamic organization of raft-assocd. glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (∼200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiol. expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts contg. up to four CD59 mols., which triggered intracellular Ca2+ responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains contg. GPI-APs.
- 22Zhang, W.; Jiang, Y.; Wang, Q.; Ma, X.; Xiao, Z.; Zuo, W.; Fang, X.; Chen, Y. G. Single-Molecule Imaging Reveals Transforming Growth Factor-Induced Type II Receptor Dimerization. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 15679– 15683, DOI: 10.1073/pnas.090827910622Single-molecule imaging reveals transforming growth factor-β-induced type II receptor dimerizationZhang, Wei; Jiang, Yaxin; Wang, Qiang; Ma, Xinyong; Xiao, Zeyu; Zuo, Wei; Fang, Xiaohong; Chen, Ye-GuangProceedings of the National Academy of Sciences of the United States of America (2009), 106 (37), 15679-15683, S15679/1-S15679/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Transforming growth factor-β (TGF-β) elicits its signals through two transmembrane serine/threonine kinase receptors, type II (TOSRII) and type I receptors. It is generally believed that the initial receptor dimerization is an essential event for receptor activation. However, previous studies suggested that TGF-β signals by binding to the preexisting TβRII homodimer. Here, using single mol. microscopy to image green fluorescent protein (GFP)-labeled TβRII on the living cell surface, we demonstrated that the receptor could exist as monomers at the low expression level in resting cells and dimerize upon TGF-β stimulation. This work reveals a model in which the activation of serine-threonine kinase receptors is also accomplished via dimerization of monomers, suggesting that receptor dimerization is a general mechanism for ligand-induced receptor activation.
- 23Kasai, R. S.; Kusumi, A. Single-Molecule Imaging Revealed Dynamic GPCR Dimerization. Curr. Opin. Cell Biol. 2014, 27, 78– 86, DOI: 10.1016/j.ceb.2013.11.00823Single-molecule imaging revealed dynamic GPCR dimerizationKasai, Rinshi S.; Kusumi, AkihiroCurrent Opinion in Cell Biology (2014), 27 (), 78-86CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)A review. Single fluorescent-mol. video imaging and tracking in living cells are revolutionizing our understanding of mol. interactions in the plasma membrane and intracellular membrane systems. They have revealed that mol. interactions occur surprisingly dynamically on much shorter time scales («1 s) than those expected from the results by conventional techniques, such as pull-down assays (minutes to hours). Single-mol. imaging has unequivocally showed that G-protein-coupled receptors (GPCRs) undergo dynamic equil. between monomers and dimers, by enabling the detn. of the 2D monomer-dimer equil. const., the dimer dissocn. rate const. (typically ∼10 s-1), and the formation rate const. Within one second, GPCRs typically undergo several cycles of monomer and homo-dimer formation with different partners.
- 24Courty, S.; Luccardini, C.; Bellaiche, Y.; Cappello, G.; Dahan, M. Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot Imaging. Nano Lett. 2006, 6, 1491– 1495, DOI: 10.1021/nl060921t24Tracking Individual Kinesin Motors in Living Cells Using Single Quantum-Dot ImagingCourty, Sebastien; Luccardini, Camilla; Bellaiche, Yohanns; Cappello, Giovanni; Dahan, MaximeNano Letters (2006), 6 (7), 1491-1495CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. The authors characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-mol. measurements provided important parameters of the motor, such as its velocity and processivity, as well as an est. of the force necessary to carry a QD. The authors' measurements demonstrate the importance of single-mol. expts. in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.
- 25Nishikawa, S.; Arimoto, I.; Ikezaki, K.; Sugawa, M.; Ueno, H.; Komori, T.; Iwane, A. H.; Yanagida, T. Switch between Large Hand-over-Hand and Small Inchworm-Like Steps in Myosin VI. Cell 2010, 142, 879– 888, DOI: 10.1016/j.cell.2010.08.03325Switch between Large Hand-Over-Hand and Small Inchworm-like Steps in Myosin VINishikawa, So; Arimoto, Ikuo; Ikezaki, Keigo; Sugawa, Mitsuhiro; Ueno, Hiroshi; Komori, Tomotaka; Iwane, Atsuko H.; Yanagida, ToshioCell (Cambridge, MA, United States) (2010), 142 (6), 879-888CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Many biol. motor mols. move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.
- 26Pierobon, P.; Achouri, S.; Courty, S.; Dunn, A. R.; Spudich, J. A.; Dahan, M.; Cappello, G. Velocity, Processivity, and Individual Steps of Single Myosin V Molecules in Live Cells. Biophys. J. 2009, 96, 4268– 4275, DOI: 10.1016/j.bpj.2009.02.04526Velocity, processivity, and individual steps of single myosin V molecules in live cellsPierobon, Paolo; Achouri, Sarra; Courty, Sebastien; Dunn, Alexander R.; Spudich, James A.; Dahan, Maxime; Cappello, GiovanniBiophysical Journal (2009), 96 (10), 4268-4275CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)We report the tracking of single myosin V mols. in their natural environment, the cell. Myosin V mols., labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single mol. level with high spatial and temporal resoln. We perform an intracellular measurement of key parameters of this mol. transporter: velocity, processivity, step size, and dwell time. Our expts. bridge the gap between in vitro single mol. assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.
- 27Liu, S. L.; Wang, Z. G.; Hu, Y.; Xin, Y.; Singaram, I.; Gorai, S.; Zhou, X.; Shim, Y.; Min, J. H.; Gong, L. W. Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of Akt. Mol. Cell 2018, 71, 1092– 1104, e5 DOI: 10.1016/j.molcel.2018.07.03527Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of AktLiu, Shu-Lin; Wang, Zhi-Gang; Hu, Yusi; Xin, Yao; Singaram, Indira; Gorai, Sukhamoy; Zhou, Xin; Shim, Yoonjung; Min, Jung-Hyun; Gong, Liang-Wei; Hay, Nissim; Zhang, Jin; Cho, WonhwaMolecular Cell (2018), 71 (6), 1092-1104.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quant. imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling mols. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.
- 28Sheng, R.; Chen, Y.; Yung Gee, H.; Stec, E.; Melowic, H. R.; Blatner, N. R.; Tun, M. P.; Kim, Y.; Kallberg, M.; Fujiwara, T. K. Cholesterol Modulates Cell Signaling and Protein Networking by Specifically Interacting with PDZ Domain-Containing Scaffold Proteins. Nat. Commun. 2012, 3, 1249, DOI: 10.1038/ncomms222128Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteinsSheng Ren; Chen Yong; Yung Gee Heon; Stec Ewa; Melowic Heather R; Blatner Nichole R; Tun Moe P; Kim Yonjung; Kallberg Morten; Fujiwara Takahiro K; Hye Hong Ji; Pyo Kim Kwang; Lu Hui; Kusumi Akihiro; Goo Lee Min; Cho WonhwaNature communications (2012), 3 (), 1249 ISSN:.Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.
- 29Komura, N.; Suzuki, K. G.; Ando, H.; Konishi, M.; Koikeda, M.; Imamura, A.; Chadda, R.; Fujiwara, T. K.; Tsuboi, H.; Sheng, R. Raft-Based Interactions of Gangliosides with a GPI-Anchored Receptor. Nat. Chem. Biol. 2016, 12, 402– 410, DOI: 10.1038/nchembio.205929Raft-based interactions of gangliosides with a GPI-anchored receptorKomura, Naoko; Suzuki, Kenichi G. N.; Ando, Hiromune; Konishi, Miku; Koikeda, Machi; Imamura, Akihiro; Chadda, Rahul; Fujiwara, Takahiro K.; Tsuboi, Hisae; Sheng, Ren; Cho, Wonhwa; Furukawa, Koichi; Furukawa, Keiko; Yamauchi, Yoshio; Ishida, Hideharu; Kusumi, Akihiro; Kiso, MakotoNature Chemical Biology (2016), 12 (6), 402-410CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Gangliosides, glycosphingolipids contg. one or more sialic acid(s) in the glyco-chain, are involved in various important physiol. and pathol. processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or prepns. Single-fluorescent-mol. imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside mols. were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.
- 30Liu, S. L.; Wang, Z. G.; Zhang, Z. L.; Pang, D. W. Tracking Single Viruses Infecting Their Host Cells Using Quantum Dots. Chem. Soc. Rev. 2016, 45, 1211– 1224, DOI: 10.1039/C5CS00657K30Tracking single viruses infecting their host cells using quantum dotsLiu, Shu-Lin; Wang, Zhi-Gang; Zhang, Zhi-Ling; Pang, Dai-WenChemical Society Reviews (2016), 45 (5), 1211-1224CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Single-virus tracking (SVT) technique, which uses microscopy to monitor the behaviors of viruses, is a vital tool to study the real-time and in situ infection dynamics and virus-related interactions in live cells. To make SVT a more versatile tool in biol. research, the researchers have developed a quantum dot (QD)-based SVT technique, which can be utilized for long-term and highly sensitive tracking in live cells. In this review, we describe the development of a QD-based SVT technique and its biol. applications. We first discuss the advantage of QDs as tags in the SVT field by comparing the conventional tags, and then focus on the implementation of QD-based SVT expts., including the QD labeling strategy, instrumentation, and image anal. method. Next, we elaborate the recent advances of QD-based SVT in the biol. field, and mainly emphasize the representative examples to show how to use this technique to acquire more meaningful biol. information.
- 31Brandenburg, B.; Zhuang, X. Virus Trafficking–Learning from Single-Virus Tracking. Nat. Rev. Microbiol. 2007, 5, 197– 208, DOI: 10.1038/nrmicro161531Virus trafficking - learning from single-virus trackingBrandenburg, Boerries; Zhuang, XiaoweiNature Reviews Microbiology (2007), 5 (3), 197-208CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell. Single-virus tracking in living cells potentially provides the authors with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.
- 32Huang, L. L.; Xie, H. Y. Progress on the Labeling and Single-Particle Tracking Technologies of Viruses. Analyst 2014, 139, 3336– 3346, DOI: 10.1039/C4AN00038B32Progress on the labeling and single-particle tracking technologies of virusesHuang, Li-Li; Xie, Hai-YanAnalyst (Cambridge, United Kingdom) (2014), 139 (13), 3336-3346CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)A review. Understanding and unravelling the invasion mechanisms of virus infection is of high importance for preventing and treating viral diseases. Single-virion tracking is a powerful way for exploring the mechanisms of viral infection. For successful tracking, the virus and cellular structures of interest must be fluorescently labeled; the microscope imaging technol. must be sufficiently powerful for real-time single virion or viral component tracking. All fields of scientists have made great efforts and improvements. Here we will review the recent advances in virus labeling and the emerging fluorescence imaging technologies used in the imaging and tracking of viruses.
- 33Parveen, N.; Borrenberghs, D.; Rocha, S.; Hendrix, J. Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication. Viruses 2018, 10, 250, DOI: 10.3390/v1005025033Single viruses on the fluorescence microscope: imaging molecular mobility, interactions and structure sheds new light on viral replicationParveen, Nagma; Borrenberghs, Doortje; Rocha, Susana; Hendrix, JelleViruses (2018), 10 (5), 250/1-250/21CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virol. Indeed, viral replication is often not particularly efficient, prone to errors or contg. parallel routes. Here, we review different single-mol. sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-mol. F.ovrddot.orster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resoln. microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virol. practice.
- 34Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics. Biophys. J. 1976, 16, 1055– 1069, DOI: 10.1016/S0006-3495(76)85755-434Mobility measurement by analysis of fluorescence photobleaching recovery kineticsAxelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W.Biophysical Journal (1976), 16 (9), 1055-69CODEN: BIOJAU; ISSN:0006-3495.Fluorescence photobleaching recovery (FPR) denotes a method for measuring 2-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled mols. in ∼10 μm2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. The theor. basis and some practical guidelines are presented for simple, rigorous anal. of FPR expts. Information obtainable from FPR expts. includes: identification of transport process type, i.e., the admixt. of random diffusion and uniform directed flow; detn. of the abs. mobility coeff., i.e., the diffusion const. and (or) flow velocity; and the fraction of total fluorophore that is mobile. To illustrate the exptl. method and to verify the theory for diffusion, exptl. models are described comprising aq. solns. of rhodamine 6G.
- 35Simons, K.; Gerl, M. J. Revitalizing Membrane Rafts: New Tools and Insights. Nat. Rev. Mol. Cell Biol. 2010, 11, 688– 699, DOI: 10.1038/nrm297735Revitalizing membrane rafts: New tools and insightsSimons, Kai; Gerl, Mathias J.Nature Reviews Molecular Cell Biology (2010), 11 (10), 688-699CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Ten years ago, the authors wrote a review on lipid rafts and signaling. At the time, this field was suffering from ambiguous methodol. and imprecise nomenclature. Now, however, new techniques are deepening the insight into the dynamics of membrane organization. Here, the authors discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol, and proteins that can be stabilized into platforms that are important in signaling, viral infection, and membrane trafficking.
- 36Magde, D.; Elson, E.; Webb, W. W. Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy. Phys. Rev. Lett. 1972, 29, 705– 708, DOI: 10.1103/PhysRevLett.29.70536Thermodynamic fluctations in a reacting system. Measurement by fluorescence correlation spectroscopyMagde, Douglas; Elson, Elliot; Webb, W. W.Physical Review Letters (1972), 29 (11), 705-8CODEN: PRLTAO; ISSN:0031-9007.Correlations of thermodynamic concn. fluctuations were measured in a chem. reactive system at equil. by observing fluctuations of the fluorescence of a reaction producct. The expt. yields the chem. rate consts. and diffusion coeffs. and shows the coupling among them. Data are reported for binding of ethidium bromide to DNA.
- 37Hess, S. T.; Huang, S.; Heikal, A. A.; Webb, W. W. Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review. Biochemistry 2002, 41, 697– 705, DOI: 10.1021/bi011851237Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A ReviewHess, Samuel T.; Huang, Shaohui; Heikal, Ahmed A.; Webb, Watt W.Biochemistry (2002), 41 (3), 697-705CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review on historical perspective and theor. background of fluorescence correlation spectroscopy (FCS), exptl. setup for FCS, FCS applications in soln., and cellular applications of FCS.
- 38Haustein, E.; Schwille, P. Ultrasensitive Investigations of Biological Systems by Fluorescence Correlation Spectroscopy. Methods 2003, 29, 153– 166, DOI: 10.1016/S1046-2023(02)00306-738Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopyHaustein, Elke; Schwille, PetraMethods (San Diego, CA, United States) (2003), 29 (2), 153-166CODEN: MTHDE9; ISSN:1046-2023. (Elsevier Science)A review. Fluorescence correlation spectroscopy (FCS) exts. information about mol. dynamics from the tiny fluctuations that can be obsd. in the emission of small ensembles of fluorescent mols. in thermodn. equil. Employing a confocal setup in conjunction with highly dil. samples, the av. no. of fluorescent particles simultaneously within the measurement vol. (∼1 fl) is minimized. Among the multitude of chem. and phys. parameters accessible by FCS are local concns., mobility coeffs., rate consts. for assocn. and dissocn. processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resoln. of less than 0.5 μm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local mol. dynamics provides access to environmental parameters such as local oxygen concns., pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quant. assessment of interactions and dynamics of small mol. quantities in biol. relevant systems.
- 39Berg, H. C. How to Track Bacteria. Rev. Sci. Instrum. 1971, 42, 868– 871, DOI: 10.1063/1.168524639How to track bacteriaBerg H CThe Review of scientific instruments (1971), 42 (6), 868-71 ISSN:0034-6748.There is no expanded citation for this reference.
- 40Berg, H. C.; Brown, D. A. Chemotaxis in Escherichia Coli Analysed by Three-Dimensional Tracking. Nature 1972, 239, 500– 504, DOI: 10.1038/239500a040Chemotaxis in Escherichia coli analyzed by three-dimensional trackingBerg, Howard C.; Brown, Douglas A.Nature (London, United Kingdom) (1972), 239 (5374), 500-4CODEN: NATUAS; ISSN:0028-0836.If serine is added to suspensions (no gradients), the run-length distributions are exponential but shift toward longer runs, and twiddles are suppressed. The shift does not occur with aspartate. The shift is not a metabolic effect. The bacteria runs are longer in the up-gradient swim than down-gradient and with less frequent direction changes. For serine, the distribution of runs down the gradient is similar to the distribution in a 9 μM isotropic soln. and for aspartate it is indistinguishable from that of the control. The statistics are Poisson. When a bacterium swims down the gradient, there is no functional relation between the length of a run and the derivs. of the concn. with respect to space or time. This is true both for serine and aspartate.
- 41Barak, L. S.; Webb, W. W. Diffusion of Low Density Lipoprotein-Receptor Complex on Human Fibroblasts. J. Cell Biol. 1982, 95, 846– 852, DOI: 10.1083/jcb.95.3.84641Diffusion of low density lipoprotein-receptor complex on human fibroblastsBarak L S; Webb W WThe Journal of cell biology (1982), 95 (3), 846-52 ISSN:0021-9525.Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.
- 42De Brabander, M.; Geuens, G.; Nuydens, R.; Moeremans, M.; De Mey, J. Probing Microtubule-Dependent Intracellular Motility with Nanometre Particle Video Ultramicroscopy (Nanovid Ultramicroscopy). Cytobios 1985, 43, 273– 28342Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy)De Brabander M; Geuens G; Nuydens R; Moeremans M; De Mey JCytobios (1985), 43 (174S), 273-83 ISSN:0011-4529.Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.
- 43Lee, G. M.; Ishihara, A.; Jacobson, K. A. Direct Observation of Brownian Motion of Lipids in a Membrane. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 6274– 6278, DOI: 10.1073/pnas.88.14.627443Direct observation of Brownian motion of lipids in a membraneLee, Greta M.; Ishihara, Akira; Jacobson, Ken A.Proceedings of the National Academy of Sciences of the United States of America (1991), 88 (14), 6274-8CODEN: PNASA6; ISSN:0027-8424.Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual mols. in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid mols. were followed in a supported, planar bilayer contg. fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepd. by conjugating only anti-Fl to the gold. Paucivalent probes were prepd. by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an av. lateral diffusion coeff. (D) of 0.26 × 10-8 cm2/s, and paucivalent probes had an av. D of 0.73 × 10-8 cm2/s. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 × 10-8 cm2/s, which, after allowance for stochastic variation, are consistent with the D of 1.3 × 10-8 cm2/s measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diam., resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results, nanovid microscopy is a valid method for analyzing the movements of single or small groups of mols. within membranes.
- 44Gelles, J.; Schnapp, B. J.; Sheetz, M. P. Tracking Kinesin-Driven Movements with Nanometre-Scale Precision. Nature 1988, 331, 450– 453, DOI: 10.1038/331450a044Tracking kinesin-driven movements with nanometer-scale precisionGelles, Jeff; Schnapp, Bruce J.; Sheetz, Michael P.Nature (London, United Kingdom) (1988), 331 (6155), 450-3CODEN: NATUAS; ISSN:0028-0836.Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (≤10) kinesin mols. may be sufficient to drive either bead movement or organelle transport. A method is described for detg. precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Measurements reveal basic mech. features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible mol. mechanisms of movement.
- 45Geerts, H.; de Brabander, M.; Nuydens, R. Nanovid Microscopy. Nature 1991, 351, 765– 766, DOI: 10.1038/351765a045Nanovid microscopyGeerts H; de Brabander M; Nuydens RNature (1991), 351 (6329), 765-6 ISSN:0028-0836.By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied.
- 46Geerts, H.; De Brabander, M.; Nuydens, R.; Geuens, S.; Moeremans, M.; De Mey, J.; Hollenbeck, P. Nanovid Tracking: A New Automatic Method for the Study of Mobility in Living Cells Based on Colloidal Gold and Video Microscopy. Biophys. J. 1987, 52, 775– 782, DOI: 10.1016/S0006-3495(87)83271-X46Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopyGeerts H; De Brabander M; Nuydens R; Geuens S; Moeremans M; De Mey J; Hollenbeck PBiophysical journal (1987), 52 (5), 775-82 ISSN:0006-3495.We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.
- 47Inoué, S. Imaging of Unresolved Objects, Superresolution, and Precision of Distance Measurement with Video Microscopy. Methods Cell Biol. 1989, 30, 85– 112, DOI: 10.1016/S0091-679X(08)60976-047Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopyInoue SMethods in cell biology (1989), 30 (), 85-112 ISSN:0091-679X.There is no expanded citation for this reference.
- 48Saxton, M. J. Single-Particle Tracking: Models of Directed Transport. Biophys. J. 1994, 67, 2110– 2019, DOI: 10.1016/S0006-3495(94)80694-048Single-particle tracking: models of directed transportSaxton, Michael J.Biophysical Journal (1994), 67 (5), 2110-19CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Single-particle tracking techniques make it possible to measure motion of individual particles on the cell surface. In these expts., individual trajectories are obsd., so the data anal. must take into account the randomness of individual random walks. Methods of data anal. are discussed for models combining diffusion and directed motion. In the uniform flow model, a tracer simultaneously diffuses and undergoes directed motion. In the conveyor belt model, a tracer binds and unbinds to a uniform conveyor belt moving with const. velocity. If a trcer is bound, it moves at the velocity of the conveyor belt; if it is unbound, it diffuses freely. Trajectories are analyzed using parameters that measure the extent and asymmetry of the trajectory. A method of assessing the usefulness of such parameters is presented, and pitfalls in data anal. are discussed. Joint probability distributions of pairs of extent and asymmetry parameters are obtained for a pure random walk. These distributions can be used to show that a trajectory is not likely to have resulted from a pure random walk.
- 49Kao, H. P.; Verkman, A. S. Tracking of Single Fluorescent Particles in 3 Dimensions-Use of Cylindrical Optics to Encode Particle Position. Biophys. J. 1994, 67, 1291– 1300, DOI: 10.1016/S0006-3495(94)80601-049Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle positionKao H P; Verkman A SBiophysical journal (1994), 67 (3), 1291-300 ISSN:0006-3495.We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.
- 50Patwardhan, A. Subpixel Position Measurement Using 1D, 2D and 3D Centroid Algorithms with Emphasis on Applications in Confocal Microscopy. J. Microsc. 1997, 186, 246– 257, DOI: 10.1046/j.1365-2818.1997.1970761.xThere is no corresponding record for this reference.
- 51Peters, I. M.; van Kooyk, Y.; van Vliet, S. J.; de Grooth, B. G.; Figdor, C. G.; Greve, J. 3D Single-Particle Tracking and Optical Trap Measurements on Adhesion Proteins. Cytometry 1999, 36, 189– 194, DOI: 10.1002/(SICI)1097-0320(19990701)36:3<189::AID-CYTO7>3.0.CO;2-3513D single-particle tracking and optical trap measurements on adhesion proteinsPeters, Inge M.; Van Kooyk, Yvette; Van Vliet, Sandra J.; De Grooth, Bart G.; Figdor, Carl G.; Greve, JanCytometry (1999), 36 (3), 189-194CODEN: CYTODQ; ISSN:0196-4763. (Wiley-Liss, Inc.)A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an α and a β chain. Thus far, only the cytoplasmic tail of the β chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete β cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the β chain.
- 52Bacher, C. P.; Reichenzeller, M.; Athale, C.; Herrmann, H.; Eils, R. 4-D Single Particle Tracking of Synthetic and Proteinaceous Microspheres Reveals Preferential Movement of Nuclear Particles Along Chromatin-Poor Tracks. BMC Cell Biol. 2004, 5, 45, DOI: 10.1186/1471-2121-5-45524-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracksBacher Christian P; Reichenzeller Michaela; Athale Chaitanya; Herrmann Harald; Eils RolandBMC cell biology (2004), 5 (), 45 ISSN:.BACKGROUND: The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. RESULTS: We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. CONCLUSIONS: Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.
- 53Genovesio, A.; Liedl, T.; Emiliani, V.; Parak, W. J.; Coppey-Moisan, M.; Olivo-Marin, J. C. Multiple Particle Tracking in 3-D+T Microscopy: Method and Application to the Tracking of Endocytosed Quantum Dots. IEEE Trans. Image Process. 2006, 15, 1062– 1070, DOI: 10.1109/TIP.2006.87232353Multiple particle tracking in 3-D+t microscopy: method and application to the tracking of endocytosed quantum dotsGenovesio Auguste; Liedl Tim; Emiliani Valentina; Parak Wolfgang J; Coppey-Moisan Maite; Olivo-Marin Jean-ChristopheIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2006), 15 (5), 1062-70 ISSN:1057-7149.We propose a method to detect and track multiple moving biological spot-like particles showing different kinds of dynamics in image sequences acquired through multidimensional fluorescence microscopy. It enables the extraction and analysis of information such as number, position, speed, movement, and diffusion phases of, e.g., endosomal particles. The method consists of several stages. After a detection stage performed by a three-dimensional (3-D) undecimated wavelet transform, we compute, for each detected spot, several predictions of its future state in the next frame. This is accomplished thanks to an interacting multiple model (IMM) algorithm which includes several models corresponding to different biologically realistic movement types. Tracks are constructed, thereafter, by a data association algorithm based on the maximization of the likelihood of each IMM. The last stage consists of updating the IMM filters in order to compute final estimations for the present image and to improve predictions for the next image. The performances of the method are validated on synthetic image data and used to characterize the 3-D movement of endocytic vesicles containing quantum dots.
- 54Ram, S.; Kim, D.; Ward, E. S.; Ober, R. J. Fast 3D Single Molecule Tracking with Multifocal Plane Microscopy in Polarized Epithelia Reveals a Novel Cellular Process of Intercellular Transfer. Biophys. J. 2013, 104, 535a, DOI: 10.1016/j.bpj.2012.11.2962There is no corresponding record for this reference.
- 55Ram, S.; Kim, D.; Ober, R. J.; Ward, E. S. 3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers. Biophys. J. 2012, 103, 1594– 1603, DOI: 10.1016/j.bpj.2012.08.054553D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell BarriersRam, Sripad; Kim, Dongyoung; Ober, Raimund J.; Ward, E. SallyBiophysical Journal (2012), 103 (7), 1594-1603CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single mol. tracking of rapid protein dynamics in a 10 μ thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf mols. between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf mols. at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.
- 56Ram, S.; Prabhat, P.; Chao, J.; Ward, E. S.; Ober, R. J. High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells. Biophys. J. 2008, 95, 6025– 6043, DOI: 10.1529/biophysj.108.14039256High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cellsRam, Sripad; Prabhat, Prashant; Chao, Jerry; Ward, E. Sally; Ober, Raimund J.Biophysical Journal (2008), 95 (12), 6025-6043CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biol. insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to det. the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and exptl. data and show that the 3D position of quantum dots can be detd. over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be detd. Our anal. shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody mols. undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.
- 57Levi, V.; Ruan, Q.; Kis-Petikova, K.; Gratton, E. Scanning FCS, a Novel Method for Three-Dimensional Particle Tracking. Biochem. Soc. Trans. 2003, 31, 997– 1000, DOI: 10.1042/bst031099757Scanning FCS, a novel method for three-dimensional particle trackingLevi, V.; Ruan, Q.; Kis-Petikova, K.; Gratton, E.Biochemical Society Transactions (2003), 31 (5), 997-1000CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)We describe a novel method to track fluorescent particles in three dimensions with nanometer precision and millisecond time resoln. In this method, we use our two-photon excitation microscope. The galvomotor-driven x-y scanning mirrors allow the laser beam to move repetitively in a circular path with a radius of half the width of the point spread function of the laser. When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-x plane can be detd. by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective was used to change the laser focus at two planes (half width of the point spread function apart). The difference of the fluorescence intensity in the two planes is used to calc. the z-position of the fluorescent particle. The laser beam is allowed to scan multiple circular orbits before it is moved to the other plane, thus improving the signal to noise ratio. With a fast feedback mechanism, the position of the laser beam is directed to the center of the fluorescent particle, thus allowing us to track a particle in three dimensions. In this contribution we describe some calibration expts. performed to test the three-dimensional tracking capability of our system over a large range.
- 58Dupont, A.; Lamb, D. C. Nanoscale Three-Dimensional Single Particle Tracking. Nanoscale 2011, 3, 4532– 4541, DOI: 10.1039/c1nr10989h58Nanoscale three-dimensional single particle trackingDupont, Aurelie; Lamb, Don C.Nanoscale (2011), 3 (11), 4532-4541CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. Single particle tracking (SPT) in biol. systems is a quickly growing field. Many new technologies are being developed providing new tracking capabilities, which also lead to higher demands and expectations for SPT. Following a single biomol. as it performs its function provides quant. mechanistic information that cannot be obtained in classical ensemble methods. From the 3D trajectory, information is available over the diffusional behavior of the particle and precise position information can also be used to elucidate interactions of the tracked particle with its surroundings. Thus, three-dimensional (3D) SPT is a very valuable tool for investigating cellular processes. This review presents recent progress in 3D SPT, from image-based techniques toward more sophisticated feedback approaches. We focus mainly on the feedback technique known as orbital tracking. We present here a modified version of the original orbital tracking in which the intensities from two z-planes are simultaneously measured allowing a concomitant wide-field imaging. The system can track single particles with a precision down to 5 nm in the x-y plane and 7 nm in the axial direction. The capabilities of the system are demonstrated using single virus tracing to follow the infection pathway of Prototype Foamy Virus in living cells.
- 59Ruthardt, N.; Lamb, D. C.; Brauchle, C. Single-Particle Tracking as a Quantitative Microscopy-Based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical Nanoparticles. Mol. Ther. 2011, 19, 1199– 1211, DOI: 10.1038/mt.2011.10259Single-particle Tracking as a Quantitative Microscopy-based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical NanoparticlesRuthardt, Nadia; Lamb, Don C.; Braeuchle, ChristophMolecular Therapy (2011), 19 (7), 1199-1211CODEN: MTOHCK; ISSN:1525-0016. (Nature Publishing Group)A review. Highly sensitive fluorescence microscopy techniques allow single nanoparticles to be tracked during their uptake into living cells with high temporal and spatial resoln. From anal. of the trajectories, random motion can be discriminated from active transport and the av. transport velocity and/or diffusion coeff. detd. Such an anal. provides important information regarding the uptake pathway and location of viruses and nanoparticles. In this review, we give an introduction into single-particle tracking (SPT) and detn. of the mean-squared displacement. We also give an overview of recent advances in SPT. These include millisecond alternating-laser excitation for removal of spectral crosstalk, alternating wide-field (WF), and total internal reflection fluorescence (TIRF) microscopy for sensitive expts. at the plasma membrane and 3-dimensional tracking strategies. Throughout the review, we highlight recent advances regarding the entry (and egress) of natural and artificial viruses obtained via SPT.
- 60Hou, S.; Johnson, C.; Welsher, K. Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells. Molecules 2019, 24, 2826, DOI: 10.3390/molecules2415282660Real-time 3D single particle tracking: towards active feedback single molecule spectroscopy in live cellsHou, Shangguo; Johnson, Courtney; Welsher, KevinMolecules (2019), 24 (15), 2826CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)A review. Single mol. fluorescence spectroscopy has been largely implemented using methods which require tethering of mols. to a substrate in order to make high temporal resoln. measurements. However, the act of tethering a mol. requires that the mol. be removed from its environment. This is esp. perturbative when measuring biomols. such as enzymes, which may rely on the non-equil. and crowded cellular environment for normal function. A method which may be able to un-tether single mol. fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resoln. over large axial ranges. This gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single mol. tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.
- 61Montiel, D.; Yang, H. Real-Time Three-Dimensional Single-Particle Tracking Spectroscopy for Complex Systems. Laser Photonics Rev. 2010, 4, 374– 385, DOI: 10.1002/lpor.20091001261Real-time three-dimensional single-particle tracking spectroscopy for complex systemsMontiel, Daniel; Yang, HawLaser & Photonics Reviews (2010), 4 (3), 374-385CODEN: LPRAB8; ISSN:1863-8880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Complex systems are characterized by dynamical processes spread over multiple time and length scales. At a given instant, these systems can display spatial heterogeneities in which the local phys. and chem. properties are nonuniform, depending on the location. They can also exhibit dynamical heterogeneities in which the local dynamical characteristics vary with time. These types of systems pose unique exptl. challenges for their characterization and test of theor. ideas. Recently, real-time three-dimensional (3D) single-particle tracking spectroscopy has been developed to address these kinds of problems. With this approach, in principle, one can follow how a system evolves spatially as well as temporally. This article attempts to provide an introduction to this promising new technique by discussing the aims of studying a complex system and recent exptl. advances towards this goal.
- 62Walsh, E. E.; Hruska, J. Monoclonal Antibodies to Respiratory Syncytial Virus Proteins: Identification of the Fusion Protein. J. Virol. 1983, 47, 171– 177, DOI: 10.1128/JVI.47.1.171-177.198362Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion proteinWalsh, Edward E.; Hruska, JeromeJournal of Virology (1983), 47 (1), 171-7CODEN: JOVIAM; ISSN:0022-538X.Six monoclonal antibodies directed against respiratory syncytial virus proteins were produced. Each was characterized by immunopptn. and indirect immunofluorescence. One was directed against the nucleocapsid protein, NP 44, 2 were directed against a 37,000-dalton protein, 2 were directed against the major envelope glycoprotein, GP 90, and 1 was directed against the 70,000-dalton envelope protein, VP 70. Indirect immunofluorescence stain patterns of infected HEp-2 cells defined GP 90 and VP 70 as viral proteins expressed on the cell surface, whereas NP 44 and the 37,000-dalton protein were detected as intracytoplasmic inclusions. One of the anti-GP 90 antibodies neutralized virus only in the presence of complement but did not inhibit cell-cell fusion. The anti-VP 70 antibody neutralized virus without complement and inhibited cell-cell fusion of previously infected HEp-2, thus identifying VP 70 as the fusion protein.
- 63Matlin, K. S.; Reggio, H.; Helenius, A.; Simons, K. Infectious Entry Pathway of Influenza Virus in a Canine Kidney Cell Line. J. Cell Biol. 1981, 91, 601– 613, DOI: 10.1083/jcb.91.3.60163Infectious entry pathway of influenza virus in a canine kidney cell lineMatlin K S; Reggio H; Helenius A; Simons KThe Journal of cell biology (1981), 91 (3 Pt 1), 601-13 ISSN:0021-9525.The entry of fowl plague virus, and avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase-resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.
- 64Bächi, T. Direct Observation of the Budding and Fusion of an Enveloped Virus by Video Microscopy of Viable Cells. J. Cell Biol. 1988, 107, 1689– 1695, DOI: 10.1083/jcb.107.5.168964Direct observation of the budding and fusion of an enveloped virus by video microscopy of viable cellsBachi TThe Journal of cell biology (1988), 107 (5), 1689-95 ISSN:0021-9525.Video-enhanced microscopy and digital image processing were used to observe the assembly, budding, and fusion of Respiratory Syncytial virus. Viral filaments were seen to bud from the plasma membrane of viable infected cells to a final length of 5-10 micron with an average speed of elongation of 110-250 nm/s. The rapidity of viral assembly and its synchronous occurrence (leading to the production of several viral particles per minute from the same surface domain) suggests a directed process of recruitment of viral components to an area selected for virus maturation. Virions were also seen to adsorb to the cell surface, and to fuse with the plasma membrane. These are the first real time observations of viral morphogenesis and penetration which are crucial events in the infectious cycle of enveloped viruses.
- 65Lowy, R. J.; Sarkar, D. P.; Chen, Y.; Blumenthal, R. Observation of Single Influenza Virus-Cell Fusion and Measurement by Fluorescence Video Microscopy. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 1850– 1854, DOI: 10.1073/pnas.87.5.185065Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopyLowy, R. J.; Sarkar, D. P.; Chen, Y.; Blumenthal, R.Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (5), 1850-4CODEN: PNASA6; ISSN:0027-8424.Intensified video fluorescence microscopy and digital image processing were used to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) experienced fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quant. intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calc. the fraction of R18 dye mols. transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approx. twice as fast as pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was obsd. after the pH changes, suggesting that activation processes are faster than the time resoln., 1-5 s, of the current method.
- 66Georgi, A.; Mottola-Hartshorn, C.; Warner, A.; Fields, B.; Chen, L. B. Detection of Individual Fluorescently Labeled Reovirions in Living Cells. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 6579– 6583, DOI: 10.1073/pnas.87.17.657966Detection of individual fluorescently labeled reovirions in living cellsGeorgi, Ann; Mottola-Hartshorn, Cristina; Warner, Angeline; Fields, Bernard; Chen, Lan BoProceedings of the National Academy of Sciences of the United States of America (1990), 87 (17), 6579-83CODEN: PNASA6; ISSN:0027-8424.Reovirus serotype 1 (Lang) can be conjugated with rhodamine B or fluorescein isothiocyanate in a way that preserves viral infectivity. Epifluorescence microscopy was used to detect individual virions bound to the surface of cells and to follow in real time the early stages of reovirus infection in living mouse fibroblast cells. Following uptake of the virus into endocytic vesicles, the movement was inhibited by nocodazole or colchicine, which was consistent with previous findings that the movement of intracellular vesicles is often microtubule-based.
- 67Pelkmans, L.; Kartenbeck, J.; Helenius, A. Caveolar Endocytosis of Simian Virus 40 Reveals a New Two-Step Vesicular-Transport Pathway to the ER. Nat. Cell Biol. 2001, 3, 473– 483, DOI: 10.1038/3507453967Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ERPelkmans, Lucas; Kartenbeck, Jurgen; Helenius, AriNature Cell Biology (2001), 3 (5), 473-483CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)SV40 virus is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-color, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After assocg. with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-contg. vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-pos., smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.
- 68Lakadamyali, M.; Rust, M. J.; Babcock, H. P.; Zhuang, X. Visualizing Infection of Individual Influenza Viruses. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9280– 9285, DOI: 10.1073/pnas.083226910068Visualizing infection of individual influenza virusesLakadamyali, Melike; Rust, Michael J.; Babcock, Hazen P.; Zhuang, XiaoweiProceedings of the National Academy of Sciences of the United States of America (2003), 100 (16), 9280-9285CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cell's constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.
- 69Seisenberger, G.; Ried, M. U.; Endress, T.; Buning, H.; Hallek, M.; Brauchle, C. Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus. Science 2001, 294, 1929– 1932, DOI: 10.1126/science.106410369Real-time single-molecule imaging of the infection pathway of an adeno-associated virusSeisenberger, Georg; Ried, Martin U.; Endress, Thomas; Buening, Hildegard; Hallek, Michael; Brauchle, ChristophScience (Washington, DC, United States) (2001), 294 (5548), 1929-1932CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We describe a method, based on single-mol. imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye mol. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resoln. show various modes of motion of adeno-assocd. viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally obsd. so far.
- 70Suomalainen, M.; Nakano, M. Y.; Keller, S.; Boucke, K.; Stidwill, R. P.; Greber, U. F. Microtubule-Dependent Plus- and Minus End-Directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus. J. Cell Biol. 1999, 144, 657– 672, DOI: 10.1083/jcb.144.4.65770Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirusSuomalainen, Maarit; Nakano, Michel Y.; Keller, Stephan; Boucke, Karin; Stidwill, Robert P.; Greber, Urs F.Journal of Cell Biology (1999), 144 (4), 657-672CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was obsd. in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was obsd. in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end-directed cytoplasmic dynein and an unknown plus end-directed activity.
- 71Coller, K. E.; Berger, K. L.; Heaton, N. S.; Cooper, J. D.; Yoon, R.; Randall, G. RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis. PLoS Pathog. 2009, 5, e1000702 DOI: 10.1371/journal.ppat.1000702There is no corresponding record for this reference.
- 72Rust, M. J.; Lakadamyali, M.; Zhang, F.; Zhuang, X. Assembly of Endocytic Machinery around Individual Influenza Viruses During Viral Entry. Nat. Struct. Mol. Biol. 2004, 11, 567– 573, DOI: 10.1038/nsmb76972Assembly of endocytic machinery around individual influenza viruses during viral entryRust, Michael J.; Lakadamyali, Melike; Zhang, Feng; Zhuang, XiaoweiNature Structural & Molecular Biology (2004), 11 (6), 567-573CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. The authors have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. The results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.
- 73van der Schaar, H. M.; Rust, M. J.; Waarts, B. L.; van der Ende-Metselaar, H.; Kuhn, R. J.; Wilschut, J.; Zhuang, X.; Smit, J. M. Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking. J. Virol. 2007, 81, 12019– 12028, DOI: 10.1128/JVI.00300-0773Characterization of the early events in Dengue virus cell entry by biochemical assays and single-virus trackingvan der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaar, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.Journal of Virology (2007), 81 (21), 12019-12028CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concn. of virus particles measured by biochem. assays was found to be substantially higher than the no. of infectious particles detd. by infectivity assays, leading to an infectious unit-to-particle ratio of approx. 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface d. of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was obsd. that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was obsd. in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.
- 74Chen, C.; Zhuang, X. Epsin 1 Is a Cargo-Specific Adaptor for the Clathrin-Mediated Endocytosis of the Influenza Virus. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11790– 11795, DOI: 10.1073/pnas.080371110574Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virusChen, Chen; Zhuang, XiaoweiProceedings of the National Academy of Sciences of the United States of America (2008), 105 (33), 11790-11795,CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)During clathrin-mediated endocytosis, adaptor proteins recognize specific internalization signals on cargo receptors, either recruiting cargos into clathrin-coated pits (CCPs) or initiating clathrin-coat assembly around the cargo mols. Here, we identify epsin 1, a clathrin-, ubiquitin-, and phospholipid-interacting protein, as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. Using live-cell imaging to monitor the entry of individual virus particles, we obsd. recruitment of epsin 1 to the binding sites of influenza viruses in synchrony with the assembly of CCPs. Epsin 1 knockdown by siRNA significantly inhibited the clathrin-mediated endocytosis of the influenza virus and caused the majority of the virus particles to enter through a clathrin-independent pathway. The same treatment did not affect the entry of several classical ligands for clathrin-mediated endocytosis, including transferrin, LDL, and EGF. Overexpression of the dominant-neg. epsin 1 mutant lacking the ubiquitin-interaction motifs nearly completely blocked the clathrin-mediated entry of the influenza virus without affecting transferrin uptake. These results suggest that epsin 1 functions as a cargo-specific adaptor for the clathrin-mediated entry of the influenza virus.
- 75van der Schaar, H. M.; Rust, M. J.; Chen, C.; van der Ende-Metselaar, H.; Wilschut, J.; Zhuang, X.; Smit, J. M. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells. PLoS Pathog. 2008, 4, e1000244 DOI: 10.1371/journal.ppat.100024475Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cellsvan der Schaar Hilde M; Rust Michael J; Chen Chen; van der Ende-Metselaar Heidi; Wilschut Jan; Zhuang Xiaowei; Smit Jolanda MPLoS pathogens (2008), 4 (12), e1000244 ISSN:.Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.
- 76Tsien, R. Y. The Green Fluorescent Protein. Annu. Rev. Biochem. 1998, 67, 509– 544, DOI: 10.1146/annurev.biochem.67.1.50976The green fluorescent proteinTsien, Roger Y.Annual Review of Biochemistry (1998), 67 (), 509-544CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review, with ∼114 refs. In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochem. and cell biol. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resoln. crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiol. indicators, biosensors, and photochem. memories.
- 77Baulcombe, D. C.; Chapman, S.; Santa Cruz, S. Jellyfish Green Fluorescent Protein as a Reporter for Virus Infections. Plant J. 1995, 7, 1045– 1053, DOI: 10.1046/j.1365-313X.1995.07061045.x77Jellyfish green fluorescent protein as a reporter for virus infectionsBaulcombe, David C.; Chapman, Sean; Cruz, Simon SantaPlant Journal (1995), 7 (6), 1045-53CODEN: PLJUED; ISSN:0960-7412. (Blackwell)The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Prodn. of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analyzed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of exts. from infected plants. To illustrate the potential application of this reporter gene in virol. studies a deriv. of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.
- 78Elliott, G.; O’Hare, P. Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection. J. Virol. 1999, 73, 4110– 4119, DOI: 10.1128/JVI.73.5.4110-4119.199978Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infectionElliott, Gillian; O'Hare, PeterJournal of Virology (1999), 73 (5), 4110-4119CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here, the authors describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). This virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, the authors have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, the authors have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.
- 79Cruz, S. S.; Chapman, S.; Roberts, A. G.; Roberts, I. M.; Prior, D.; Oparka, K. J. Assembly and Movement of a Plant Virus Carrying a Green Fluorescent Protein Overcoat. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 6286– 6290, DOI: 10.1073/pnas.93.13.628679Assembly and movement of a plant virus carrying a green fluorescent protein overcoatCruz S S; Chapman S; Roberts A G; Roberts I M; Prior D A; Oparka K JProceedings of the National Academy of Sciences of the United States of America (1996), 93 (13), 6286-90 ISSN:0027-8424.Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.
- 80Desai, P.; Person, S. Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid. J. Virol. 1998, 72, 7563– 7568, DOI: 10.1128/JVI.72.9.7563-7568.199880Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsidDesai, Prashant; Person, StanleyJournal of Virology (1998), 72 (9), 7563-7568CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An anal. of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) anal. of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was obsd., confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
- 81Moradpour, D.; Evans, M. J.; Gosert, R.; Yuan, Z.; Blum, H. E.; Goff, S. P.; Lindenbach, B. D.; Rice, C. M. Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes. J. Virol. 2004, 78, 7400– 7409, DOI: 10.1128/JVI.78.14.7400-7409.200481Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexesMoradpour, Darius; Evans, Matthew J.; Gosert, Rainer; Yuan, Zhenghong; Blum, Hubert E.; Goff, Stephen P.; Lindenbach, Brett D.; Rice, Charles M.Journal of Virology (2004), 78 (14), 7400-7409CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Hepatitis C virus (HCV) replicates its genome in a membrane-assocd. replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. The authors describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
- 82McDonald, D.; Vodicka, M. A.; Lucero, G.; Svitkina, T. M.; Borisy, G. G.; Emerman, M.; Hope, T. J. Visualization of the Intracellular Behavior of HIV in Living Cells. J. Cell Biol. 2002, 159, 441– 452, DOI: 10.1083/jcb.20020315082Visualization of the intracellular behavior of HIV in living cellsMcDonald, David; Vodicka, Marie A.; Lucero, Ginger; Svitkina, Tatyana M.; Borisy, Gary G.; Emerman, Michael; Hope, Thomas J.Journal of Cell Biology (2002), 159 (3), 441-452CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to det. the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resoln. imaging of microtubule-assocd. structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.
- 83Miyauchi, K.; Kim, Y.; Latinovic, O.; Morozov, V.; Melikyan, G. B. HIV Enters Cells via Endocytosis and Dynamin-Dependent Fusion with Endosomes. Cell 2009, 137, 433– 444, DOI: 10.1016/j.cell.2009.02.04683HIV enters cells via endocytosis and dynamin-dependent fusion with endosomesMiyauchi, Kosuke; Kim, Yuri; Latinovic, Olga; Morozov, Vladimir; Melikyan, Gregory B.Cell (Cambridge, MA, United States) (2009), 137 (3), 433-444CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Enveloped viruses that rely on a low pH-dependent step for entry initiate infection by fusing with acidic endosomes, whereas the entry sites for pH-independent viruses, such as HIV-1, have not been defined. These viruses have long been assumed to fuse directly with the plasma membrane. Here, the authors used population-based measurements of the viral content delivery into the cytosol and time-resolved imaging of single viruses to demonstrate that complete HIV-1 fusion occurred in endosomes. In contrast, viral fusion with the plasma membrane did not progress beyond the lipid mixing step. HIV-1 underwent receptor-mediated internalization long before endosomal fusion, thus minimizing the surface exposure of conserved viral epitopes during fusion and reducing the efficacy of inhibitors targeting these epitopes. The authors also show that, strikingly, endosomal fusion is sensitive to a dynamin inhibitor, dynasore. These findings imply that HIV-1 infects cells via endocytosis and envelope glycoprotein- and dynamin-dependent fusion with intracellular compartments.
- 84Koch, P.; Lampe, M.; Godinez, W. J.; Müller, B.; Rohr, K.; Kräusslich, H.-G.; Lehmann, M. J. Visualizing Fusion of Pseudotyped HIV-1 Particles in Real Time by Live Cell Microscopy. Retrovirology 2009, 6, 84, DOI: 10.1186/1742-4690-6-8484Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopyKoch Peter; Lampe Marko; Godinez William J; Muller Barbara; Rohr Karl; Krausslich Hans-Georg; Lehmann Maik JRetrovirology (2009), 6 (), 84 ISSN:.BACKGROUND: Most retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown. RESULTS: We generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env. CONCLUSION: These results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.
- 85Endreß, T.; Lampe, M.; Briggs, J. A. G.; Kräusslich, H.-G.; Bräuchle, C.; Müller, B.; Lamb, D. C. HIV-1–Cellular Interactions Analyzed by Single Virus Tracing. Eur. Biophys. J. 2008, 37, 1291– 1301, DOI: 10.1007/s00249-008-0322-z85HIV-1-cellular interactions analyzed by single virus tracingEndress, Thomas; Lampe, Marko; Briggs, John A. G.; Kraeusslich, Hans-Georg; Braeuchle, Christoph; Mueller, Barbara; Lamb, Don C.European Biophysics Journal (2008), 37 (8), 1291-1301CODEN: EBJOE8; ISSN:0175-7571. (Springer)Single virus tracing (SVT) allows the direct investigation of the entry pathway of viruses into living cells. Using fluorescently labeled virus-like particles (VLPs) and SVT, we have studied the interaction between human immunodeficiency virus type 1 (HIV-1) and the plasma membrane of living cells. From the trajectories of freely diffusing VLPs in soln., we established that the particle prepn. was homogeneous and the particles had a hydrodynamic radius of 86 ± 5 nm, consistent with the size of single HI viruses. The VLPs that come in contact with the cell surface either become immobilized or rapidly dissoc. from the cell surface. The fraction of virions that become immobilized on the plasma membrane correlates with the surface heparan sulfate linked proteoglycans (HSPG) concn. of the cell line tested. The particles that are not immobilized make an av. of 1.5 contacts with the cell surface before diffusing away. For most cell lines investigated, the contact duration follows an exponential distribution with a lifetime between 20 and 50 ms depending on the cell type.
- 86Jolly, C.; Kashefi, K.; Hollinshead, M.; Sattentau, Q. J. HIV-1 Cell to Cell Transfer Across an Env-Induced, Actin-Dependent Synapse. J. Exp. Med. 2004, 199, 283– 293, DOI: 10.1084/jem.2003064886HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapseJolly, Clare; Kashefi, Kirk; Hollinshead, Michael; Sattentau, Quentin J.Journal of Experimental Medicine (2004), 199 (2), 283-293CODEN: JEMEAV; ISSN:0022-1007. (Rockefeller University Press)Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4+/CXCR4+ target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-assocd. antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane mols. into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was obsd. by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV-receptors and adhesion mols. to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.
- 87Arhel, N.; Genovesio, A.; Kim, K. A.; Miko, S.; Perret, E.; Olivo-Marin, J. C.; Shorte, S.; Charneau, P. Quantitative Four-Dimensional Tracking of Cytoplasmic and Nuclear HIV-1 Complexes. Nat. Methods 2006, 3, 817– 824, DOI: 10.1038/nmeth92887Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexesArhel, Nathalie; Genovesio, Auguste; Kim, Kyeong-Ae; Miko, Sarah; Perret, Emmanuelle; Olivo-Marin, Jean-Christophe; Shorte, Spencer; Charneau, PierreNature Methods (2006), 3 (10), 817-824CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein deriv. FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quant. particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined vol. once assocd. with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.
- 88Hubner, W.; McNerney, G. P.; Chen, P.; Dale, B. M.; Gordon, R. E.; Chuang, F. Y.; Li, X. D.; Asmuth, D. M.; Huser, T.; Chen, B. K. Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses. Science 2009, 323, 1743– 1747, DOI: 10.1126/science.116752588Quantitative 3D video microscopy of HIV transfer across T cell virological synapsesHubner Wolfgang; McNerney Gregory P; Chen Ping; Dale Benjamin M; Gordon Ronald E; Chuang Frank Y S; Li Xiao-Dong; Asmuth David M; Huser Thomas; Chen Benjamin KScience (New York, N.Y.) (2009), 323 (5922), 1743-7 ISSN:.The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized "buttons" containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.
- 89Dahan, M.; Levi, S.; Luccardini, C.; Rostaing, P.; Riveau, B.; Triller, A. Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking. Science 2003, 302, 442– 445, DOI: 10.1126/science.108852589Diffusion dynamics of glycine receptors revealed by single-quantum dot trackingDahan, Maxime; Levi, Sabine; Luccardini, Camilla; Rostaing, Philippe; Riveau, Beatrice; Triller, AntoineScience (Washington, DC, United States) (2003), 302 (5644), 442-445CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biol. imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was obsd. and further confirmed by electron microscopy imaging of QD-tagged receptors.
- 90Bonneau, S.; Dahan, M.; Cohen, L. D. Single Quantum Dot Tracking Based on Perceptual Grouping Using Minimal Paths in a Spatiotemporal Volume. IEEE T. Image Process 2005, 14, 1384– 1395, DOI: 10.1109/TIP.2005.85279490Single quantum dot tracking based on perceptual grouping using minimal paths in a spatiotemporal volumeBonneau Stephane; Dahan Maxime; Cohen Laurent DIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2005), 14 (9), 1384-95 ISSN:1057-7149.Semiconductor quantum dots (QDs) are new fluorescent probes with great promise for ultrasensitive biological imaging. When detected at the single-molecule level, QD-tagged molecules can be observed and tracked in the membrane of live cells over unprecedented durations. The motion of these individual molecules, recorded in sequences of fluorescence images, can reveal aspects of the dynamics of cellular processes that remain hidden in conventional ensemble imaging. Due to QD complex optical properties, such as fluorescence intermittency, the quantitative analysis of these sequences is, however, challenging and requires advanced algorithms. We present here a novel approach, which, instead of a frame by frame analysis, is based on perceptual grouping in a spatiotemporal volume. By applying a detection process based on an image fluorescence model, we first obtain an unstructured set of points. Individual molecular trajectories are then considered as minimal paths in a Riemannian metric derived from the fluorescence image stack. These paths are computed with a variant of the fast marching method and few parameters are required. We demonstrate the ability of our algorithm to track intermittent objects both in sequences of synthetic data and in experimental measurements obtained with individual QD-tagged receptors in the membrane of live neurons. While developed for tracking QDs, this method can, however, be used with any fluorescent probes.
- 91Bachir, A. I.; Durisic, N.; Hebert, B.; Grütter, P.; Wiseman, P. W. Characterization of Blinking Dynamics in Quantum Dot Ensembles Using Image Correlation Spectroscopy. J. Appl. Phys. 2006, 99, 064503 DOI: 10.1063/1.217547091Characterization of blinking dynamics in quantum dot ensembles using image correlation spectroscopyBachir, Alexia I.; Durisic, Nela; Hebert, Benedict; Grutter, Peter; Wiseman, Paul W.Journal of Applied Physics (2006), 99 (6), 064503/1-064503/7CODEN: JAPIAU; ISSN:0021-8979. (American Institute of Physics)Quantum dots (QDs) are being increasingly applied as luminescent labels in optical studies for biophys. and cell biol. applications due to their unique spectroscopic properties. However, their fluorescence blinking characteristics that follow power law statistics make it difficult to use QDs in some quant. biophys. applications. The authors present image correlation spectroscopy (ICS) in combination with total internal reflection fluorescence microscopy as a tool to characterize blinking dynamics in QDs. The rate of decay of the ICS measured ensemble correlation function reflects variation in blinking dynamics and can be used to distinguish different blinking distribution regimes. To test and confirm hypothesis, the authors also analyze image time series simulations of ensembles of point emitters with set blinking statistics. Optimization of the temporal sampling and the no. of QDs sampled is essential for detecting changes in blinking dynamics with ICS. Probably this exptl. characterization of the QD blinking statistics can actually serve as a sensitive reporter for certain quant. biol. applications.
- 92Durisic, N.; Bachir, A. I.; Kolin, D. L.; Hebert, B.; Lagerholm, B. C.; Grutter, P.; Wiseman, P. W. Detection and Correction of Blinking Bias in Image Correlation Transport Measurements of Quantum Dot Tagged Macromolecules. Biophys. J. 2007, 93, 1338– 1346, DOI: 10.1529/biophysj.107.10686492Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromoleculesDurisic, Nela; Bachir, Alexia I.; Kolin, David L.; Hebert, Benedict; Lagerholm, B. Christoffer; Grutter, Peter; Wiseman, Paul W.Biophysical Journal (2007), 93 (4), 1338-1346CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biol. applications. Here the authors demonstrate that fluorescence fluctuation anal. of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation anal. of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coeffs. can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and anal. of computer simulated image time series verified that the effect the authors observe is caused by fluorescence intermittency. The authors show that reciprocal space image correlation anal. can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. The authors also demonstrate application of the image correlation methods for measurement of the diffusion coeff. of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts.
- 93He, K.; Luo, W.; Zhang, Y.; Liu, F.; Liu, D.; Xu, L.; Qin, L.; Xiong, C.; Lu, Z.; Fang, X. Intercellular Transportation of Quantum Dots Mediated by Membrane Nanotubes. ACS Nano 2010, 4, 3015– 3022, DOI: 10.1021/nn100219893Intercellular Transportation of Quantum Dots Mediated by Membrane NanotubesHe, Kangmin; Luo, Wangxi; Zhang, Yuliang; Liu, Fei; Liu, Da; Xu, Li; Qin, Lei; Xiong, Chunyang; Lu, Zhizhen; Fang, Xiaohong; Zhang, YouyiACS Nano (2010), 4 (6), 3015-3022CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)In this work, we reported that the quantum dot (QD) nanoparticles could be actively transported in the membrane nanotubes between cardiac myocytes. Single particle imaging and tracking of QDs revealed that most QDs moved in a bidirectional mode along the membrane nanotubes with a mean velocity of 1.23 μm/s. The results suggested that QDs moving in the nanotubes were coordinately motivated by mol. motors. It provides new information for the study of the intercellular transportation of nanoparticles.
- 94Chang, Y.-P.; Pinaud, F.; Antelman, J.; Weiss, S. Tracking Bio-Molecules in Live Cells Using Quantum Dots. J. Biophotonics 2008, 1, 287– 298, DOI: 10.1002/jbio.20081002994Tracking bio-molecules in live cells using quantum dotsChang, Yun-Pei; Pinaud, Fabien; Antelman, Joshua; Weiss, ShimonJournal of Biophotonics (2008), 1 (4), 287-298CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Single particle tracking (SPT) techniques were developed to explore bio-mols. dynamics in live cells at single mol. sensitivity and nanometer spatial resoln. Recent developments in quantum dots (Qdots) surface coating and bio-conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell expts.
- 95Pinaud, F.; Clarke, S.; Sittner, A.; Dahan, M. Probing Cellular Events, One Quantum Dot at a Time. Nat. Methods 2010, 7, 275– 285, DOI: 10.1038/nmeth.144495Probing cellular events, one quantum dot at a timePinaud, Fabien; Clarke, Samuel; Sittner, Assa; Dahan, MaximeNature Methods (2010), 7 (4), 275-285CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Monitoring the behavior of single mols. in living cells is a powerful approach to investigate the details of cellular processes. Owing to their optical, chem. and biofunctional properties, semiconductor quantum dot (QD) probes promise to be tools of choice in this endeavor. Here, the authors review recent advances that allow ever more controlled expts. at the single-nanoparticle level in live cells. Several examples, related to membrane dynamics, cell signaling, or intracellular transport, illustrate how single QD tracking can be readily used to decipher complex biol. processes and address key concepts that underlie cellular organization and dynamics.
- 96Wang, Z. G.; Liu, S. L.; Tian, Z. Q.; Zhang, Z. L.; Tang, H. W.; Pang, D. W. Myosin-Driven Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer Cells. ACS Nano 2012, 6, 10033– 10041, DOI: 10.1021/nn303729r96Myosin-Driven Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer CellsWang, Zhi-Gang; Liu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Tang, Hong-Wu; Pang, Dai-WenACS Nano (2012), 6 (11), 10033-10041CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Membrane nanotubes can facilitate direct intercellular communication between cells and provide a unique channel for intercellular transfer of cellular contents. However, the transport mechanisms of membrane nanotubes remain poorly understood between cancer cells. Also largely unknown is the transport pattern mediated by membrane nanotubes. In this work, wheat germ agglutinin (WGA), a widely used drug carrier and potential antineoplastic drug, was labeled with quantum dots (QDs-WGA) as a model for exploring the intercellular transportation via membrane nanotubes. We found that membrane nanotubes allowed effective transfer of QDs-WGA. Long-term single-particle tracking indicated that the movements of QDs-WGA exhibited a slow and directed motion pattern in nanotubes. Significantly, the transport of QDs-WGA was driven by myosin mol. motors in an active and unidirectional manner. These results contribute to a better understanding of cell-to-cell communication for cancer research.
- 97Liu, S. L.; Zhang, Z. L.; Sun, E. Z.; Peng, J.; Xie, M.; Tian, Z. Q.; Lin, Y.; Pang, D. W. Visualizing the Endocytic and Exocytic Processes of Wheat Germ Agglutinin by Quantum Dot-Based Single-Particle Tracking. Biomaterials 2011, 32, 7616– 7624, DOI: 10.1016/j.biomaterials.2011.06.04697Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle trackingLiu, Shu-Lin; Zhang, Zhi-Ling; Sun, En-Ze; Peng, Jun; Xie, Min; Tian, Zhi-Quan; Lin, Yi; Pang, Dai-WenBiomaterials (2011), 32 (30), 7616-7624CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Wheat germ agglutinin (WGA) is a paradigm for understanding intracellular transport of lectins. As a protein exploiting the receptor-mediated endocytosis for internalization, WGA is also a valuable model system for exploring the endocytic and exocytic pathway. In this study, quantum dot-based single-particle tracking was performed to investigate the transport of WGA in live cells, revealing firstly that the endocytic and exocytic processes of WGA were both actin- and microtubule-dependent, each including five stages. The vesicle fusion event occurred near the cytomembrane, followed by two destinies with WGA: shedding to the extracellular or reversing to the cytoplasm. These findings suggest a distinct and dynamic scenario for the transport of lectins following a receptor-mediated endo/exocytic pathway in live cells. This is important for the application of lectins as drug carriers and antineoplastic drugs in medicine, and also offers insights into the pathway of endocytosis and exocytosis.
- 98Joo, K.-I.; Lei, Y.; Lee, C.-L.; Lo, J.; Xie, J.; Hamm-Alvarez, S. F.; Wang, P. Site-Specific Labeling of Enveloped Viruses with Quantum Dots for Single Virus Tracking. ACS Nano 2008, 2, 1553– 1562, DOI: 10.1021/nn800213698Site-Specific Labeling of Enveloped Viruses with Quantum Dots for Single Virus TrackingJoo, Kye-Il; Lei, Yuning; Lee, Chi-Lin; Lo, Jonathon; Xie, Jiansong; Hamm-Alvarez, Sarah F.; Wang, PinACS Nano (2008), 2 (8), 1553-1562CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. It also allows for the live cell imaging of the trafficking of labeled virus to the Rab5+ endosomal compartments. This study further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway. The studies monitoring HIV particles using QD-labeling showed that the authors could detect single virions on the surface of target cells expressing either CD4/CCR5 or DC-SIGN. Further internalization studies of QD-HIV evidently showed that the clathrin pathway is the major route for DC-SIGN-mediated uptake of viruses. Taken together, the authors' data demonstrate the potential of this QD-labeling for visualizing the dynamic interactions between viruses and target cell structures.
- 99Liu, S. L.; Tian, Z. Q.; Zhang, Z. L.; Wu, Q. M.; Zhao, H. S.; Ren, B.; Pang, D. W. High-Efficiency Dual Labeling of Influenza Virus for Single-Virus Imaging. Biomaterials 2012, 33, 7828– 7833, DOI: 10.1016/j.biomaterials.2012.07.02699High-efficiency dual labeling of influenza virus for single-virus imagingLiu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Wu, Qiu-Mei; Zhao, Hai-Su; Ren, Bin; Pang, Dai-WenBiomaterials (2012), 33 (31), 7828-7833CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Many viruses invade host cells by entering the cells and releasing their genome for replication, which are remarkable incidents for viral infection. Therefore, the viral internal and external components should be simultaneously labeled and dynamically tracked at single-virus level for further understanding viral infection mechanisms. However, most of the previously reported methods have very low labeling efficiency and require considerable time and effort, which is laborious and inconvenient for researchers. In this work, we report a general strategy to high-efficiently label viral envelope and genome for single-virus imaging with quantum dots (QDs) and Syto 82, resp. It was found that nearly all viral envelopes could be labeled with QDs with superior stability, which makes it possible to realize global and long-term tracking of single virus in individual cells. Effectively labeling their genome with Syto 82, about 90% of QDs-labeled viruses could be used to monitor the viral genome signal, which may provide valuable information for deeply studying viral genome transport. This is very important and meaningful to investigate the viral infection mechanism. Our labeling strategy has advantage in commonality, convenience and efficiency, which is expected to be widely used in biol. research.
- 100Lv, C.; Lin, Y.; Liu, A. A.; Hong, Z. Y.; Wen, L.; Zhang, Z.; Zhang, Z. L.; Wang, H.; Pang, D. W. Labeling Viral Envelope Lipids with Quantum Dots by Harnessing the Biotinylated Lipid-Self-Inserted Cellular Membrane. Biomaterials 2016, 106, 69– 77, DOI: 10.1016/j.biomaterials.2016.08.013100Labeling viral envelope lipids with quantum dots by harnessing the biotinylated lipid-self-inserted cellular membraneLv, Cheng; Lin, Yi; Liu, An-An; Hong, Zheng-Yuan; Wen, Li; Zhang, Zhenfeng; Zhang, Zhi-Ling; Wang, Hanzhong; Pang, Dai-WenBiomaterials (2016), 106 (), 69-77CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Highly efficient labeling of viruses with quantum dots (QDs) is the prerequisite for the long-term tracking of virus invasion at the single virus level to reveal mechanisms of virus infection. As one of the structural components of viruses, viral envelope lipids are hard to be labeled with QDs due to the lack of efficient methods to modify viral envelope lipids. Moreover, it is still a challenge to maintain the intactness and infectivity of labeled viruses. Herein, a mild method has been developed to label viral envelope lipids with QDs by harnessing the biotinylated lipid-self-inserted cellular membrane. Biotinylated lipids can spontaneously insert in cellular membranes of host cells during culture and then be naturally assembled on progeny Pseudorabies virus (PrV) via propagation. The biotinylated PrV can be labeled with streptavidin-conjugated QDs, with a labeling efficiency of ∼90%. Such a strategy to label lipids with QDs can retain the intactness and infectivity of labeled viruses to the largest extent, facilitating the study of mechanisms of virus infection at the single virus level.
- 101Hong, Z. Y.; Lv, C.; Liu, A. A.; Liu, S. L.; Sun, E. Z.; Zhang, Z. L.; Lei, A. W.; Pang, D. W. Clicking Hydrazine and Aldehyde: The Way to Labeling of Viruses with Quantum Dots. ACS Nano 2015, 9, 11750– 11760, DOI: 10.1021/acsnano.5b03256101Clicking Hydrazine and Aldehyde: The Way to Labeling of Viruses with Quantum DotsHong, Zheng-Yuan; Lv, Cheng; Liu, An-An; Liu, Shu-Lin; Sun, En-Ze; Zhang, Zhi-Ling; Lei, Ai-Wen; Pang, Dai-WenACS Nano (2015), 9 (12), 11750-11760CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Real-time tracking of fluorophore-tagged viruses in living cells can help uncover virus infection mechanisms. Certainly, the indispensable prerequisite for virus-tracking is to label viruses with some bright and photostable beacons such as quantum dots (QDs) via an appropriate labeling strategy. Herein, the authors devise a convenient hydrazine-aldehyde based strategy to label viruses with QDs through the conjugation of 4-formylbenzoate (4FB) modified QDs to 6-hydrazinonicotinate acetone hydrazone (HyNic) modified viruses under mild conditions. On the basis of this strategy, viruses can be successfully labeled with QDs with high selectivity, stable conjugation, good reproducibility, high labeling efficiency of 92-93% and max. retention of both fluorescence properties of QDs and infectivity of viruses, which is very meaningful to tracking and statistical anal. of virus infection processes. By further comparing with the most widely used labeling strategy based on the Biotin-SA system, this new strategy has advantages of both high labeling efficiency and good retention of virus infectivity, thus offering a promising alternative for virus-labeling. Moreover, due to the ubiquitous presence of exposed amino groups on the surface of various viruses, this selective, efficient, reproducible and biofriendly strategy should have good universality for labeling both enveloped and nonenveloped viruses.
- 102Wen, L.; Lin, Y.; Zheng, Z. H.; Zhang, Z. L.; Zhang, L. J.; Wang, L. Y.; Wang, H. Z.; Pang, D. W. Labeling the Nucleocapsid of Enveloped Baculovirus with Quantum Dots for Single-Virus Tracking. Biomaterials 2014, 35, 2295– 2301, DOI: 10.1016/j.biomaterials.2013.11.069102Labeling the nucleocapsid of enveloped baculovirus with quantum dots for single-virus trackingWen, Li; Lin, Yi; Zheng, Zhen-Hua; Zhang, Zhi-Ling; Zhang, Li-Juan; Wang, Li-Ying; Wang, Han-Zhong; Pang, Dai-WenBiomaterials (2014), 35 (7), 2295-2301CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Utilization of quantum dots (QDs) for single-virus tracking is highly important for understanding virus infection mechanism. However, QD labeling site of real enveloped viruses has been confined to the external envelope so far, causing the impossibility to monitor the late infection events after the loss of envelope. Herein, a strategy to label the internal nucleocapsid of enveloped virus with QDs was proposed. The nucleocapsid of enveloped baculovirus was self-biotinylated during virus replication process in host cells and subsequently labeled with streptavidin-conjugated QDs (SA-QDs). Such host cell-assisted QD labeling was proved to be reliable, specific, efficient and capable of maintaining virus infectivity. Based on such labeling, crit. infection events before and after the envelope loss were monitored in real time, including single virus interacting with late endosomes and the subsequent nucleocapsid transporting into cell nucleus. Thus our established QD labeling of enveloped virus nucleocapsid with QDs enables the comprehensive single-virus tracking for deeply understanding virus infection mechanism.
- 103Xie, M.; Luo, K.; Huang, B.-H.; Liu, S.-L.; Hu, J.; Cui, D.; Zhang, Z.-L.; Xiao, G.-F.; Pang, D.-W. PEG-Interspersed Nitrilotriacetic Acid-Functionalized Quantum Dots for Site-Specific Labeling of Prion Proteins Expressed on Cell Surfaces. Biomaterials 2010, 31, 8362– 8370, DOI: 10.1016/j.biomaterials.2010.07.063103PEG-interspersed nitrilotriacetic acid-functionalized quantum dots for site-specific labeling of prion proteins expressed on cell surfacesXie, Min; Luo, Kan; Huang, Bi-Hai; Liu, Shu-Lin; Hu, Jun; Cui, Di; Zhang, Zhi-Ling; Xiao, Geng-Fu; Pang, Dai-WenBiomaterials (2010), 31 (32), 8362-8370CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)A strategy has been put forward to fabricate PEG-interspersed nitrilotriacetic acid (NTA)-functionalized QDs by one-step self-assembly using a mixt. of self-synthesized NTA-terminated amphiphilic polymer and 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(Polyethylene Glycol)2000] (DSPE-PEG-COOH). The process was highly reproducible for facile functionalization of QDs via simultaneous self-assembly of biocompatible PEG mols. onto their surface. An optimized molar ratio of NTA-terminated amphiphilic polymer to DSPE-PEG-COOH was used to obtain NTA-functionalized QDs for site-specific labeling of prion proteins (PrPC) expressed on cell surfaces. Fabricated NTA-functionalized QDs can be a good candidate used for real-time visualization of PrPC in single live cells to clarify the nosogenesis of pathogenic scrapie prion protein (PrPSc).
- 104Wen, L.; Zheng, Z. H.; Liu, A. A.; Lv, C.; Zhang, L. J.; Ao, J.; Zhang, Z. L.; Wang, H. Z.; Lin, Y.; Pang, D. W. Tracking Single Baculovirus Retrograde Transportation in Host Cell via Quantum Dot-Labeling of Virus Internal Component. J. Nanobiotechnol. 2017, 15, 37, DOI: 10.1186/s12951-017-0270-9104Tracking single baculovirus retrograde transportation in host cell via quantum dot-labeling of virus internal componentWen, Li; Zheng, Zhen-Hua; Liu, An-An; Lv, Cheng; Zhang, Li-Juan; Ao, Jian; Zhang, Zhi-Ling; Wang, Han-Zhong; Lin, Yi; Pang, Dai-WenJournal of Nanobiotechnology (2017), 15 (), 37/1-37/10CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)Background: Quantum dot (QD)-based single virus tracking has become a powerful tool for dissecting virus infection mechanism. However, only virus behaviors at the early stage of retrograde trafficking have been dynamically tracked so far. Monitoring of comprehensive virus retrograde transportation remains a challenge. Results: Based on the superior fluorescence properties of QDs and their labeling of virus internal component, the dynamic interactions between baculoviruses and all key transportation-related cellular structures, including vesicles, acidic endosomes, actins, nuclear pores and nuclei, were visualized at the single-virus level. Detailed scenarios and dynamic information were provided for these crit. interaction processes. Conclusions: A comprehensive model of baculovirus retrograde trafficking involving virus endocytosis, fusion with acidic endosome, translocation to nuclear periphery, internalization into nucleus, and arriving at the destination in nucleus was proposed. Thus the whole retrograde transportation of baculovirus in live host cells was elucidated at the single-virus level for the first time.
- 105Hao, J.; Huang, L. L.; Zhang, R.; Wang, H. Z.; Xie, H. Y. A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click Chemistry. Anal. Chem. 2012, 84, 8364– 8370, DOI: 10.1021/ac301918t105A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click ChemistryHao, Jian; Huang, Li-Li; Zhang, Rui; Wang, Han-Zhong; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2012), 84 (19), 8364-8370CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Real-time tracking of the dynamic process of virus invasion is crucial to understanding the infection mechanism. For successful tracking, efficient labeling methods are indispensable. The authors report a mild and reliable method for labeling viruses, esp. with regard to easily disabled enveloped viruses. The copper-free click chem. has been used to label enveloped viruses with quantum dots (QDs) by linking virions modified with azide to the QDs derived with dibenzocyclooctynes (DBCO). Both vaccinia virus (VACV) and avian influenza A virus (H9N2) can be specifically and rapidly labeled under mild conditions, with a labeling efficiency of >80%. The labeled virions were of intact infectivity, and their fluorescence was strong enough to realize single-virion tracking. Compared to previously reported methods, the authors' method is less destructive, reliable, and universal, without specific requirements for the type and structure of viruses to be labeled, which has laid the foundation for long-term dynamic visualization of virus infection process.
- 106Zhang, P.; Liu, S.; Gao, D.; Hu, D.; Gong, P.; Sheng, Z.; Deng, J.; Ma, Y.; Cai, L. Click-Functionalized Compact Quantum Dots Protected by Multidentate-Imidazole Ligands: Conjugation-Ready Nanotags for Living-Virus Labeling and Imaging. J. Am. Chem. Soc. 2012, 134, 8388– 8391, DOI: 10.1021/ja302367s106Click-Functionalized Compact Quantum Dots Protected by Multidentate-Imidazole Ligands: Conjugation-Ready Nanotags for Living-Virus Labeling and ImagingZhang, Pengfei; Liu, Shuhui; Gao, Duyang; Hu, Dehong; Gong, Ping; Sheng, Zonghai; Deng, Jizhe; Ma, Yifan; Cai, LintaoJournal of the American Chemical Society (2012), 134 (20), 8388-8391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We synthesized a new class of multifunctional multidentate-imidazole polymer ligands by one-step reaction to produce conjugation-ready QDs with great stability and compact size. Furthermore, combined with strain-promoted click chem., we developed a general strategy for efficient labeling of living-viruses with QD probes.
- 107Liu, H.; Liu, Y.; Liu, S.; Pang, D. W.; Xiao, G. Clathrin-Mediated Endocytosis in Living Host Cells Visualized through Quantum Dot Labeling of Infectious Hematopoietic Necrosis virus. J. Virol. 2011, 85, 6252– 6262, DOI: 10.1128/JVI.00109-11107Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virusLiu, Haibin; Liu, Yi; Liu, Shulin; Pang, Dai-Wen; Xiao, GengfuJournal of Virology (2011), 85 (13), 6252-6262CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, the authors found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked redn. in viral entry. The authors also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymn. is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compd. that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments.
- 108Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Zhao, H. S.; Liu, H.; Sun, E. Z.; Xiao, G. F.; Zhang, W.; Wang, H. Z.; Pang, D. W. Effectively and Efficiently Dissecting the Infection of Influenza Virus by Quantum-Dot-Based Single-Particle Tracking. ACS Nano 2012, 6, 141– 150, DOI: 10.1021/nn2031353108Effectively and Efficiently Dissecting the Infection of Influenza Virus by Quantum-Dot-Based Single-Particle TrackingLiu, Shu-Lin; Zhang, Zhi-Ling; Tian, Zhi-Quan; Zhao, Hai-Su; Liu, Haibin; Sun, En-Ze; Xiao, Geng Fu; Zhang, Wanpo; Wang, Han-Zhong; Pang, Dai-WenACS Nano (2012), 6 (1), 141-150CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Exploring the virus infection mechanisms is significant for defending against virus infection and providing a basis for studying endocytosis mechanisms. Single-particle tracking technique is a powerful tool to monitor virus infection in real time for obtaining dynamic information. In this study, the authors reported a quantum-dot-based single-particle tracking technique to efficiently and globally research the virus infection behaviors in individual cells. It was obsd. that many influenza viruses were moving rapidly, converging to the microtubule organizing center (MTOC), interacting with acidic endosomes, and finally entering the target endosomes for genome release, which provides a vivid portrayal of the five-stage virus infection process. This report settles a long-pending question of how viruses move and interact with acidic endosomes before genome release in the perinuclear region and also finds that influenza virus infection is likely to be a "MTOC rescue" model for genome release. The systemic technique developed in this report is expected to be widely used for studying the mechanisms of virus infection and uncovering the secrets of endocytosis.
- 109Luo, K.; Li, S.; Xie, M.; Wu, D.; Wang, W.; Chen, R.; Huang, L.; Huang, T.; Pang, D.; Xiao, G. Real-Time Visualization of Prion Transport in Single Live Cells Using Quantum Dots. Biochem. Biophys. Res. Commun. 2010, 394, 493– 497, DOI: 10.1016/j.bbrc.2010.02.159109Real-time visualization of prion transport in single live cells using quantum dotsLuo, Kan; Li, Shu; Xie, Min; Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao; Pang, Daiwen; Xiao, GengfuBiochemical and Biophysical Research Communications (2010), 394 (3), 493-497CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrPC to the infectious scrapie isoform PrPSc. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, the authors have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addn., using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, the authors show that lipid rafts play an important role in locating cellular PrPC to the cell membrane and in initiating PrPC endocytosis.
- 110Liu, S. L.; Zhang, L. J.; Wang, Z. G.; Zhang, Z. L.; Wu, Q. M.; Sun, E. Z.; Shi, Y. B.; Pang, D. W. Globally Visualizing the Microtubule-Dependent Transport Behaviors of Influenza Virus in Live Cells. Anal. Chem. 2014, 86, 3902– 3908, DOI: 10.1021/ac500640u110Globally Visualizing the Microtubule-Dependent Transport Behaviors of Influenza Virus in Live CellsLiu, Shu-Lin; Zhang, Li-Juan; Wang, Zhi-Gang; Zhang, Zhi-Ling; Wu, Qiu-Mei; Sun, En-Ze; Shi, Yun-Bo; Pang, Dai-WenAnalytical Chemistry (Washington, DC, United States) (2014), 86 (8), 3902-3908CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.
- 111Liu, S. L.; Wu, Q. M.; Zhang, L. J.; Wang, Z. G.; Sun, E. Z.; Zhang, Z. L.; Pang, D. W. Three-Dimensional Tracking of Rab5- and Rab7-Associated Infection Process of Influenza Virus. Small 2014, 10, 4746– 4753, DOI: 10.1002/smll.201400944111Three-Dimensional Tracking of Rab5- and Rab7- Associated Infection Process of Influenza VirusLiu, Shu-Lin; Wu, Qiu-Mei; Zhang, Li-Juan; Wang, Zhi-Gang; Sun, En-Ze; Zhang, Zhi-Ling; Pang, Dai-WenSmall (2014), 10 (22), 4746-4753CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biol. problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-assocd. infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The exptl. results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-contg. vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-pos. endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.
- 112Wang, Z. G.; Liu, S. L.; Zhang, Z. L.; Tian, Z. Q.; Tang, H. W.; Pang, D. W. Exploring Sialic Acid Receptors-Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single-Particle Tracking. Small 2014, 10, 2712– 2720, DOI: 10.1002/smll.201303532112Exploring sialic acid receptors-related infection behavior of avian influenza virus in human bronchial epithelial cells by single-particle trackingWang, Zhi-Gang; Liu, Shu-Lin; Zhang, Zhi-Ling; Tian, Zhi-Quan; Tang, Hong-Wu; Pang, Dai-WenSmall (2014), 10 (13), 2712-2720CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomols. in live cells. In this work, the authors use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. The authors have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.
- 113Qin, C.; Li, W.; Li, Q.; Yin, W.; Zhang, X.; Zhang, Z.; Zhang, X. E.; Cui, Z. Real-Time Dissection of Dynamic Uncoating of Individual Influenza Viruses. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 2577– 2582, DOI: 10.1073/pnas.1812632116113Real-time dissection of dynamic uncoating of individual influenza virusesQin, Chong; Li, Wei; Li, Qin; Yin, Wen; Zhang, Xiaowei; Zhang, Zhiping; Zhang, Xian-En; Cui, ZongqiangProceedings of the National Academy of Sciences of the United States of America (2019), 116 (7), 2577-2582CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Uncoating is an obligatory step in the virus life cycle that serves as an antiviral target. Unfortunately, it is challenging to study viral uncoating due to methodol. limitations for detecting this transient and dynamic event. The uncoating of influenza A virus (IAV), which contains an unusual genome of eight segmented RNAs, is particularly poorly understood. Here, by encapsulating quantum dot (QD)-conjugated viral ribonucleoprotein complexes (vRNPs) within infectious IAV virions and applying single-particle imaging, we tracked the uncoating process of individual IAV virions. Approx. 30% of IAV particles were found to undergo uncoating through fusion with late endosomes in the "around-nucleus" region at 30 to 90 min postinfection. Inhibition of viral M2 proton channels and cellular endosome acidification prevented IAV uncoating. IAV vRNPs are released sep. into the cytosol after virus uncoating. Then, individual vRNPs undergo a three-stage movement to the cell nucleus and display two diffusion patterns when inside the nucleus. These findings reveal IAV uncoating and vRNP trafficking mechanisms, filling a crit. gap in knowledge about influenza viral infection.
- 114Sun, E. Z.; Liu, A. A.; Zhang, Z. L.; Liu, S. L.; Tian, Z. Q.; Pang, D. W. Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking. ACS Nano 2017, 11, 4395– 4406, DOI: 10.1021/acsnano.6b07853114Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus TrackingSun, En-Ze; Liu, An-An; Zhang, Zhi-Ling; Liu, Shu-Lin; Tian, Zhi-Quan; Pang, Dai-WenACS Nano (2017), 11 (5), 4395-4406CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Entry is the 1st crit. step for the infection of influenza A virus and of great significance for the research and development of antiflu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of a single-virus tracking technique and quantum dots, we have realized real-time and multicolor visualization of the endocytic process of individual viruses and comprehensive dissection of 2 distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of the virus, with a large population of events for short-lived recruitments of these 2 proteins being abortive. In addn., the differentiated durations of dynamin recruitment and responses to inhibitors in these 2 routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also participates in the maturation of a clathrin-coated pit in the clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.
- 115Wu, Q. M.; Liu, S. L.; Chen, G.; Zhang, W.; Sun, E. Z.; Xiao, G. F.; Zhang, Z. L.; Pang, D. W. Uncovering the Rab5-Independent Autophagic Trafficking of Influenza A Virus by Quantum-Dot-Based Single-Virus Tracking. Small 2018, 14, e1702841 DOI: 10.1002/smll.201702841There is no corresponding record for this reference.
- 116Ma, Y.; Wang, M.; Li, W.; Zhang, Z.; Zhang, X.; Tan, T.; Zhang, X. E.; Cui, Z. Live Cell Imaging of Single Genomic Loci with Quantum Dot-Labeled TALEs. Nat. Commun. 2017, 8, 15318, DOI: 10.1038/ncomms15318116Live cell imaging of single genomic loci with quantum dot-labeled talesMa, Yingxin; Wang, Mingxiu; Li, Wei; Zhang, Zhiping; Zhang, Xiaowei; Tan, Tianwei; Zhang, Xian-En; Cui, ZongqiangNature Communications (2017), 8 (), 15318CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labeling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different color quantum dots (QDs). These QD-labeled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different colored QD-labeled TALEs, we det. and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.
- 117Li, Q.; Yin, W.; Li, W.; Zhang, Z.; Zhang, X.; Zhang, X. E.; Cui, Z. Encapsulating Quantum Dots within HIV-1 Virions through Site-Specific Decoration of the Matrix Protein Enables Single Virus Tracking in Live Primary Macrophages. Nano Lett. 2018, 18, 7457– 7468, DOI: 10.1021/acs.nanolett.8b02800117Encapsulating Quantum Dots within HIV-1 Virions through Site-Specific Decoration of the Matrix Protein Enables Single Virus Tracking in Live Primary MacrophagesLi, Qin; Yin, Wen; Li, Wei; Zhang, Zhiping; Zhang, Xiaowei; Zhang, Xian-En; Cui, ZongqiangNano Letters (2018), 18 (12), 7457-7468CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Labeling and imaging with quantum dots (QDs) provides powerful tools to visualize viral infection in living cells. Encapsulating QDs within virions represents a novel strategy for virus labeling. Here, the authors developed infectious HIV-1 virions encapsulating QDs through site-specific decoration of the viral matrix protein (MA) and used them to visualize early infection events in human primary macrophages by single-particle imaging. The MA protein was fused to a biotin acceptor peptide (BAP) tag, biotinylated, complexed with streptavidin-conjugated QDs in live cells, and incorporated into virions during virus assembly. The QD-encapsulated virions were tracked during infection of macrophages at a single particle level. The dynamic dissocn. of MA and Vpr was also tracked in real time, and MA has multiple dynamic behaviors and functions during virus entry. More importantly, the authors tracked the dynamic interplay of QD-encapsulated virions with cellular mitochondria in live primary macrophages. Also HIV-1 can induce fission of mitochondria during the early phases of infection. In summary, the authors have constructed a type of QD-encapsulated virus particle and used this technol. to further the authors' understanding of the early events of HIV-1 infection.
- 118Zhang, L. J.; Xia, L.; Liu, S. L.; Sun, E. Z.; Wu, Q. M.; Wen, L.; Zhang, Z. L.; Pang, D. W. A ″Driver Switchover″ Mechanism of Influenza Virus Transport from Microfilaments to Microtubules. ACS Nano 2018, 12, 474– 484, DOI: 10.1021/acsnano.7b06926118A "Driver Switchover" Mechanism of Influenza Virus Transport from Microfilaments to MicrotubulesZhang, Li-Juan; Xia, Li; Liu, Shu-Lin; Sun, En-Ze; Wu, Qiu-Mei; Wen, Li; Zhang, Zhi-Ling; Pang, Dai-WenACS Nano (2018), 12 (1), 474-484CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.
- 119Muller, B.; Daecke, J.; Fackler, O. T.; Dittmar, M. T.; Zentgraf, H.; Krausslich, H. G. Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative. J. Virol. 2004, 78, 10803– 10813, DOI: 10.1128/JVI.78.19.10803-10813.2004119Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivativeMuller Barbara; Daecke Jessica; Fackler Oliver T; Dittmar Matthias T; Zentgraf Hanswalter; Krausslich Hans-GeorgJournal of virology (2004), 78 (19), 10803-13 ISSN:0022-538X.The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.
- 120Carlson, L. A.; Briggs, J. A. G.; Glass, B.; Riches, J. D.; Simon, M. N.; Johnson, M. C.; Muller, B.; Grunewald, K.; Krausslich, H. G. Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis. Cell Host Microbe 2008, 4, 592– 599, DOI: 10.1016/j.chom.2008.10.013120Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesisCarlson, Lars-Anders; Briggs, John A. G.; Glass, Baerbel; Riches, James D.; Simon, Martha N.; Johnson, Marc C.; Mueller, Barbara; Gruenewald, Kay; Kraeusslich, Hans-GeorgCell Host & Microbe (2008), 4 (6), 592-599CODEN: CHMECB; ISSN:1931-3128. (Cell Press)Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomog. and scanning transmission electron microscopy, we find that virions have a morphol. and compn. distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release-akin to its role in vesicle formation-and is not restricted to severing the thin membrane tether.
- 121Sakin, V.; Paci, G.; Lemke, E. A.; Muller, B. Labeling of Virus Components for Advanced, Quantitative Imaging Analyses. FEBS Lett. 2016, 590, 1896– 1914, DOI: 10.1002/1873-3468.12131121Labeling of virus components for advanced, quantitative imaging analysesSakin, Volkan; Paci, Giulia; Lemke, Edward A.; Mueller, BarbaraFEBS Letters (2016), 590 (13), 1896-1914CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)In recent years, investigation of virus-cell interactions has moved from ensemble measurements to imaging analyses at the single-particle level. Advanced fluorescence microscopy techniques provide single-mol. sensitivity and subdiffraction spatial resoln., allowing observation of subviral details and individual replication events to obtain detailed quant. information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virol. questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging.
- 122Goncalves, M. S. Fluorescent Labeling of Biomolecules with Organic Probes. Chem. Rev. 2009, 109, 190– 212, DOI: 10.1021/cr0783840122Fluorescent Labeling of Biomolecules with Organic ProbesGoncalves, M. Sameiro T.Chemical Reviews (Washington, DC, United States) (2009), 109 (1), 190-212CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review looking at org. h labels with emissions up to 500 nm and org. labels with emissions beyond 500 nm.
- 123Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Quantum Dots versus Organic Dyes as Fluorescent Labels. Nat. Methods 2008, 5, 763– 775, DOI: 10.1038/nmeth.1248123Quantum dots versus organic dyes as fluorescent labelsResch-Genger, Ute; Grabolle, Markus; Cavaliere-Jaricot, Sara; Nitschke, Roland; Nann, ThomasNature Methods (2008), 5 (9), 763-775CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Suitable labels are at the core of luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technol. is the emerging development of quantum dots (QDs) - inorg. nanocrystals with unique optical and chem. properties but complicated surface chem. - as in vitro and in vivo fluorophores. Here the authors compare and evaluate the differences in physicochem. properties of common fluorescent labels, focusing on traditional org. dyes and QDs. The authors' aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.
- 124Miyawaki, A.; Sawano, A.; Kogure, T. Lighting Up Cells: Labelling Proteins with Fluorophores. Nat. Cell Biol. 2003, S1– S7124Lighting up cells: Labelling proteins with fluorophoresMiyawaki, Atsushi; Sawano, Asako; Kogure, TakakoNature Cell Biology (2003), (Suppl.), S1-S7CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)A review. During the past decade, rapid improvements have been made in the tools available for labeling proteins within cells, which has increased our ability to unravel the finer details of cellular events. One significant reason for these advances has been the development of fluorescent proteins that can be incorporated into proteins by genetic fusion to produce a fluorescent label. In addn., new techniques have made it possible to label proteins with small org. fluorophores and semiconductor nanocrystals.
- 125Sivaraman, D.; Biswas, P.; Cella, L. N.; Yates, M. V.; Chen, W. Detecting RNA Viruses in Living Mammalian Cells by Fluorescence Microscopy. Trends Biotechnol. 2011, 29, 307– 313, DOI: 10.1016/j.tibtech.2011.02.006125Detecting RNA viruses in living mammalian cells by fluorescence microscopySivaraman, Divya; Biswas, Payal; Cella, Lakshmi N.; Yates, Marylynn V.; Chen, WilfredTrends in Biotechnology (2011), 29 (7), 307-313CODEN: TRBIDM; ISSN:0167-7799. (Elsevier B.V.)A review. Traditional methods that rely on viral isolation and culture techniques continue to be the gold stds. used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.
- 126Wang, I. H.; Burckhardt, C. J.; Yakimovich, A.; Greber, U. F. Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton. Viruses 2018, 10, 166, DOI: 10.3390/v10040166126Imaging, tracking and computational analyses of virus entry and egress with the cytoskeletonWang, I-Hsuan; Burckhardt, Christoph J.; Yakimovich, Artur; Greber, Urs F.Viruses (2018), 10 (4), 166/1-166/29CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Viruses have a dual nature: particles are "passive substances" lacking chem. energy transformation, whereas infected cells are "active substances" turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and mol. biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and assocd. motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and assocd. motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in assocn. with cellular organelles. This review explores how the anal. of viral trajectories informs about mechanisms of infection. We tdiscuss the methodol. enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodol., we highlight the role of actin filaments and microtubules, and their assocd. motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolns. thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function.
- 127Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Fluorescence Imaging in Vivo: Recent Advances. Curr. Opin. Biotechnol. 2007, 18, 17– 25, DOI: 10.1016/j.copbio.2007.01.003127Fluorescence imaging in vivo: recent advancesRao, Jianghong; Dragulescu-Andrasi, Anca; Yao, HequanCurrent Opinion in Biotechnology (2007), 18 (1), 17-25CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-IR (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addn. of fluorescent org., inorg. and biol. nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resoln., multimodality and lifetime-based in vivo fluorescence imaging.
- 128Lakadamyali, M.; Rust, M. J.; Zhuang, X. Ligands for Clathrin-Mediated Endocytosis Are Differentially Sorted into Distinct Populations of Early Endosomes. Cell 2006, 124, 997– 1009, DOI: 10.1016/j.cell.2005.12.038128Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomesLakadamyali, Melike; Rust, Michael J.; Zhuang, XiaoweiCell (Cambridge, MA, United States) (2006), 124 (5), 997-1009CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Cells rely on the correct sorting of endocytic ligands and receptors for proper function. Early endosomes have been considered as the initial sorting station where cargos for degrdn. sep. from those for recycling. Using live-cell imaging to monitor individual endosomes and ligand particles in real time, we have discovered a sorting mechanism that takes place prior to early endosome entry. We show that early endosomes are in fact comprised of two distinct populations: a dynamic population that is highly mobile on microtubules and matures rapidly toward late endosomes and a static population that matures much more slowly. Several cargos destined for degrdn. are preferentially targeted to the dynamic endosomes, whereas the recycling ligand transferrin is nonselectively delivered to all early endosomes and effectively enriched in the larger, static population. This pre-early endosome sorting process begins at clathrin-coated vesicles, depends on microtubule-dependent motility, and appears to involve endocytic adaptors.
- 129Babcock, H. P.; Chen, C.; Zhuang, X. Using Single-Particle Tracking to Study Nuclear Trafficking of Viral Genes. Biophys. J. 2004, 87, 2749– 2758, DOI: 10.1529/biophysj.104.042234129Using single-particle tracking to study nuclear trafficking of viral genesBabcock, Hazen P.; Chen, Chen; Zhuang, XiaoweiBiophysical Journal (2004), 87 (4), 2749-2758CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)The question of how genetic materials are trafficked in and out of the cell nucleus is a problem of great importance not only for understanding viral infections but also for advancing gene-delivery technol. Here we demonstrate a phys. technique that allows gene trafficking to be studied at the single-gene level by combining sensitive fluorescence microscopy with microinjection. As a model system, we investigate the nuclear import of influenza genes, in the form of ribonucleoproteins (vRNPs), by imaging single vRNPs in living cells in real time. Our single-particle trajectories show that vRNPs are transported to the nuclear envelope by diffusion. We have obsd. heterogeneous interactions between the vRNPs and nuclear pore complexes with dissocn. rate consts. spanning two orders of magnitude. Our single-particle tracking expts. also provided new insights into the regulation mechanisms for the nuclear import of vRNPs: the influenza M1 protein, a regulatory protein for the import process, downregulates the nuclear import of vRNPs by inhibiting the interactions between vRNPs and nuclear pore complexes but has no significant effect on the transport properties of vRNPs. We expect this single-particle tracking approach to find broad application in investigations of genetic trafficking.
- 130Vaughan, J. C.; Brandenburg, B.; Hogle, J. M.; Zhuang, X. Rapid Actin-Dependent Viral Motility in Live Cells. Biophys. J. 2009, 97, 1647– 1656, DOI: 10.1016/j.bpj.2009.07.011130Rapid actin-dependent viral motility in live cellsVaughan, Joshua C.; Brandenburg, Boerries; Hogle, James M.; Zhuang, XiaoweiBiophysical Journal (2009), 97 (6), 1647-1656CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased anal. of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was obsd. in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymn. or by myosin motor proteins.
- 131Brandenburg, B.; Lee, L. Y.; Lakadamyali, M.; Rust, M. J.; Zhuang, X.; Hogle, J. M. Imaging Poliovirus Entry in Live Cells. PLoS Biol. 2007, 5, e183 DOI: 10.1371/journal.pbio.0050183There is no corresponding record for this reference.
- 132Xu, H.; Hao, X.; Wang, S.; Wang, Z.; Cai, M.; Jiang, J.; Qin, Q.; Zhang, M.; Wang, H. Real-Time Imaging of Rabies Virus Entry into Living Vero Cells. Sci. Rep. 2015, 5, 11753, DOI: 10.1038/srep11753132Real-time Imaging of Rabies Virus Entry into Living Vero cellsXu Haijiao; Hao Xian; Wang Zhiyong; Cai Mingjun; Jiang Junguang; Wang Shaowen; Qin Qiwei; Zhang Maolin; Wang HongdaScientific reports (2015), 5 (), 11753 ISSN:.Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.
- 133Vonderheit, A.; Helenius, A. Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes. PLoS Biol. 2005, 3, e233 DOI: 10.1371/journal.pbio.0030233133Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomesVonderheit Andreas; Helenius AriPLoS biology (2005), 3 (7), e233 ISSN:.Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.
- 134Johannsdottir, H. K.; Mancini, R.; Kartenbeck, J.; Amato, L.; Helenius, A. Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry. J. Virol. 2009, 83, 440– 453, DOI: 10.1128/JVI.01864-08134Host cell factors and functions involved in vesicular stomatitis virus entryJohannsdottir, Hrefna Kristin; Mancini, Roberta; Kartenbeck, Jurgen; Amato, Lea; Helenius, AriJournal of Virology (2009), 83 (1), 440-453CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although assocd. with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-assocd. proteins and cellular functions.
- 135Engel, S.; Heger, T.; Mancini, R.; Herzog, F.; Kartenbeck, J.; Hayer, A.; Helenius, A. Role of Endosomes in Simian Virus 40 Entry and Infection. J. Virol. 2011, 85, 4198– 4211, DOI: 10.1128/JVI.02179-10135Role of endosomes in simian virus 40 entry and infectionEngel, Sabrina; Heger, Thomas; Mancini, Roberta; Herzog, Fabian; Kartenbeck, Jurgen; Hayer, Arnold; Helenius, AriJournal of Virology (2011), 85 (9), 4198-4211CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was esp. sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.
- 136Joo, K. I.; Fang, Y.; Liu, Y.; Xiao, L.; Gu, Z.; Tai, A.; Lee, C. L.; Tang, Y.; Wang, P. Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates. ACS Nano 2011, 5, 3523– 3535, DOI: 10.1021/nn102651p136Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot ConjugatesJoo, Kye-Il; Fang, Yun; Liu, Yarong; Xiao, Liang; Gu, Zhen; Tai, April; Lee, Chi-Lin; Tang, Yi; Wang, PinACS Nano (2011), 5 (5), 3523-3535CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The unique spectral properties of semiconductor quantum dots (QDs) enable long-term live-cell imaging and ultrasensitive detection of viral particles, which in turn can potentially provide a practical means for detailed anal. of the underlying mol. mechanisms of virus entry. In this study, the authors report a general method of labeling adeno-assocd. virus serotype 2 (AAV2) with QDs for enhanced visualization of the intracellular behavior of viruses in living target cells. It was found that the mild conditions required for this QD conjugation reaction allowed for the retention of viral infectivity of AAV2. Furthermore, quant. anal. of viral motility in living cells suggested that QD-labeling had no significant effect on the intracellular transport properties of AAV2 particles compared to those of conventional org. dye-labeled AAV2. The authors' imaging study demonstrated that QD-AAV2 was internalized mainly through a clathrin-dependent pathway and then trafficked through various endosomes. It was also obsd. that QD-AAV2 particles exploit the cytoskeleton network to facilitate their transport within cells, and the labeling study provided evidence that the ubiquitin-proteasome system was likely involved in the intracellular trafficking of AAV2, at least at the level of nuclear transport. Taken together, the authors' findings reveal the potential of this QD-labeling method for monitoring the intracellular dynamics of virus-host cell interactions and interrogating the mol. mechanisms of viral infection in greater detail.
- 137Schelhaas, M.; Ewers, H.; Rajamaki, M. L.; Day, P. M.; Schiller, J. T.; Helenius, A. Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions. PLoS Pathog. 2008, 4, e1000148 DOI: 10.1371/journal.ppat.1000148137Human papillomavirus type 16 entry: retrograde cell surface transport along actin-rich protrusionsSchelhaas Mario; Ewers Helge; Rajamaki Minna-Liisa; Day Patricia M; Schiller John T; Helenius AriPLoS pathogens (2008), 4 (9), e1000148 ISSN:.The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30-60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing.
- 138Martin-Acebes, M. A.; Vazquez-Calvo, A.; Gonzalez-Magaldi, M.; Sobrino, F. Foot-and-Mouth Disease Virus Particles Inactivated with Binary Ethylenimine Are Efficiently Internalized into Cultured Cells. Vaccine 2011, 29, 9655– 9662, DOI: 10.1016/j.vaccine.2011.10.031138Foot-and-mouth disease virus particles inactivated with binary ethylenimine are efficiently internalized into cultured cellsMartin-Acebes, Miguel A.; Vazquez-Calvo, Angela; Gonzalez-Magaldi, Monica; Sobrino, FranciscoVaccine (2011), 29 (52), 9655-9662CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Conventional foot-and-mouth disease (FMD) vaccines are produced from virus grown in cell culture that is chem. inactivated by using binary ethylenimide (BEI). Here, we show that BEI treatment preserves both the architecture of FMDV particles, as inactivated viral particles showed by electron microscopy characteristics similar to those of infectious virions, as well as the general features of infectious virus internalization. Binding of inactivated particles to BHK-21 cells was blocked by preincubation with either a FMDV-specific monoclonal antibody or a synthetic peptide spanning the integrin-binding viral motif Arg-Gly-Asp (RGD). In addn., these particles were internalized into cultured cells through endocytosis, being directed to early endosomes, as indicated by their colocalization with the marker protein Rab5. When purified BEI-inactivated virions were labeled and their interaction with live cultured cells analyzed by time-lapse fluorescence microscopy, a major subpopulation of virus particles, about 80%, was shown to undergo internalization into a static endosome population, insensitive to the microtubule depolymn. exerted by nocodazole, while the remaining subpopulation (about 20%) was dynamic and sensitive to this drug. Thus, BEI-inactivated particles provide an interesting tool to study early steps in FMDV-cell interactions enabling a distinction between FMDV internalization and productive infection. Possible implications for FMDV immune response elicited following vaccine administration are discussed.
- 139Ewers, H.; Smith, A. E.; Sbalzarini, I. F.; Lilie, H.; Koumoutsakos, P.; Helenius, A. Single-Particle Tracking of Murine Polyoma Virus-Like Particles on Live Cells and Artificial Membranes. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 15110– 15115, DOI: 10.1073/pnas.0504407102139Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranesEwers, Helge; Smith, Alicia E.; Sbalzarini, Ivo F.; Lilie, Hauke; Koumoutsakos, Petros; Helenius, AriProceedings of the National Academy of Sciences of the United States of America (2005), 102 (42), 15110-15115CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, anal. of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diam.), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside mols. by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.
- 140Cureton, D. K.; Massol, R. H.; Whelan, S. P.; Kirchhausen, T. The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly During Clathrin-Dependent Endocytosis. PLoS Pathog. 2010, 6, e1001127 DOI: 10.1371/journal.ppat.1001127There is no corresponding record for this reference.
- 141Pelkmans, L.; Puntener, D.; Helenius, A. Local Actin Polymerization and Dynamin Recruitment in SV40-Induced Internalization of Caveolae. Science 2002, 296, 535– 539, DOI: 10.1126/science.1069784141Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolaePelkmans, Lucas; Puntener, Daniel; Helenius, AriScience (Washington, DC, United States) (2002), 296 (5567), 535-539CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. The authors found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.
- 142Cureton, D. K.; Harbison, C. E.; Cocucci, E.; Parrish, C. R.; Kirchhausen, T. Limited Transferrin Receptor Clustering Allows Rapid Diffusion of Canine Parvovirus into Clathrin Endocytic Structures. J. Virol. 2012, 86, 5330– 5340, DOI: 10.1128/JVI.07194-11142Limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structuresCureton, David K.; Harbison, Carole E.; Cocucci, Emanuele; Parrish, Colin R.; Kirchhausen, TomJournal of Virology (2012), 86 (9), 5330-5340CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here, the authors used fluorescence microscopy to directly visualize the assocn. of single canine parvovirus (CPV) capsids with cellular transferring receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. They found that most capsids assocd. with fewer than five TfRs and that ∼25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissocd. from the cell surface with a half-life of ∼30 s. Thus, the results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.
- 143Ehrlich, M.; Boll, W.; Van Oijen, A.; Hariharan, R.; Chandran, K.; Nibert, M. L.; Kirchhausen, T. Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits. Cell 2004, 118, 591– 605, DOI: 10.1016/j.cell.2004.08.017143Endocytosis by random initiation and stabilization of clathrin-coated pitsEhrlich, Marcelo; Boll, Werner; van Oijen, Antoine; Hariharan, Ramesh; Chandran, Kartik; Nibert, Max L.; Kirchhausen, TomasCell (Cambridge, MA, United States) (2004), 118 (5), 591-605CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approx. const. growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.
- 144Damm, E. M.; Pelkmans, L.; Kartenbeck, J.; Mezzacasa, A.; Kurzchalia, T.; Helenius, A. Clathrin- and Caveolin-1-Independent Endocytosis: Entry of Simian Virus 40 into Cells Devoid of Caveolae. J. Cell Biol. 2005, 168, 477– 488, DOI: 10.1083/jcb.200407113144Clathrin- and caveolin-1-independent endocytosis: Entry of simian virus 40 into cells devoid of caveolaeDamm, Eva-Maria; Pelkmans, Lucas; Kartenbeck, Juergen; Mezzacasa, Anna; Kurzchalia, Teymuras; Helenius, AriJournal of Cell Biology (2005), 168 (3), 477-488CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol- and tyrosine kinase- dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.
- 145Meier, R.; Franceschini, A.; Horvath, P.; Tetard, M.; Mancini, R.; Von Mering, C.; Helenius, A.; Lozach, P.-Y. Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration. J. Virol. 2014, 88, 8565– 8578, DOI: 10.1128/JVI.00388-14145Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetrationMeier, Roger; Franceschini, Andrea; Horvath, Peter; Tetard, Marilou; Mancini, Roberta; von Mering, Christian; Helenius, Ari; Lozach, Pierre-YvesJournal of Virology (2014), 88 (15), 8565-8578, 15 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used 2 genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence anal. of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but addnl. genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was assocd. with the VAMP3+ virus-contg. endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-neg. compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network.
- 146Kalin, S.; Amstutz, B.; Gastaldelli, M.; Wolfrum, N.; Boucke, K.; Havenga, M.; DiGennaro, F.; Liska, N.; Hemmi, S.; Greber, U. F. Macropinocytotic Uptake and Infection of Human Epithelial Cells with Species B2 Adenovirus Type 35. J. Virol. 2010, 84, 5336– 5350, DOI: 10.1128/JVI.02494-09146Macropinocytotic uptake and infection of human epithelial cells with species B2 adenovirus type 35Kalin, Stefan; Amstutz, Beat; Gastaldelli, Michele; Wolfrum, Nina; Boucke, Karin; Havenga, Menzo; Di Gennaro, Fabienne; Liska, Nicole; Hemmi, Silvio; Greber, Urs F.Journal of Virology (2010), 84 (10), 5336-5350CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here, the authors show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chems. against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-neg. proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were pos. for CD46, αν integrins, and also CtBP1. The results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.
- 147Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B. Cyanines During the 1990s: A Review. Chem. Rev. 2000, 100, 1973– 2011, DOI: 10.1021/cr990402t147Cyanines during the 1990s: A ReviewMishra, Amaresh; Behera, Rajani K.; Behera, Pradipta K.; Mishra, Bijaya K.; Behera, Gopa B.Chemical Reviews (Washington, D. C.) (2000), 100 (6), 1973-2011CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 401 refs. on synthesis, self-aggregation, nonlinear optical properties, adsorption, photodimerization and -isomerization, photodynamic therapy, dyes in organized systems, and as fluorescent ion sensors.
- 148Kvach, M. V.; Ustinov, A. V.; Stepanova, I. A.; Malakhov, A. D.; Skorobogatyi, M. V.; Shmanai, V. V.; Korshun, V. A. A Convenient Synthesis of Cyanine Dyes: Reagents for the Labeling of Biomolecules. Eur. J. Org. Chem. 2008, 2008, 2107– 2117, DOI: 10.1002/ejoc.200701190There is no corresponding record for this reference.
- 149Yang, X.; Shi, C.; Tong, R.; Qian, W.; Zhau, H. E.; Wang, R.; Zhu, G.; Cheng, J.; Yang, V. W.; Cheng, T. Near IR Heptamethine Cyanine Dye-Mediated Cancer Imaging. Clin. Cancer Res. 2010, 16, 2833– 2844, DOI: 10.1158/1078-0432.CCR-10-0059149Near IR Heptamethine Cyanine Dye-Mediated Cancer ImagingYang, Xiaojian; Shi, Chunmeng; Tong, Rong; Qian, Weiping; Zhau, Haiyen E.; Wang, Ruoxiang; Zhu, Guodong; Cheng, Jianjun; Yang, Vincent W.; Cheng, Tianmin; Henary, Maged; Strekowski, Lucjan; Chung, Leland W. K.Clinical Cancer Research (2010), 16 (10), 2833-2844CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Near-IR fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we show the preferential uptake and retention of two heptamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues. IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts, and spontaneous mouse tumors in transgenic animals. Time- and concn.-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein, a competitive inhibitor of org. anion transporting peptides. These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood. These near-IR heptamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to conc. in the mitochondria and lysosomes of cancer cells, probably through org. anion transporting peptides, because the dye uptake and retention in cancer cells can be blocked completely by bromosulfophthalein. These dyes, when injected to mice, did not cause systemic toxicity. These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis, and treatment.
- 150Brauchle, C.; Seisenberger, G.; Endress, T.; Ried, M. U.; Buning, H.; Hallek, M. Single Virus Tracing: Visualization of the Infection Pathway of a Virus into a Living Cell. ChemPhysChem 2002, 3, 299– 303, DOI: 10.1002/1439-7641(20020315)3:3<299::AID-CPHC299>3.0.CO;2-R150Single virus tracing: visualization of the infection pathway of a virus into a living cellBrauchle, Christoph; Seisenberger, Georg; Endress, Thomas; Ried, Martin U.; Buning, Hildegard; Hallek, MichaelChemPhysChem (2002), 3 (3), 299-303CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH)The novel technique, known as single virus tracing (SVT), allows the visualization of the infection pathway of an individual virus labeled with only one fluorescent dye mol. into a living cell. The fluorescence of the marker mol. is imaged with single-mol. techniques and used to follow the pathway of the virus with high spatial (>40 nm) and time (>10 ms) resoln. An overview of the different steps, which can be visualized and kinetically characterized by SVT, is presented for the model system, the adeno-assocd. virus (AAV). Since the capsid of the AAV becomes internalized into the cell, a fluorescent label can be attached either to the protein capsid, the DNA genome, or both. When this labeled virus is given to a living cell, a sequence of events can be monitored by SVT starting with the virus approaching the cell surface.
- 151Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.; Filanoski, B. J.; Telford, W. G.; Yue, S.; Liu, J. X.; Cheung, C. Y. Quantitative Comparison of Long-Wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates. J. Histochem. Cytochem. 2003, 51, 1699– 1712, DOI: 10.1177/002215540305101214151Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: Fluorescence of the dyes and their bioconjugatesBerlier, Judith E.; Rothe, Anca; Buller, Gayle; Bradford, Jolene; Gray, Diane R.; Filanoski, Brian J.; Telford, William G.; Yue, Stephen; Liu, Jixiang; Cheung, Ching-Ying; Chang, Wesley; Hirsch, James D.; Beechem, Joseph M.; Haugland, Rosaria P.; Haugland, Richard P.Journal of Histochemistry and Cytochemistry (2003), 51 (12), 1699-1712CODEN: JHCYAS; ISSN:0022-1554. (Histochemical Society, Inc.)Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coeffs. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepd. from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously obsd. for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, esp. at high degrees of labeling. The results from our flow cytometry, immunocytochem., and immunohistochem. expts. demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.
- 152Mahmoudian, J.; Hadavi, R.; Jeddi-Tehrani, M.; Mahmoudi, A. R.; Bayat, A. A.; Shaban, E.; Vafakhah, M.; Darzi, M.; Tarahomi, M.; Ghods, R. Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568-and Fluorescein Isothiocyanate- Conjugated Antibody. Cell J. 2011, 13, 169– 172152Comparison of the photobleaching and photostability traits of Alexa fluor 568- and fluorescein isothiocyanate- conjugated antibodyMahmoudian, Jafar; Hadavi, Reza; Jeddi-Tehrani, Mahmood; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Shaban, Elham; Vafakhah, Mohtaram; Darzi, Maryam; Tarahomi, Majid; Ghods, RoyaCell Journal (2011), 13 (3), 169-172,, 1 plateCODEN: CJEOAD; ISSN:2228-5806. (Royan Institute)Objective: Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe mols. Based on dye chem. structures, their photobleaching and photostability indexes are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleaching profiles than FITC. Materials and Methods: In this exptl. study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indexes. Then, the fluorophore/antibody ratios were calcd. using a spectrophotometer. The photobleaching profiles and photostability indexes of conjugated antibodies were subsequently studied by immunocytochem. (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Results: Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Conclusion: Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.
- 153Hoekstra, D.; de Boer, T.; Klappe, K.; Wilschut, J. Fluorescence Method for Measuring the Kinetics of Fusion between Biological Membranes. Biochemistry 1984, 23, 5675– 5681, DOI: 10.1021/bi00319a002153Fluorescence method for measuring the kinetics of fusion between biological membranesHoekstra, Dick; De Boer, Tiny; Klappe, Karin; Wilschut, JanBiochemistry (1984), 23 (24), 5675-81CODEN: BICHAW; ISSN:0006-2960.An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biol. membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concns. up to 9 mol % with respect to total lipid, the efficiency of self-quenching is proportional to its surface d. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface d. of the fluorophores results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quant. measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were the same as those detd. with a fusion assay based on resonance energy transfer (Struck, D. K. et al., 1981). Octadecyl Rhodamine B chloride can be readily inserted into native biol. membranes by addn. of an ethanolic soln. of the probe. Evidence is presented showing that the diln. of the fluorophore, occurring when octadecyl Rhodamine contg. influenza viruses are mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Diln. of the probe was not obsd. upon prior treatment of fluorescently labeled Sendai virus with trypsin. Virus-induced fusion between fluorescently tagged ghosts and ghosts devoid of the probe was only obsd. (at 37°) after a low-temp. preincubation; no fluorescence developed was seen during virus-induced aggregation at low temp. nor when ghosts and the virus were directly mixed at 37°. Apparently, spontaneous intermembrane transfer of the fluorophore did not occur. This technique may be of considerable value for investigating fusion between biol. membranes and, hence, provides an important tool in elucidating the mechanism of fusion in such systems.
- 154Floyd, D. L.; Ragains, J. R.; Skehel, J. J.; Harrison, S. C.; van Oijen, A. M. Single-Particle Kinetics of Influenza Virus Membrane Fusion. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15382– 15387, DOI: 10.1073/pnas.0807771105154Single-particle kinetics of influenza virus membrane fusionFloyd, Daniel L.; Ragains, Justin R.; Skehel, John J.; Harrison, Stephen C.; van Oijen, Antoine M.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (40), 15382-15387CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed anal. of the sequence of the mol. steps involved. The authors have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Anal. of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. The authors interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.
- 155Jha, N. K.; Latinovic, O.; Martin, E.; Novitskiy, G.; Marin, M.; Miyauchi, K.; Naughton, J.; Young, J. A.; Melikyan, G. B. Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion. PLoS Pathog. 2011, 7, e1001260 DOI: 10.1371/journal.ppat.1001260155Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusionJha, Naveen K.; Latinovic, Olga; Martin, Erik; Novitskiy, Gennadiy; Marin, Mariana; Miyauchi, Kosuke; Naughton, John; Young, John A. T.; Melikyan, Gregory B.PLoS Pathogens (2011), 7 (1), e1001260CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A crit. barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor d. and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a crit. role in establishing low pH-dependent virus entry from within acidic endosomes.
- 156Hoornweg, T. E.; van Duijl-Richter, M. K. S.; Ayala Nunez, N. V.; Albulescu, I. C.; van Hemert, M. J.; Smit, J. M. Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells. J. Virol. 2016, 90, 4745– 4756, DOI: 10.1128/JVI.03184-15156Dynamics of chikungunya virus cell entry unraveled by single-virus tracking in living cellsHoornweg, Tabitha E.; van Duijl-Richter, Mareike K. S.; Nunez, Nilda V. Ayala; Albulescu, Irina C.; van Hemert, Martijn J.; Smit, Jolanda M.Journal of Virology (2016), 90 (9), 4745-4756CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We obsd. that almost all particles fused within 20 min after addn. to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The av. time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was obsd. predominantly from within Rab5-pos. endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes.
- 157Li, Q.; Li, W.; Yin, W.; Guo, J.; Zhang, Z. P.; Zeng, D.; Zhang, X.; Wu, Y.; Zhang, X. E.; Cui, Z. Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages. ACS Nano 2017, 11, 3890– 3903, DOI: 10.1021/acsnano.7b00275157Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary MacrophagesLi, Qin; Li, Wei; Yin, Wen; Guo, Jia; Zhang, Zhi-Ping; Zeng, Dejun; Zhang, Xiaowei; Wu, Yuntao; Zhang, Xian-En; Cui, ZongqiangACS Nano (2017), 11 (4), 3890-3903CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and obsd. that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-pos. endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Addnl., we obsd. that a dynamic actin cytoskeleton is crit. for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.
- 158Nanbo, A.; Imai, M.; Watanabe, S.; Noda, T.; Takahashi, K.; Neumann, G.; Halfmann, P.; Kawaoka, Y. Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner. PLoS Pathog. 2010, 6, e1001121 DOI: 10.1371/journal.ppat.1001121There is no corresponding record for this reference.
- 159Hao, X.; Shang, X.; Wu, J. Z.; Shan, Y. P.; Cai, M. J.; Jiang, J. G.; Huang, Z.; Tang, Z. Y.; Wang, H. D. Single-Particle Tracking of Hepatitis B Virus-Like Vesicle Entry into Cells. Small 2011, 7, 1212– 1218, DOI: 10.1002/smll.201002020159Single-Particle Tracking of Hepatitis B Virus-like Vesicle Entry into CellsHao, Xian; Shang, Xin; Wu, Jiazhen; Shan, Yuping; Cai, Mingjun; Jiang, Junguang; Huang, Zhong; Tang, Zhiyong; Wang, HongdaSmall (2011), 7 (9), 1212-1218CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)HBsAg, the surface antigen of the hepatitis B virus (HBV), is used as a model to study the mechanisms and dynamics of a single-enveloped virus infecting living cells by imaging and tracking at the single-particle level. By monitoring the fluorescent indicator of HBsAg particles, it is found that HBsAg enters cells via a caveolin-mediated endocytic pathway. Tracking of individual HBsAg particles in living cells reveals the anomalously actin-dependent but not microtubule-dependent motility of the internalized HBsAg particle. The motility of HBsAg particles in living cells is also analyzed quant. These results may settle the long-lasting debate of whether HBV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell, and how the virus moves in the cell.
- 160Le Blanc, I.; Luyet, P. P.; Pons, V.; Ferguson, C.; Emans, N.; Petiot, A.; Mayran, N.; Demaurex, N.; Faure, J.; Sadoul, R. Endosome-to-Cytosol Transport of Viral Nucleocapsids. Nat. Cell Biol. 2005, 7, 653– 664, DOI: 10.1038/ncb1269160Endosome-to-cytosol transport of viral nucleocapsidsLe Blanc, Isabelle; Luyet, Pierre-Philippe; Pons, Veronique; Ferguson, Charles; Emans, Neil; Petiot, Anne; Mayran, Nathalie; Demaurex, Nicolas; Faure, Julien; Sadoul, Remy; Parton, Robert G.; Gruenberg, J.Nature Cell Biology (2005), 7 (7), 653-664CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns(3)P) signaling via the PtdIns(3)P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns(3)P and their effectors.
- 161Lozach, P.-Y.; Kühbacher, A.; Meier, R.; Mancini, R.; Bitto, D.; Bouloy, M.; Helenius, A. DC-SIGN as a Receptor for Phleboviruses. Cell Host Microbe 2011, 10, 75– 88, DOI: 10.1016/j.chom.2011.06.007161DC-SIGN as a Receptor for PhlebovirusesLozach, Pierre-Yves; Kuehbacher, Andreas; Meier, Roger; Mancini, Roberta; Bitto, David; Bouloy, Michele; Helenius, AriCell Host & Microbe (2011), 10 (1), 75-88CODEN: CHMECB; ISSN:1931-3128. (Cell Press)Summary: During natural transmission, bunyaviruses are introduced into the skin through arthropod bites, and dermal dendritic cells (DCs) are the first to encounter incoming viruses. DC-SIGN is a C-type lectin highly expressed on the surface of dermal DCs. We found that several arthropod-borne phleboviruses (Bunyaviridae), including Rift Valley fever and Uukuniemi viruses, exploit DC-SIGN to infect DCs and other DC-SIGN-expressing cells. DC-SIGN binds the virus directly via interactions with high-mannose N-glycans on the viral glycoproteins and is required for virus internalization and infection. In live cells, virus-induced clustering of cell surface DC-SIGN could be visualized. An endocytosis-defective mutant of DC-SIGN was unable to mediate virus uptake, indicating that DC-SIGN is an authentic receptor required for both attachment and endocytosis. After internalization, viruses sepd. from DC-SIGN and underwent trafficking to late endosomes. Our study provides real-time visualization of virus-receptor interactions on the cell surface and establishes DC-SIGN as a phlebovirus entry receptor.
- 162Ayala-Nuñez, N. V.; Wilschut, J.; Smit, J. M. Monitoring Virus Entry into Living Cells Using DiD-Labeled Dengue Virus Particles. Methods 2011, 55, 137– 143, DOI: 10.1016/j.ymeth.2011.07.009162Monitoring virus entry into living cells using DiD-labeled dengue virus particlesAyala-Nunez, Nilda V.; Wilschut, Jan; Smit, Jolanda M.Methods (Amsterdam, Netherlands) (2011), 55 (2), 137-143CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)A review. A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addn. - and using the same exptl. setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data anal., and hints for troubleshooting are described throughout the paper.
- 163Crowther, D.; Melnick, J. L. The Incorporation of Neutral Red and Acridine Orange into Developing Poliovirus Particles Making Them Photosensitive. Virology 1961, 14, 11– 21, DOI: 10.1016/0042-6822(61)90127-1163Incorporation of neutral red and Acridine Orange into developing poliovirus particles making them photosensitiveCrowther, Derek; Melnick, Joseph L.Virology (1961), 14 (1), 11-21CODEN: VIRLAX; ISSN:0042-6822.Mature, infective virus incubated with neutral red and Acridine orange for one hour in the absence of cells and then exposed to white light showed no redn. in titer. Virus grown in cells contg. neutral red or Acridine Orange may be subsequently inactivated by light, thus indicating an incorporation of the dye into the developing virus. The effect of neutral red and light was the same for the virulent and the attenuated strains. The biol. and chem. similarities between the quinonimide dyes (neutral red, toluidine blue) and the acridine (Acridine Orange, proflavine) are discussed.
- 164Wilson, J. N.; Cooper, P. D. Aspects of the Growth of Poliovirus as Revealed by the Photodynamic Effects of Neutral Red and Acridine Orange. Virology 1963, 21, 135– 145, DOI: 10.1016/0042-6822(63)90249-6164ASPECTS OF THE GROWTH OF POLIOVIRUS AS REVEALED BY THE PHOTODYNAMIC EFFECTS OF NEUTRAL RED AND ACRIDINE ORANGEWILSON J N; COOPER P DVirology (1963), 21 (), 135-45 ISSN:0042-6822.There is no expanded citation for this reference.
- 165Glynn, T. J.; Power, S.; Ryder, A. G.; Morrison, J. J. Time-Domain Measurement of Fluorescence Lifetime Variation with pH. Proc. SPIE-Int. Soc. Opt. Eng.; Society of Photo-optical Instrumentation Engineers: 2001.There is no corresponding record for this reference.
- 166Kremser, L.; Okun, V. M.; Nicodemou, A.; Blaas, D.; Kenndler, E. Binding of Fluorescent Dye to Genomic RNA inside Intact Human Rhinovirus after Viral Capsid Penetration Investigated by Capillary Electrophoresis. Anal. Chem. 2004, 76, 882– 887, DOI: 10.1021/ac034898x166Binding of Fluorescent Dye to Genomic RNA Inside Intact Human Rhinovirus after Viral Capsid Penetration Investigated by Capillary ElectrophoresisKremser, Leopold; Okun, Vadim M.; Nicodemou, Andreas; Blaas, Dieter; Kenndler, ErnstAnalytical Chemistry (2004), 76 (4), 882-887CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)RiboGreen is used for concn. measurements of RNA. Upon binding to the RNA, an ∼1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is obsd. In the present work, we demonstrate that this dye can penetrate in a time- and temp.-dependent manner the intact viral capsids of human rhinovirus serotypes 2 and 14, where it forms a fluorescent complex with the viral RNA. Capillary electrophoresis with laser-induced fluorescence detection of virus incubated with RiboGreen shows that the electrophoretic mobility of the viruses remained unchanged upon dye-binding. As shown for human rhinovirus serotype 2, its native conformation was conserved, since it still bound a recombinant sol. receptor fragment derived from the very low d. lipoprotein receptor. The labeled RNA was released by heat-induced uncoating of the virus, and the RNA-dye complex could be directly detected if degrdn. was prevented with an RNase inhibitor. This in vitro labeling of viral RNA encased within a protein shell demonstrates the virion's dynamic nature that temporarily allows access of a low-mol.-mass compd. to the otherwise protected RNA. It might be of great value for expts. requiring fluorescent viral particles with an unmodified surface, such as investigations of endocytosis and viral uncoating on the single mol. level.
- 167Kremser, L.; Petsch, M.; Blaas, D.; Kenndler, E. Labeling of Capsid Proteins and Genomic RNA of Human Rhinovirus with Two Different Fluorescent Dyes for Selective Detection by Capillary Electrophoresis. Anal. Chem. 2004, 76, 7360– 7365, DOI: 10.1021/ac048999m167Labeling of Capsid Proteins and Genomic RNA of Human Rhinovirus with Two Different Fluorescent Dyes for Selective Detection by Capillary ElectrophoresisKremser, Leopold; Petsch, Martina; Blaas, Dieter; Kenndler, ErnstAnalytical Chemistry (2004), 76 (24), 7360-7365CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)During uncoating of human rhinoviruses, the innermost capsid protein VP4 and the genomic RNA are released from the viral protein shell. This process gives rise to subviral particles that are composed of the remaining three capsid proteins VP1, VP2, and VP3. The process is believed to take place in a sequential manner in that first VP4 is expelled resulting in A-particles sedimenting at 135S followed by the RNA resulting in B-particles sedimenting at 80S. Aiming at ultimately analyzing this process in vivo, we introduced two different fluorophores into the RNA and the viral capsid proteins, resp. Incubation of the virus with RiboGreen resulted in formation of a RNA-dye complex with λex/λem = 500/525 nm, whereas subsequent derivatization of the viral protein shell in the same sample with AMCA-S introduced a label with λex/λem = 345-350/440-460 nm. In this way, both viral components could be selectively detected via fluorescence in a capillary electrophoresis system. The intact virus delivers two superimposed signals in the electropherogram. Derivatization of the free amino groups of the capsid proteins partially preserved the bioaffinity of the virus toward a synthetic receptor fragment, an artificial recombinant concatemer of repeat no. 3 of the very low d. lipoprotein receptor. Between 10 and 20% of the infectivity were recovered after labeling when compared to native virus. In addn. to anal. of factors influencing the stability of the virus by CE, double-labeled virions might be useful for the investigation of the uncoating process by real-time confocal fluorescence microscopy.
- 168Jones, L. J.; Yue, S. T.; Cheung, C. Y.; Singer, V. L. RNA Quantitation by Fluorescence-Based Solution Assay: RiboGreen Reagent Characterization. Anal. Biochem. 1998, 265, 368– 374, DOI: 10.1006/abio.1998.2914168RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterizationJones, Laurie J.; Yue, Stephen T.; Cheung, Ching-Ying; Singer, Victoria L.Analytical Biochemistry (1998), 265 (2), 368-374CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)We described the development of a sensitive fluorescence-based soln. assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits > 1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coeff. (67,000 cm-1 M-1). The RiboGreen assay allows detection of as little as 1.0 ng/mL RNA in a std. fluorometer, filter fluorometer, or fluorescence microplate reader-surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range for RiboGreen reagent extends over three orders of magnitude in RNA concn. Using 750 nM RiboGreen reagent, we quantitated 20 ng/mL to 1.0 μg/mL RNA. By dilg. the reagent to 75 nM, we could quantitate 1.0 to 50 ng/mL RNA. Both assay ranges exhibited linear fluorescence increases vs. RNA concn. (r2 = 0.999). Assay linearity was maintained in the presence of salts, protein, urea, ethanol, chloroform, agarose, and some detergents. Several different RNA types yielded similar signal intensities and detection sensitivities. The assay is easy to use, rapid, and readily adaptable for automation. (c) 1998 Academic Press.
- 169Lin, Y.-W.; Chiu, T.-C.; Chang, H.-T. Laser-Induced Fluorescence Technique for DNA and Proteins Separated by Capillary Electrophoresis. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2003, 793, 37– 48, DOI: 10.1016/S1570-0232(03)00363-5169Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresisLin, Yang-Wei; Chiu, Tai-Chia; Chang, Huan-TsungJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2003), 793 (1), 37-48CODEN: JCBAAI; ISSN:1570-0232. (Elsevier Science B.V.)A review. Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (max. excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chems. can be carried out prior to, during, or after sepns. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the anal. of polymerase chain reaction products, DNA sequencing, trace anal. of proteins, and single cell anal. were presented.
- 170Wen, L.; Lin, Y.; Zhang, Z. L.; Lu, W.; Lv, C.; Chen, Z. L.; Wang, H. Z.; Pang, D. W. Intracellular Self-Assembly Based Multi-Labeling of Key Viral Components: Envelope, Capsid and Nucleic Acids. Biomaterials 2016, 99, 24– 33, DOI: 10.1016/j.biomaterials.2016.04.038170Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acidsWen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-WenBiomaterials (2016), 99 (), 24-33CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissocn. and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment.
- 171Zhou, P.; Zheng, Z.; Lu, W.; Zhang, F.; Zhang, Z.; Pang, D.; Hu, B.; He, Z.; Wang, H. Multicolor Labeling of Living-Virus Particles in Live Cells. Angew. Chem., Int. Ed. 2012, 51, 670– 674, DOI: 10.1002/anie.201105701171Multicolor Labeling of Living-Virus Particles in Live CellsZhou, Peng; Zheng, Zhenhua; Lu, Wen; Zhang, Fuxian; Zhang, Zhenfeng; Pang, Daiwen; Hu, Bin; He, Zhike; Wang, HanzhongAngewandte Chemie, International Edition (2012), 51 (3), 670-674, S670/1-S670/3CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Herein, the authors describe a new strategy for synchronous multicolor labeling of a living virus in live cells. The viral genomes were labeled by using an in vivo virus self-assembly system in combination with a novel nucleic acid probe based on a ruthenium complex, and the viral envelope was labeled by fusing a characteristic external viral protein with green fluorescent protein (GFP). Multicolor labeling of distinct viral components can be done during the viral replication in host cells. More importantly, labeling a virus in this manner can overcome the photobleaching and self-quenching effects between dye mols. and does not affect the viral infectivity.
- 172Huang, L. L.; Zhou, P.; Wang, H. Z.; Zhang, R.; Hao, J.; Xie, H. Y.; He, Z. K. A New Stable and Reliable Method for Labeling Nucleic Acids of Fully Replicative Viruses. Chem. Commun. 2012, 48, 2424– 2426, DOI: 10.1039/c2cc17069h172A new stable and reliable method for labeling nucleic acids of fully replicative virusesHuang, Li-Li; Zhou, Peng; Wang, Han-Zhong; Zhang, Rui; Hao, Jian; Xie, Hai-Yan; He, Zhi-KeChemical Communications (Cambridge, United Kingdom) (2012), 48 (18), 2424-2426CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Efficiently labeling nucleic acids of fully replicative viruses is a challenge. In this work, a mol. light switch complex [Ru(phen)2(dppz)]2+, where phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2',3'-c]phenazine, has been exploringly used to label vaccinia virus nucleic acid. The labeled virions exhibited strong and stable fluorescence and could be imaged at the single-virion level. Moreover, they were fully infectious and can be used to study the behaviors of invasion into their host cells. The method is general and suitable for labeling various DNA viruses.
- 173Shimomura, O.; Johnson, F. H.; Saiga, Y. Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. J. Cell. Comp. Physiol. 1962, 59, 223– 239, DOI: 10.1002/jcp.1030590302173Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, AequoreaShimomura, Osamu; Johnson, Frank H.; Saiga, YoJournal of Cellular and Comparative Physiology (1962), 59 (), 223-39CODEN: JCCPAY; ISSN:0095-9898.cf. CA 57, 1392b. The material obtained by squeezing marginal strips of Aequorea through cloth was filtered with filtercel and the cake extd. with di-Na ethylenediaminetetraacetate (EDTA). Pptn. with (NH4)2SO4 and chromatography on diethylaminoethyl cellulose gave aequorin (I) with the properties of a protein of mol. wt. approx. 35,000. Addn. of Ca++ in an amt. at least the molar equiv. to that of EDTA present produced a flash of light. No other ion could replace Ca++. The rate of light emission followed 1st order reaction kinetics; O was not necessary and only a single component (in addn. to Ca++) could be identified. Total light produced decreased with rise in temp. from 0° to 40° and was independent of pH between 5.1 and 8.3. The rate of emission was nearly const. between pH 6.5 and 7.5, decreasing at lower and increasing at higher values. Malonate, EDTA, and a series of aliphatic aldehydes (C3-C7) reversibly inhibited the rate but not the total of light emission. Aromatic aldehydes, inorg. reducing or strong oxidizing agents, p-ClHgC6H4CO2H, HgCl2, N1-methylnicotinamide chloride, hydroquinone, benzoquinone, histidine-HCl, dinitrofluorobenzene-NaHCO3, and NCCH2CO2H reduced the total light without affecting the rate of emission; H2O2 had no effect. Aliphatic alcs. increased light emission; C6H5CH2OH caused a slight decrease. The absorption spectrum of I shows a max. at 280 mμ with a bulge at 310 mμ. In the luminescent reaction the bulge is replaced by a new max. at 333 mμ. These changes suggest the presence of a reduced pyridinium deriv. combined with the protein.
- 174Chalfie, M. Green Fluorescent Protein as a Marker for Gene Expression. Trends Genet. 1994, 10, 151, DOI: 10.1016/0168-9525(94)90088-4There is no corresponding record for this reference.
- 175Lippincott-Schwartz, J.; Patterson, G. H. Development and Use of Fluorescent Protein Markers in Living Cells. Science 2003, 300, 87– 91, DOI: 10.1126/science.1082520175Development and use of fluorescent protein markers in living cellsLippincott-Schwartz, Jennifer; Patterson, George H.Science (Washington, DC, United States) (2003), 300 (5616), 87-91CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. The ability to visualize, track, and quantify mols. and events in living cells with high spatial and temporal resoln. is essential for understanding biol. systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technol. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.
- 176Day, R. N.; Davidson, M. W. The Fluorescent Protein Palette: Tools for Cellular Imaging. Chem. Soc. Rev. 2009, 38, 2887– 2921, DOI: 10.1039/b901966a176The fluorescent protein palette: Tools for cellular imagingDay, Richard N.; Davidson, Michael W.Chemical Society Reviews (2009), 38 (10), 2887-2921CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)This crit. review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biol. and physiol. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quant. measurements in living systems (227 refs.).
- 177Wiedenmann, J.; Oswald, F.; Nienhaus, G. U. Fluorescent Proteins for Live Cell Imaging: Opportunities, Limitations, and Challenges. IUBMB Life 2009, 61, 1029– 1042, DOI: 10.1002/iub.256177Fluorescent proteins for live cell imaging: opportunities, limitations, and challengesWiedenmann, Jorg; Oswald, Franz; Nienhaus, Gerd UlrichIUBMB Life (2009), 61 (11), 1029-1042CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons Inc.)A review. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse mol. properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.
- 178Shaner, N. C.; Campbell, R. E.; Steinbach, P. A.; Giepmans, B. N.; Palmer, A. E.; Tsien, R. Y. Improved Monomeric Red, Orange and Yellow Fluorescent Proteins Derived from Discosoma sp. Red Fluorescent Protein. Nat. Biotechnol. 2004, 22, 1567– 1572, DOI: 10.1038/nbt1037178Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteinShaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y.Nature Biotechnology (2004), 22 (12), 1567-1572CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biol. and biotechnol. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
- 179Shkrob, M. A.; Yanushevich, Y. G.; Chudakov, D. M.; Gurskaya, N. G.; Labas, Y. A.; Poponov, S. Y.; Mudrik, N. N.; Lukyanov, S.; Lukyanov, K. A. Far-Red Fluorescent Proteins Evolved From a Blue Chromoprotein from Actinia Equina. Biochem. J. 2005, 392, 649– 654, DOI: 10.1042/BJ20051314179Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equinaShkrob, Maria A.; Yanushevich, Yurii G.; Chudakov, Dmitriy M.; Gurskaya, Nadya G.; Labas, Yulii A.; Poponov, Sergey Y.; Mudrik, Nikolay N.; Lukyanov, Sergey; Lukyanov, Konstantin A.Biochemical Journal (2005), 392 (3), 649-654CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-colored Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disk. A novel blue chromoprotein, aeCP597, with an absorption max. at 597 nm detg. the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chem. identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is detd. by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm resp. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.
- 180Nienhaus, K.; Nienhaus, G. U. Fluorescent Proteins for Live-Cell Imaging with Super-Resolution. Chem. Soc. Rev. 2014, 43, 1088– 1106, DOI: 10.1039/C3CS60171D180Fluorescent proteins for live-cell imaging with super-resolutionNienhaus, Karin; Ulrich Nienhaus, G.Chemical Society Reviews (2014), 43 (4), 1088-1106CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradn. at specific wavelengths. They enable regional optical marking in pulse-chase expts. on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resoln. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chem. reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resoln. microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many labs., there is still room for further improvement of these important markers for live cell imaging.
- 181Shcherbakova, D. M.; Verkhusha, V. V. Near-Infrared Fluorescent Proteins for Multicolor in Vivo Imaging. Nat. Methods 2013, 10, 751– 754, DOI: 10.1038/nmeth.2521181Near-infrared fluorescent proteins for multicolor in vivo imagingShcherbakova, Daria M.; Verkhusha, Vladislav V.Nature Methods (2013), 10 (8), 751-754CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Near-IR fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-IR FPs-iRFP670, iRFP682, iRFP702 and iRFP720-from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with std. approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.
- 182Shcherbakova, D. M.; Subach, O. M.; Verkhusha, V. V. Red Fluorescent Proteins: Advanced Imaging Applications and Future Design. Angew. Chem., Int. Ed. 2012, 51, 10724– 10738, DOI: 10.1002/anie.201200408182Red Fluorescent Proteins: Advanced Imaging Applications and Future DesignShcherbakova, Daria M.; Subach, Oksana M.; Verkhusha, Vladislav V.Angewandte Chemie, International Edition (2012), 51 (43), 10724-10738CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. In the past few years a large series of the advanced red shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biol. processes at the levels ranging from single mols. to whole organisms. Herein the relation between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chem. transformations of red chromophores allows the future RFP phenotypes and their resp. novel imaging applications to be foreseen.
- 183Tomosugi, W.; Matsuda, T.; Tani, T.; Nemoto, T.; Kotera, I.; Saito, K.; Horikawa, K.; Nagai, T. An Ultramarine Fluorescent Protein with Increased Photostability and pH Insensitivity. Nat. Methods 2009, 6, 351– 353, DOI: 10.1038/nmeth.1317183An ultramarine fluorescent protein with increased photostability and pH insensitivityTomosugi, Wataru; Matsuda, Tomoki; Tani, Tomomi; Nemoto, Tomomi; Kotera, Ippei; Saito, Kenta; Horikawa, Kazuki; Nagai, TakeharuNature Methods (2009), 6 (5), 351-353CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biol. events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concn. and caspase-3 activation in the same apoptotic cells.
- 184Ai, H. W.; Shaner, N. C.; Cheng, Z.; Tsien, R. Y.; Campbell, R. E. Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins. Biochemistry 2007, 46, 5904– 5910, DOI: 10.1021/bi700199g184Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent ProteinsAi, Hui-Wang; Shaner, Nathan C.; Cheng, Zihao; Tsien, Roger Y.; Campbell, Robert E.Biochemistry (2007), 46 (20), 5904-5910CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein the authors report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".
- 185Subach, O. M.; Gundorov, I. S.; Yoshimura, M.; Subach, F. V.; Zhang, J.; Grüenwald, D.; Souslova, E. A.; Chudakov, D. M.; Verkhusha, V. V. Conversion of Red Fluorescent Protein into a Bright Blue Probe. Chem. Biol. 2008, 15, 1116– 1124, DOI: 10.1016/j.chembiol.2008.08.006185Conversion of Red Fluorescent Protein into a Bright Blue ProbeSubach, Oksana M.; Gundorov, Illia S.; Yoshimura, Masami; Subach, Fedor V.; Zhang, Jinghang; Gruenwald, David; Souslova, Ekaterina A.; Chudakov, Dmitriy M.; Verkhusha, Vladislav V.Chemistry & Biology (Cambridge, MA, United States) (2008), 15 (10), 1116-1124CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochem. and photochem. anal. indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Foerster resonance energy transfer applications.
- 186Goedhart, J.; van Weeren, L.; Hink, M. A.; Vischer, N. O.; Jalink, K.; Gadella, T. W., Jr. Bright Cyan Fluorescent Protein Variants Identified by Fluorescence Lifetime Screening. Nat. Methods 2010, 7, 137– 139, DOI: 10.1038/nmeth.1415186Bright cyan fluorescent protein variants identified by fluorescence lifetime screeningGoedhart, Joachim; van Weeren, Laura; Hink, Mark A.; Vischer, Norbert O. E.; Jalink, Kees; Gadella, Theodorus W. J., Jr.Nature Methods (2010), 7 (2), 137-139CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.
- 187Ai, H. W.; Henderson, J. N.; Remington, S. J.; Campbell, R. E. Directed Evolution of a Monomeric, Bright and Photostable Version of Clavularia Cyan Fluorescent Protein: Structural Characterization and Applications in Fluorescence Imaging. Biochem. J. 2006, 400, 531– 540, DOI: 10.1042/BJ20060874187Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imagingAi, Hui-wang; Henderson, J. Nathan; Remington, S. James; Campbell, Robert E.Biochemical Journal (2006), 400 (3), 531-540CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochem. and cell biol. research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, the authors report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, the authors identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, the authors arrived at an optimized variant that the authors have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addn., the fluorescence is insensitive to physiol. relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 Å crystal structure (1 Å = 0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As the authors exptl. demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
- 188Samarkina, O. N.; Popova, A. G.; Gvozdik, E. Y.; Chkalina, A. V.; Zvyagin, I. V.; Rylova, Y. V.; Rudenko, N. V.; Lusta, K. A.; Kelmanson, I. V.; Gorokhovatsky, A. Y. Universal and Rapid Method for Purification of GFP-Like Proteins by the Ethanol Extraction. Protein Expression Purif. 2009, 65, 108– 113, DOI: 10.1016/j.pep.2008.11.008188Universal and rapid method for purification of GFP-like proteins by the ethanol extractionSamarkina, Olga N.; Popova, Anastasia G.; Gvozdik, Elena Yu.; Chkalina, Anna V.; Zvyagin, Ivan V.; Rylova, Yulia V.; Rudenko, Natalia V.; Lusta, Konstantin A.; Kelmanson, Ilya V.; Gorokhovatsky, Andrey Yu.; Vinokurov, Leonid M.Protein Expression & Purification (2009), 65 (1), 108-113CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)GFP-like fluorescent proteins (FPs) are crucial in biol. and biomedical studies. The majority of FP purifn. techniques either include multiple time-consuming chromatog. steps with a low yield of the desired product or require prior protein modification (addn. of special tags). In the present work, the authors propose an alternative ethanol extn.-based technique previously used for GFP purifn. and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purifn. of any amt. of protein and requires no expensive reagents and equipment.
- 189Ai, H. W.; Olenych, S. G.; Wong, P.; Davidson, M. W.; Campbell, R. E. Hue-Shifted Monomeric Variants of Clavularia Cyan Fluorescent Protein: Identification of the Molecular Determinants of Color and Applications in Fluorescence Imaging. BMC Biol. 2008, 6, 13, DOI: 10.1186/1741-7007-6-13189Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imagingAi Hui-wang; Olenych Scott G; Wong Peter; Davidson Michael W; Campbell Robert EBMC biology (2008), 6 (), 13 ISSN:.BACKGROUND: In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. RESULTS: Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. CONCLUSION: Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.
- 190Shaner, N. C.; Lambert, G. G.; Chammas, A.; Ni, Y.; Cranfill, P. J.; Baird, M. A.; Sell, B. R.; Allen, J. R.; Day, R. N.; Israelsson, M. A Bright Monomeric Green Fluorescent Protein Derived from Branchiostoma Lanceolatum. Nat. Methods 2013, 10, 407– 409, DOI: 10.1038/nmeth.2413190A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatumShaner, Nathan C.; Lambert, Gerard G.; Chammas, Andrew; Ni, Yuhui; Cranfill, Paula J.; Baird, Michelle A.; Sell, Brittney R.; Allen, John R.; Day, Richard N.; Israelsson, Maria; Davidson, Michael W.; Wang, JiwuNature Methods (2013), 10 (5), 407-409CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. MNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to the authors' knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-mol. superresoln. imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
- 191Miyawaki, A.; Griesbeck, O.; Heim, R.; Tsien, R. Y. Dynamic and Quantitative Ca2+ Measurements Using Improved Cameleons. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 2135– 2140, DOI: 10.1073/pnas.96.5.2135191Dynamic and quantitative Ca2+ measurements using improved cameleonsMiyawaki, Atsushi; Griesbeck, Oliver; Heim, Roger; Tsien, Roger Y.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (5), 2135-2140CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+ -buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.
- 192Griesbeck, O.; Baird, G. S.; Campbell, R. E.; Zacharias, D. A.; Tsien, R. Y. Reducing the Environmental Sensitivity of Yellow Fluorescent Protein Mechanism and Applications. J. Biol. Chem. 2001, 276, 29188– 29194, DOI: 10.1074/jbc.M102815200192Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applicationsGriesbeck, Oliver; Baird, Geoffrey S.; Campbell, Robert E.; Zacharias, David A.; Tsien, Roger Y.Journal of Biological Chemistry (2001), 276 (31), 29188-29194CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37°. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pKα (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37° and in organelles. The halide resistance is explained by a 2.2-Å x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca2+] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiol. signals.
- 193Sakaue-Sawano, A.; Kurokawa, H.; Morimura, T.; Hanyu, A.; Hama, H.; Osawa, H.; Kashiwagi, S.; Fukami, K.; Miyata, T.; Miyoshi, H. Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell 2008, 132, 487– 498, DOI: 10.1016/j.cell.2007.12.033193Visualizing spatiotemporal dynamics of multicellular cell-cycle progressionSakaue-Sawano, Asako; Kurokawa, Hiroshi; Morimura, Toshifumi; Hanyu, Aki; Hama, Hiroshi; Osawa, Hatsuki; Kashiwagi, Saori; Fukami, Kiyoko; Miyata, Takaki; Miyoshi, Hiroyuki; Imamura, Takeshi; Ogawa, Masaharu; Masai, Hisao; Miyawaki, AtsushiCell (Cambridge, MA, United States) (2008), 132 (3), 487-498CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resoln. to help us better understand how the cell cycle is coordinated with various biol. events.
- 194Merzlyak, E. M.; Goedhart, J.; Shcherbo, D.; Bulina, M. E.; Shcheglov, A. S.; Fradkov, A. F.; Gaintzeva, A.; Lukyanov, K. A.; Lukyanov, S.; Gadella, T. W. Bright Monomeric Red Fluorescent Protein with an Extended Fluorescence Lifetime. Nat. Methods 2007, 4, 555– 557, DOI: 10.1038/nmeth1062194Bright monomeric red fluorescent protein with an extended fluorescence lifetimeMerzlyak, Ekaterina M.; Goedhart, Joachim; Shcherbo, Dmitry; Bulina, Mariya E.; Shcheglov, Aleksandr S.; Fradkov, Arkady F.; Gaintzeva, Anna; Lukyanov, Konstantin A.; Lukyanov, Sergey; Gadella, Theodorus W. J.; Chudakov, Dmitriy M.Nature Methods (2007), 4 (7), 555-557CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Fluorescent proteins have become extremely popular tools for in vivo imaging and esp. for the study of localization, motility and interaction of proteins in living cells. Here the authors report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
- 195Lam, A. J.; St-Pierre, F.; Gong, Y.; Marshall, J. D.; Cranfill, P. J.; Baird, M. A.; McKeown, M. R.; Wiedenmann, J.; Davidson, M. W.; Schnitzer, M. J. Improving FRET Dynamic Range with Bright Green and Red Fluorescent Proteins. Nat. Methods 2012, 9, 1005– 1012, DOI: 10.1038/nmeth.2171195Improving FRET dynamic range with bright green and red fluorescent proteinsLam, Amy J.; St-Pierre, Francois; Gong, Yiyang; Marshall, Jesse D.; Cranfill, Paula J.; Baird, Michelle A.; McKeown, Michael R.; Wiedenmann, Joerg; Davidson, Michael W.; Schnitzer, Mark J.; Tsien, Roger Y.; Lin, Michael Z.Nature Methods (2012), 9 (10), 1005-1012CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A variety of genetically encoded reporters use changes in fluorescence (or Foerster) resonance energy transfer (FRET) to report on biochem. processes in living cells. The std. genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Foerster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochem. events such as neuronal action-potential firing and RhoA activation in growth cones.
- 196Shcherbo, D.; Murphy, C. S.; Ermakova, G. V.; Solovieva, E. A.; Chepurnykh, T. V.; Shcheglov, A. S.; Verkhusha, V. V.; Pletnev, V. Z.; Hazelwood, K. L.; Roche, P. M. Far-Red Fluorescent Tags for Protein Imaging in Living Tissues. Biochem. J. 2009, 418, 567– 574, DOI: 10.1042/BJ20081949196Far-red fluorescent tags for protein imaging in living tissuesShcherbo, Dmitry; Murphy, Christopher S.; Ermakova, Galina V.; Solovieva, Elena A.; Chepurnykh, Tatiana V.; Shcheglov, Aleksandr S.; Verkhusha, Vladislav V.; Pletnev, Vladimir Z.; Hazelwood, Kristin L.; Roche, Patrick M.; Lukyanov, Sergey; Zaraisky, Andrey G.; Davidson, Michael W.; Chudakov, Dmitriy M.Biochemical Journal (2009), 418 (3), 567-574CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)A vast color palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolor labeling and whole-body imaging techniques. In the present paper, the authors report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. The authors also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.
- 197Lin, M. Z.; McKeown, M. R.; Ng, H. L.; Aguilera, T. A.; Shaner, N. C.; Campbell, R. E.; Adams, S. R.; Gross, L. A.; Ma, W.; Alber, T. Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in Mammals. Chem. Biol. 2009, 16, 1169– 1179, DOI: 10.1016/j.chembiol.2009.10.009197Autofluorescent Proteins with Excitation in the Optical Window for Intravital Imaging in MammalsLin, Michael Z.; McKeown, Michael R.; Ng, Ho-Leung; Aguilera, Todd A.; Shaner, Nathan C.; Campbell, Robert E.; Adams, Stephen R.; Gross, Larry A.; Ma, Wendy; Alber, Tom; Tsien, Roger Y.Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (11), 1169-1179CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by Hb. Fluorescent proteins that excite efficiently in the "optical window" above 600 nm are therefore highly desirable. The authors report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.
- 198Piatkevich, K. D.; Malashkevich, V. N.; Morozova, K. S.; Nemkovich, N. A.; Almo, S. C.; Verkhusha, V. V. Extended Stokes Shift in Fluorescent Proteins: Chromophore-Protein Interactions in a Near-Infrared TagRFP675 Variant. Sci. Rep. 2013, 3, 1847, DOI: 10.1038/srep01847198Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variantPiatkevich, Kiryl D.; Malashkevich, Vladimir N.; Morozova, Kateryna S.; Nemkovich, Nicolai A.; Almo, Steven C.; Verkhusha, Vladislav V.Scientific Reports (2013), 3 (), 1847, 11 pp.CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Most green fluorescent protein (GFP)-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to the rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-IR deriv. of red fluorescent protein mKate, named TagRFP675, exhibits a Stokes shift, which is 30-nm extended in comparison to that of the parental protein. In physiol. conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addn., its emission max. strongly depends on the excitation wavelength. Here, crystal structure studies of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacted with the protein matrix via an extensive network of H-bonds capable of large flexibility. Based on spectroscopic, biochem., and structural analyses, the authors suggest that the rearrangement of the H-bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.
- 199Sankaranarayanan, S.; De Angelis, D.; Rothman, J. E.; Ryan, T. A. The Use of pHluorins for Optical Measurements of Presynaptic Activity. Biophys. J. 2000, 79, 2199– 2208, DOI: 10.1016/S0006-3495(00)76468-X199The use of pHluorins for optical measurements of presynaptic activitySankaranarayanan, Sethuraman; De Angelis, Dino; Rothman, James E.; Ryan, Timothy A.Biophysical Journal (2000), 79 (4), 2199-2208CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. PHluorins have a pK ∼ 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theor. anal. of the expected signals using this approach and guidelines for future reporter development are provided.
- 200Shen, Y.; Rosendale, M.; Campbell, R. E.; Perrais, D. pHuji, A pH-Sensitive Red Fluorescent Protein for Imaging of Exo- and Endocytosis. J. Cell Biol. 2014, 207, 419– 432, DOI: 10.1083/jcb.201404107200pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosisShen, Yi; Rosendale, Morgane; Campbell, Robert E.; Perrais, DavidJournal of Cell Biology (2014), 207 (3), 419-432CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These expts. reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.
- 201Rodriguez, E. A.; Campbell, R. E.; Lin, J. Y.; Lin, M. Z.; Miyawaki, A.; Palmer, A. E.; Shu, X.; Zhang, J.; Tsien, R. Y. The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem. Sci. 2017, 42, 111– 129, DOI: 10.1016/j.tibs.2016.09.010201The Growing and Glowing Toolbox of Fluorescent and Photoactive ProteinsRodriguez, Erik A.; Campbell, Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, Amy E.; Shu, Xiaokun; Zhang, Jin; Tsien, Roger Y.Trends in Biochemical Sciences (2017), 42 (2), 111-129CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophys. and biochem. extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
- 202Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y. A Guide to Choosing Fluorescent Proteins. Nat. Methods 2005, 2, 905– 909, DOI: 10.1038/nmeth819202A guide to choosing fluorescent proteinsShaner, Nathan C.; Steinbach, Paul A.; Tsien, Roger Y.Nature Methods (2005), 2 (12), 905-909CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biol. imaging. With no unified std. for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use. Which are the brightest. What addnl. factors det. which are best for a given expt.. Although in many cases, a trial-and-error approach may still be necessary in detg. the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
- 203Chudakov, D. M.; Matz, M. V.; Lukyanov, S.; Lukyanov, K. A. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol. Rev. 2010, 90, 1103– 1163, DOI: 10.1152/physrev.00038.2009203Fluorescent proteins and their applications in imaging living cells and tissuesChudakov, Dmitriy M.; Matz, Mikhail V.; Lukyanov, Sergey; Lukyanov, Konstantin A.Physiological Reviews (2010), 90 (3), 1103-1163CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)A review. Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many labs. have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled mols. and cells in space and time and can also be used for super-resoln. imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concns. of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.
- 204Foo, Y. H.; Naredi-Rainer, N.; Lamb, D. C.; Ahmed, S.; Wohland, T. Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy. Biophys. J. 2012, 102, 1174– 1183, DOI: 10.1016/j.bpj.2012.01.040204Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation SpectroscopyFoo, Yong Hwee; Naredi-Rainer, Nikolaus; Lamb, Don C.; Ahmed, Sohail; Wohland, ThorstenBiophysical Journal (2012), 102 (5), 1174-1183CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence cross-correlation spectroscopy (FCCS) is used to det. interactions and dissocn. consts. (Kds) of biomols. The detn. of a Kd depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ∼0.5 or less for different FCCS schemes. The authors use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and det. the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation vols. for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Forster (or fluorescence) resonance energy transfer between the labels. The authors deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation vols. The authors use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the Kd.
- 205McDonald, D.; Wu, L.; Bohks, S. M.; KewalRamani, V. N.; Unutmaz, D.; Hope, T. J. Recruitment of HIV and Its Receptors to Dendritic Cell-T Cell Junctions. Science 2003, 300, 1295– 1297, DOI: 10.1126/science.1084238205Recruitment of HIV and Its Receptors to Dendritic Cell-T Cell JunctionsMcDonald, David; Wu, Li; Bohks, Stacy M.; KewalRamani, Vineet N.; Unutmaz, Derya; Hope, Thomas J.Science (Washington, DC, United States) (2003), 300 (5623), 1295-1297CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Monocyte-derived dendritic cells (MDDCs) can efficiently bind and transfer HIV infectivity without themselves becoming infected. Using live-cell microscopy, we found that HIV was recruited to sites of cell contact in MDDCs. Anal. of conjugates between MDDCs and T cells revealed that, in the absence of antigen-specific signaling, the HIV receptors CD4, CCR5, and CXCR4 on the T cell were recruited to the interface while the MDDCs concd. HIV to the same region. We propose that contact between dendritic cells and T cells facilitates transmission of HIV by locally concg. virus, receptor, and coreceptor during the formation of an infectious synapse.
- 206Zacharias, D. A.; Tsien, R. Y. Molecular Biology and Mutation of Green Fluorescent Protein. Methods Biochem. Anal. 2005, 47, 83– 120, DOI: 10.1002/0471739499.ch5There is no corresponding record for this reference.
- 207Klingen, Y.; Conzelmann, K. K.; Finke, S. Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport. J. Virol. 2008, 82, 237– 245, DOI: 10.1128/JVI.01342-07207Double-labeled rabies virus: live tracking of enveloped virus transportKlingen, Yvonne; Conzelmann, Karl-Klaus; Finke, StefanJournal of Virology (2008), 82 (1), 237-245CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addn. to G yielded infectious viruses with mosaic envelopes contg. both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addn. an enhanced green fluorescent protein-phosphoprotein fusion construct. Individual enveloped virus particles were obsd. under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.
- 208Lehmann, M. J.; Sherer, N. M.; Marks, C. B.; Pypaert, M.; Mothes, W. Actin- and Myosin-Driven Movement of Viruses along Filopodia Precedes Their Entry into Cells. J. Cell Biol. 2005, 170, 317– 325, DOI: 10.1083/jcb.200503059208Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cellsLehmann, Maik J.; Sherer, Nathan M.; Marks, Carolyn B.; Pypaert, Marc; Mothes, WaltherJournal of Cell Biology (2005), 170 (2), 317-325CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Viruses have often been obsd. in assocn. with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.
- 209Sugimoto, K.; Uema, M.; Sagara, H.; Tanaka, M.; Sata, T.; Hashimoto, Y.; Kawaguchi, Y. Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1. J. Virol. 2008, 82, 5198– 5211, DOI: 10.1128/JVI.02681-07209Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1Sugimoto, Ken; Uema, Masashi; Sagara, Hiroshi; Tanaka, Michiko; Sata, Tetsutaro; Hashimoto, Yasuhiro; Kawaguchi, YasushiJournal of Virology (2008), 82 (11), 5198-5211CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, resp. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.
- 210Liesche, J.; Ziomkiewicz, I.; Schulz, A. Super-Resolution Imaging with Pontamine Fast Scarlet 4BS Enables Direct Visualization of Cellulose Orientation and Cell Connection Architecture in Onion Epidermis Cells. BMC Plant Biol. 2013, 13, 226, DOI: 10.1186/1471-2229-13-226210Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cellsLiesche Johannes; Ziomkiewicz Iwona; Schulz AlexanderBMC plant biology (2013), 13 (), 226 ISSN:.BACKGROUND: In plants, a complex cell wall protects cells and defines their shape. Cellulose fibrils form a multilayered network inside the cell-wall matrix that plays a direct role in controlling cell expansion. Resolving the structure of this network will allow us to comprehend the relationship of cellulose fibril orientation and growth.The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal microscopy of some 200 nm in xy and 550 nm in z for green light, restricts the direct visualization of cellulose to relatively large bundles, whereas the structure of cellulose microfibrils with their diameter below 10 nm remains unresolved. Over the last decade, several so-called super-resolution microscopy approaches have been developed; in this paper we explore the potential of such approaches for the direct visualization of cellulose. RESULTS: To ensure optimal imaging we determined the spectral properties of PFS-stained tissue. PFS was found not to affect cell viability in the onion bulb scale epidermis. We present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. CONCLUSIONS: Super-resolution light microscopy of PFS-stained cellulose fibrils is possible and the increased resolution over conventional approaches makes it a valuable tool for the investigation of the cell-wall structure. This is one step in method developments that will close the gap to more invasive techniques, such as atomic force and electron microscopy.
- 211Melikyan, G. B.; Barnard, R. J.; Abrahamyan, L. G.; Mothes, W.; Young, J. A. Imaging Individual Retroviral Fusion Events: From Hemifusion to Pore Formation and Growth. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8728– 8733, DOI: 10.1073/pnas.0501864102211Imaging individual retroviral fusion events: From hemifusion to pore formation and growthMelikyan, Gregory B.; Barnard, Richard J. O.; Abrahamyan, Levon G.; Mothes, Walther; Young, John A. T.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (24), 8728-8733CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming following by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.
- 212Dixit, R.; Tiwari, V.; Shukla, D. Herpes Simplex Virus Type 1 Induces Filopodia in Differentiated P19 Neural Cells to Facilitate Viral Spread. Neurosci. Lett. 2008, 440, 113– 118, DOI: 10.1016/j.neulet.2008.05.031212Herpes simplex virus type 1 induces filopodia in differentiated P19 neural cells to facilitate viral spreadDixit, Rohan; Tiwari, Vaibhav; Shukla, DeepakNeuroscience Letters (2008), 440 (2), 113-118CODEN: NELED5; ISSN:0304-3940. (Elsevier Ireland Ltd.)Herpes simplex virus type-1 (HSV-1) is a neurotropic virus with significant potential as a viral vector for central nervous system (CNS) gene therapy. This study provides visual evidence that recombinant green fluorescent protein (GFP)-expressing HSV-1 travel down dendrites in differentiated P19 neuronal-like cells to efficiently reach the soma. The virus also promotes cytoskeletal rearrangements which facilitate viral spread in vitro, including often dramatic increases in dendritic filopodia. Viral movements, cell infection and filopodia induction were each reduced with the actin polymn. inhibitor cytochalasin D, suggesting the involvement of the actin cortex in these processes. The observation of neural cytoskeletal reorganization in response to HSV-1 may shed light on the mechanisms by which acute viral infection assocd. with herpes encephalitis produces cognitive deficits in patients.
- 213Adu-Gyamfi, E.; Digman, M. A.; Gratton, E.; Stahelin, R. V. Single-Particle Tracking Demonstrates That Actin Coordinates the Movement of the Ebola Virus Matrix Protein. Biophys. J. 2012, 103, L41– 143, DOI: 10.1016/j.bpj.2012.09.026213Single-Particle Tracking Demonstrates that Actin Coordinates the Movement of the Ebola Virus Matrix ProteinAdu-Gyamfi, Emmanuel; Digman, Michelle A.; Gratton, Enrico; Stahelin, Robert V.Biophysical Journal (2012), 103 (9), L41-L43CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to the authors' knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. The assembly and egress of VP40 at the plasma membrane of human cells were investigated using single-particle tracking. The results demonstrate that actin coordinates the movement and assembly of VP40, a crit. step in viral egress. These findings underscore the ability of single-mol. techniques to investigate the interplay of VP40 and host proteins in viral replication.
- 214Miyauchi, K.; Marin, M.; Melikyan, G. B. Visualization of Retrovirus Uptake and Delivery into Acidic Endosomes. Biochem. J. 2011, 434, 559– 569, DOI: 10.1042/BJ20101588214Visualization of retrovirus uptake and delivery into acidic endosomesMiyauchi, Kosuke; Marin, Mariana; Melikyan, Gregory B.Biochemical Journal (2011), 434 (3), 559-569CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solns. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solns. of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alk. buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alk. buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.
- 215Padilla-Parra, S.; Marin, M.; Kondo, N.; Melikyan, G. B. Pinpointing Retrovirus Entry Sites in Cells Expressing Alternatively Spliced Receptor Isoforms by Single Virus Imaging. Retrovirology 2014, 11, 47, DOI: 10.1186/1742-4690-11-47215Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imagingPadilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki; Melikyan, Gregory B.Retrovirology (2014), 11 (), 47/1-47/14CODEN: RETRBO; ISSN:1742-4690. (BioMed Central Ltd.)Background: The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacol. interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiol. conditions. Results: Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-pos. early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-pos.) or intermediate (Rab5- and Rab7-pos.) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-pos.) and intermediate (Rab5- and Rab7-pos.) compartments. Conclusions: Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are detd. by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.
- 216Mercer, J.; Helenius, A. Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 2008, 320, 531– 535, DOI: 10.1126/science.1155164216Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host CellsMercer, Jason; Helenius, AriScience (Washington, DC, United States) (2008), 320 (5875), 531-535CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles assocd. with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.
- 217Manicassamy, B.; Manicassamy, S.; Belicha-Villanueva, A.; Pisanelli, G.; Pulendran, B.; Garcia-Sastre, A. Analysis of in Vivo Dynamics of Influenza Virus Infection in Mice Using a GFP Reporter Virus. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11531– 11536, DOI: 10.1073/pnas.0914994107217Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virusManicassamy, Balaji; Manicassamy, Santhakumar; Belicha-Villanueva, Alan; Pisanelli, Giuseppe; Pulendran, Bali; Garcia-Sastre, AdolfoProceedings of the National Academy of Sciences of the United States of America (2010), 107 (25), 11531-11536, S11531/1-S11531/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to det., partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, the authors report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, the authors have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addn. to epithelial cells, the authors found GFP-pos. antigen-presenting cells, such as CD11b+CD11c-, CD11b-CD11c+, and CD11b+CD11c+, as early as 24 h after intranasal infection. In addn., a significant proportion of NK and B cells were GFP pos., suggesting active infection of these cells. The authors next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. The authors' results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening addnl. avenues of research in the influenza virus field.
- 218Bencina, M. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors. Sensors 2013, 13, 16736– 16758, DOI: 10.3390/s131216736218Illumination of the spatial order of intracellular pH by genetically encoded pH-sensitive sensorsBencina, MojcaSensors (2013), 13 (12), 16736-16758, 23 pp.CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)A review. Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiol. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.
- 219Hogue, I. B.; Bosse, J. B.; Engel, E. A.; Scherer, J.; Hu, J. R.; Del Rio, T.; Enquist, L. W. Fluorescent Protein Approaches in Alpha Herpesvirus Research. Viruses 2015, 7, 5933– 5961, DOI: 10.3390/v7112915219Fluorescent protein approaches in alpha herpesvirus researchHogue, Ian B.; Bosse, Jens B.; Engel, Esteban A.; Scherer, Julian; Hu, Jiun-Ruey; del Rio, Tony; Enquist, Lynn W.Viruses (2015), 7 (11), 5933-5961CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized mol. and cell biol., allowing us to literally see biol. processes as never before. Naturally, this revolution has extended to virol. in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats assocd. with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful expts. these tools now offer.
- 220Jouvenet, N.; Bieniasz, P. D.; Simon, S. M. Imaging the Biogenesis of Individual HIV-1 Virions in Live Cells. Nature 2008, 454, 236– 240, DOI: 10.1038/nature06998220Imaging the biogenesis of individual HIV-1 virions in live cellsJouvenet, Nolwenn; Bieniasz, Paul D.; Simon, Sanford M.Nature (London, United Kingdom) (2008), 454 (7201), 236-240CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport. However, the assembly of individual virions has never been obsd. in real time. Insights into this process have come primarily from biochem. analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial. Here we describe quant. the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivs. of Gag, the major structural component of HIV-1-which is sufficient to drive the assembly of virus-like particles-with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5-6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the detn. of parameters of viral assembly that are inaccessible with conventional techniques.
- 221Hogue, I. B.; Bosse, J. B.; Hu, J. R.; Thiberge, S. Y.; Enquist, L. W. Cellular Mechanisms of Alpha Herpesvirus Eegress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis. PLoS Pathog. 2014, 10, e1004535 DOI: 10.1371/journal.ppat.1004535221Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosisHogue, Ian B.; Bosse, Jens B.; Hu, Jiun-Ruey; Thiberge, Stephan Y.; Enquist, Lynn W.PLoS Pathogens (2014), 10 (12), e1004535/1-e1004535/12, 12 pp.CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and mol. mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement anal. to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically assocd. with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biol. and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
- 222Adam, V.; Berardozzi, R.; Byrdin, M.; Bourgeois, D. Phototransformable Fluorescent Proteins: Future Challenges. Curr. Opin. Chem. Biol. 2014, 20, 92– 102, DOI: 10.1016/j.cbpa.2014.05.016222Phototransformable fluorescent proteins: Future challengesAdam, Virgile; Berardozzi, Romain; Byrdin, Martin; Bourgeois, DominiqueCurrent Opinion in Chemical Biology (2014), 20 (), 92-102CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. In fluorescence microscopy, the photophys. properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biol. cells or organisms dynamically, quant. and with unprecedented resoln. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.
- 223Chudakov, D. M.; Verkhusha, V. V.; Staroverov, D. B.; Souslova, E. A.; Lukyanov, S.; Lukyanov, K. A. Photoswitchable Cyan Fluorescent Protein for Protein Tracking. Nat. Biotechnol. 2004, 22, 1435– 1439, DOI: 10.1038/nbt1025223Photoswitchable cyan fluorescent protein for protein trackingChudakov, Dmitriy M.; Verkhusha, Vladislav V.; Staroverov, Dmitry B.; Souslova, Ekaterina A.; Lukyanov, Sergey; Lukyanov, Konstantin A.Nature Biotechnology (2004), 22 (11), 1435-1439CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradn. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.
- 224Nemet, I.; Ropelewski, P.; Imanishi, Y. Applications of Phototransformable Fluorescent Proteins for Tracking the Dynamics of Cellular Components. Photochem. Photobiol. Sci. 2015, 14, 1787– 1806, DOI: 10.1039/C5PP00174A224Applications of phototransformable fluorescent proteins for tracking the dynamics of cellular componentsNemet, Ina; Ropelewski, Philip; Imanishi, YoshikazuPhotochemical & Photobiological Sciences (2015), 14 (10), 1787-1806CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)A review. In the past few decades, fluorescent proteins have revolutionized the field of cell biol. Phototransformable fluorescent proteins are capable of changing their excitation and emission spectra after being exposed to specific wavelength(s) of light. The majority of phototransformable fluorescent proteins have originated from marine organisms. Genetic engineering of these proteins has made available many choices for different colors, modes of conversion, and other biophys. properties. Their phototransformative property has allowed the highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. Furthermore, phototransformable fluorescent proteins have offered new methods for superresoln. fluorescence microscopy and optogenetics manipulation of proteins. One of the major advantages of phototransformable fluorescent proteins is their applicability for visualizing newly synthesized proteins that are en route to their final destinations. In this paper, we will discuss the biol. applications of phototransformable fluorescent proteins with special emphasis on the application of tracking membrane proteins in vertebrate photoreceptor cells.
- 225Ando, R.; Hama, H.; Yamamoto-Hino, M.; Mizuno, H.; Miyawaki, A. An Optical Marker Based on the UV-Induced Green-to-Red Photoconversion of a Fluorescent Protein. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12651– 12656, DOI: 10.1073/pnas.202320599225An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent proteinAndo, Ryoko; Hama, Hiroshi; Yamamoto-Hino, Miki; Mizuno, Hideaki; Miyawaki, AtsushiProceedings of the National Academy of Sciences of the United States of America (2002), 99 (20), 12651-12656CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradn. with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.
- 226Patterson, G. H.; Lippincott-Schwartz, J. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells. Science 2002, 297, 1873– 1877, DOI: 10.1126/science.1074952226A photoactivatable GFP for selective photolabeling of proteins and cellsPatterson, George H.; Lippincott-Schwartz, JenniferScience (Washington, DC, United States) (2002), 297 (5588), 1873-1877CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradn. with 413-nm light, increases fluorescence 100 times when excited by 488-nm light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated mols. that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
- 227Zhou, X. X.; Lin, M. Z. Photoswitchable Fluorescent Proteins: Ten Years of Colorful Chemistry and Exciting Applications. Curr. Opin. Chem. Biol. 2013, 17, 682– 690, DOI: 10.1016/j.cbpa.2013.05.031227Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applicationsZhou, Xin X.; Lin, Michael Z.Current Opinion in Chemical Biology (2013), 17 (4), 682-690CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Reversibly photoswitchable fluorescent proteins (RSFPs) are fluorescent proteins whose fluorescence, upon excitation at a certain wavelength, can be switched on or off by light in a reversible manner. In the last 10 years, many new RSFPs have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties. This review will describe research on the mechanisms of reversible photoswitching and recent applications using RSFPs. While cis-trans isomerization of the chromophore is believed to be the general mechanism for most RSFPs, structural studies reveal diversity in the details of photoswitching mechanisms, including different effects of protonation, chromophore planarity, and pocket flexibility. Applications of RSFPs include new types of live-cell superresoln. imaging, tracking of protein movements and interactions, information storage, and optical control of protein activity.
- 228Shroff, H.; Galbraith, C. G.; Galbraith, J. A.; White, H.; Gillette, J.; Olenych, S.; Davidson, M. W.; Betzig, E. Dual-Color Superresolution Imaging of Genetically Expressed Probes within Individual Adhesion Complexes. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 20308– 20313, DOI: 10.1073/pnas.0710517105228Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexesShroff, Hari; Galbraith, Catherine G.; Galbraith, James A.; White, Helen; Gillette, Jennifer; Olenych, Scott; Davidson, Michael W.; Betzig, EricProceedings of the National Academy of Sciences of the United States of America (2007), 104 (51), 20308-20313CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Accurate detn. of the relative positions of proteins within localized regions of the cell is essential for understanding their biol. function. Although fluorescent fusion proteins are targeted with mol. precision, the position of these genetically expressed reporters is usually known only to the resoln. of conventional optics (≈200 nm). Here, the authors report the use of two-color photoactivated localization microscopy (PALM) to det. the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resoln. in whole, fixed cells of ≈20-30 nm at acquisition times of 5-30 min. The authors apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, the authors find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resoln., and speed of the technique all suggest its potential to directly visualize mol. interactions within cellular structures at the nanometer scale.
- 229Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642– 1645, DOI: 10.1126/science.1127344229Imaging Intracellular Fluorescent Proteins at Nanometer ResolutionBetzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.Science (Washington, DC, United States) (2006), 313 (5793), 1642-1645CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The authors introduce a method for optically imaging intracellular proteins at nanometer spatial resoln. Numerous sparse subsets of photoactivatable fluorescent protein mols. were activated, localized (to ∼2 to 25 nm), and then bleached. The aggregate position information from all subsets was then assembled into a superresoln. image. The authors used this method - termed photoactivated localization microscopy - to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, the authors imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
- 230Fernandez-Suarez, M.; Ting, A. Y. Fluorescent Probes for Super-Resolution Imaging in Living Cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 929– 943, DOI: 10.1038/nrm2531230Fluorescent probes for super-resolution imaging in living cellsFernandez-Suarez, Marta; Ting, Alice Y.Nature Reviews Molecular Cell Biology (2008), 9 (12), 929-943CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resoln. imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resoln. Current research is focused on further improving spatial resoln. in an effort to reach the goal of video-rate imaging of live cells with mol. (1-5 nm) resoln. Here, the authors describe the contributions of fluorescent probes to far-field super-resoln. imaging, focusing on fluorescent proteins and org. small-mol. fluorophores. The authors describe the features of existing super-resoln. fluorophores and highlight areas of importance for future research and development.
- 231Bourgeois, D.; Adam, V. Reversible Photoswitching in Fluorescent Proteins: A Mechanistic View. IUBMB Life 2012, 64, 482– 491, DOI: 10.1002/iub.1023231Reversible photoswitching in fluorescent proteins: A mechanistic viewBourgeois, Dominique; Adam, VirgileIUBMB Life (2012), 64 (6), 482-491CODEN: IULIF8; ISSN:1521-6543. (John Wiley & Sons Inc.)A review. Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnol. On illumination with proper actinic light, phototransformable FPs are amenable to long-lived transitions between various fluorescent or nonfluorescent states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high-d. data bio-storage, photochromic FRET, and super-resoln. microscopy by either point-scanning, structured illumination, or single mol.-based wide-field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis-trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future. © 2012 IUBMB IUBMB Life, 2012.
- 232Muranyi, W.; Malkusch, S.; Muller, B.; Heilemann, M.; Krausslich, H. G. Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail. PLoS Pathog. 2013, 9, e1003198 DOI: 10.1371/journal.ppat.1003198There is no corresponding record for this reference.
- 233Muller, B.; Heilemann, M. Shedding New Light on Viruses: Super-Resolution Microscopy for Studying Human Immunodeficiency Virus. Trends Microbiol. 2013, 21, 522– 533, DOI: 10.1016/j.tim.2013.06.010233Shedding new light on viruses: super-resolution microscopy for studying human immunodeficiency virusMuller Barbara; Heilemann MikeTrends in microbiology (2013), 21 (10), 522-33 ISSN:.For more than 70 years electron microscopy (EM) techniques have played an important role in investigating structures of enveloped viruses. By contrast, use of fluorescence microscopy (FM) methods for this purpose was limited by the fact that the size of virus particles is generally around or below the diffraction limit of light microscopy. Various super-resolution (SR) fluorescence imaging techniques developed over the past two decades bypass the diffraction limit of light microscopy, allowing visualization of subviral details and bridging the gap between conventional FM and EM methods. We summarize here findings on human immunodeficiency virus (HIV-1) obtained using SR-FM techniques. Although the number of published studies is currently limited and some of the pioneering analyses also covered methodological or descriptive aspects, recent publications clearly indicate the potential to approach open questions in HIV-1 replication from a new angle.
- 234Grove, J. Super-Resolution Microscopy: A Virus’ Eye View of the Cell. Viruses 2014, 6, 1365– 1378, DOI: 10.3390/v6031365234Super-resolution microscopy: a virus' eye view of the cellGrove JoeViruses (2014), 6 (3), 1365-78 ISSN:.It is difficult to observe the molecular choreography between viruses and host cell components, as they exist on a spatial scale beyond the reach of conventional microscopy. However, novel super-resolution microscopy techniques have cast aside technical limitations to reveal a nanoscale view of virus replication and cell biology. This article provides an introduction to super-resolution imaging; in particular, localisation microscopy, and explores the application of such technologies to the study of viruses and tetraspanins, the topic of this special issue.
- 235Manley, S.; Gillette, J. M.; Patterson, G. H.; Shroff, H.; Hess, H. F.; Betzig, E.; Lippincott-Schwartz, J. High-Density Mapping of Single-Molecule Trajectories with Photoactivated Localization Microscopy. Nat. Methods 2008, 5, 155– 157, DOI: 10.1038/nmeth.1176235High-density mapping of single-molecule trajectories with photoactivated localization microscopyManley, Suliana; Gillette, Jennifer M.; Patterson, George H.; Shroff, Hari; Hess, Harald F.; Betzig, Eric; Lippincott-Schwartz, JenniferNature Methods (2008), 5 (2), 155-157CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-mol. motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of mols., sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
- 236Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. In Vivo Cancer Targeting and Imaging with Semiconductor Quantum Dots. Nat. Biotechnol. 2004, 22, 969– 976, DOI: 10.1038/nbt994236In vivo cancer targeting and imaging with semiconductor quantum dotsGao, Xiaohu; Cui, Yuanyuan; Levenson, Richard M.; Chung, Leland W. K.; Nie, ShumingNature Biotechnology (2004), 22 (8), 969-976CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both s.c. injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of mol. targets in vivo.
- 237Algar, W. R.; Susumu, K.; Delehanty, J. B.; Medintz, I. L. Semiconductor Quantum Dots in Bioanalysis: Crossing the Valley of Death. Anal. Chem. 2011, 83, 8826– 8837, DOI: 10.1021/ac201331r237Semiconductor Quantum Dots in Bioanalysis: Crossing the Valley of DeathAlgar, W. Russ; Susumu, Kimihiro; Delehanty, James B.; Medintz, Igor L.Analytical Chemistry (Washington, DC, United States) (2011), 83 (23), 8826-8837CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Colloidal semiconductor quantum dots (QDs) have evolved beyond scientific novelties and are transitioning into bona fide anal. tools. We describe the burgeoning role of QDs in many different fields of bioanalyses and highlight the advantages afforded by their unique phys. and optical properties.
- 238Wegner, K. D.; Hildebrandt, N. Quantum Dots: Bright and Versatile in Vitro and in Vivo Fluorescence Imaging Biosensors. Chem. Soc. Rev. 2015, 44, 4792– 4834, DOI: 10.1039/C4CS00532E238Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensorsWegner, K. David; Hildebrandt, NikoChemical Society Reviews (2015), 44 (14), 4792-4834CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Semiconductor quantum dots (QDs) have become important fluorescent probes for in vitro and in vivo bioimaging research. Their nanoparticle surfaces for versatile bioconjugation, their adaptable photophys. properties for multiplexed detection, and their superior stability for longer investigation times are the main advantages of QDs compared to other fluorescence imaging agents. Here, we review the recent literature dealing with the design and application of QD-bioconjugates for advanced in vitro and in vivo imaging. After a short summary of QD prepn. and their most important properties, different QD-based imaging applications will be discussed from the technol. and the biol. point of view, ranging from super-resoln. microscopy and single-particle tracking over in vitro cell and tissue imaging to in vivo investigations. A substantial part of the review will focus on multifunctional applications, in which the QD fluorescence is combined with drug or gene delivery towards theranostic approaches or with complementary technologies for multimodal imaging. We also briefly discuss QD toxicity issues and give a short outlook on future directions of QD-based bioimaging.
- 239Chen, G.; Zhu, J. Y.; Zhang, Z. L.; Zhang, W.; Ren, J. G.; Wu, M.; Hong, Z. Y.; Lv, C.; Pang, D. W.; Zhao, Y. F. Transformation of Cell-Derived Microparticles into Quantum-Dot-Labeled Nanovectors for Antitumor siRNA Delivery. Angew. Chem., Int. Ed. 2015, 54, 1036– 1040, DOI: 10.1002/anie.201410223239Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA deliveryChen, Gang; Zhu, Jun-Yi; Zhang, Zhi-Ling; Zhang, Wei; Ren, Jian-Gang; Wu, Min; Hong, Zheng-Yuan; Lv, Cheng; Pang, Dai-Wen; Zhao, Yi-FangAngewandte Chemie, International Edition (2015), 54 (3), 1036-1040CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Cell-derived microparticles (MPs) have been recently recognized as crit. intercellular information conveyors. However, further understanding of their biol. behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomol.-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors.
- 240Rosenthal, S. J.; Chang, J. C.; Kovtun, O.; McBride, J. R.; Tomlinson, I. D. Biocompatible Quantum Dots for Biological Applications. Chem. Biol. 2011, 18, 10– 24, DOI: 10.1016/j.chembiol.2010.11.013240Biocompatible Quantum Dots for Biological ApplicationsRosenthal, Sandra J.; Chang, Jerry C.; Kovtun, Oleg; McBride, James R.; Tomlinson, Ian D.Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (1), 10-24CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Semiconductor quantum dots are quickly becoming a crit. diagnostic tool for discerning cellular function at the mol. level. Their high brightness, long-lasting, size-tunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biol. oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biol. compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.
- 241Jethi, L.; Mack, T. G.; Kambhampati, P. Extending Semiconductor Nanocrystals from the Quantum Dot Regime to the Molecular Cluster Regime. J. Phys. Chem. C 2017, 121, 26102– 26107, DOI: 10.1021/acs.jpcc.7b08439241Extending Semiconductor Nanocrystals from the Quantum Dot Regime to the Molecular Cluster RegimeJethi, Lakshay; Mack, Timothy G.; Kambhampati, PatanjaliJournal of Physical Chemistry C (2017), 121 (46), 26102-26107CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)The size-dependent optical and electronic properties of semiconductor nanocrystal (NC) were exploited over decades for various applications. This size dependence involves a transition from the regime of bulk colloids of ∼100 nm radius to quantum dots (QDs) of ∼10 nm radius, the details of which are material specific. To understand the transition from the QD (∼10 nm) to the mol. cluster regimes (∼1 nm) of nanocrystals, a set of CdSe nanocrystals with sizes 0.89-1.66 nm in radius were synthesized. As the nanocrystals become small, the surface emission strongly increases in amplitude, and the core emission broadens and red shifts. These effects are rationalized in terms of coupling to ligands via electron transfer theory. The core emission spectra arise from increased vibrational coupling of ligands for very small NC. The surface emission amplitudes arise from a size-dependent surface free energy. The transition from the QD to the mol. cluster regime is at 1.2 nm radius, in contrast to the transition from the bulk to QD transition at the Bohr radius of 5.4 nm in CdSe. These size-dependent surface electronic phenomena may be used for light emission applications.
- 242Zhou, J.; Liu, Y.; Tang, J.; Tang, W. Surface Ligands Engineering of Semiconductor Quantum Dots for Chemosensory and Biological Applications. Mater. Today 2017, 20, 360– 376, DOI: 10.1016/j.mattod.2017.02.006242Surface ligands engineering of semiconductor quantum dots for chemosensory and biological applicationsZhou, Jie; Liu, Yun; Tang, Jian; Tang, WeihuaMaterials Today (Oxford, United Kingdom) (2017), 20 (7), 360-376CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)Featuring size-tunable elec. and optical properties, semiconductor quantum dots (QDs) are appealing intensive interests in developing ingenious luminescent materials for chemosensory and biol. applications. The surface modification of QDs with functional ligands not only fine-tunes the physiochem. properties and fluorescence emission behaviors, but also induces the designated interplay between analytes and probes for special detn. In this review, the fundamental principles guiding the rational design of high-efficiency luminescent sensors with surface engineering are overviewed. The state-of-the-art applications of QDs-based probes are highlighted for the sensing of mol. substrates and ionic species as well as various biol. applications, with the inherent recognition mechanisms elaborated for representative cases. The challenge and future research direction in this emerging and promising research field are also discussed.
- 243Zhou, J.; Yang, Y.; Zhang, C. Y. Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 2015, 115, 11669– 11717, DOI: 10.1021/acs.chemrev.5b00049243Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical ApplicationZhou, Juan; Yang, Yong; Zhang, Chun-yangChemical Reviews (Washington, DC, United States) (2015), 115 (21), 11669-11717CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review.
- 244Zhang, M.; Yue, J.; Cui, R.; Ma, Z.; Wan, H.; Wang, F.; Zhu, S.; Zhou, Y.; Kuang, Y.; Zhong, Y. Bright Quantum Dots Emitting at Approximately 1,600 nm in the NIR-IIb Window for Deep Tissue Fluorescence Imaging. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 6590– 6595, DOI: 10.1073/pnas.1806153115244Bright quantum dots emitting at ∼1,600 nm in the NIR-IIb window for deep tissue fluorescence imagingZhang, Mingxi; Yue, Jingying; Cui, Ran; Ma, Zhuoran; Wan, Hao; Wang, Feifei; Zhu, Shoujun; Zhou, Ying; Kuang, Yun; Zhong, Yeteng; Pang, Dai-Wen; Dai, HongjieProceedings of the National Academy of Sciences of the United States of America (2018), 115 (26), 6590-6595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aq. stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in org. phase. The CdS shell plays a crit. role of protecting the lead sulfide (PbS) core from oxidn. and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aq. stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 h and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per s (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke's shift. The aq.-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.
- 245Zhao, J. Y.; Chen, G.; Gu, Y. P.; Cui, R.; Zhang, Z. L.; Yu, Z. L.; Tang, B.; Zhao, Y. F.; Pang, D. W. Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived Microvesicles. J. Am. Chem. Soc. 2016, 138, 1893– 1903, DOI: 10.1021/jacs.5b10340245Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived MicrovesiclesZhao, Jing-Ya; Chen, Gang; Gu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Yu, Zi-Li; Tang, Bo; Zhao, Yi-Fang; Pang, Dai-WenJournal of the American Chemical Society (2016), 138 (6), 1893-1903CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Cell-derived microvesicles (MVs) are natural carriers that can transport biol. mols. between cells, which are expected to be promising delivery vehicles for therapeutic purposes. Strategies to label MVs are very important for investigation and application of MVs. Herein, ultrasmall Mn-magnetofunctionalized Ag2Se quantum dots (Ag2Se@Mn QDs) integrated with excellent near-IR (NIR) fluorescence and magnetic resonance (MR) imaging capabilities have been developed for instant efficient labeling of MVs for their in vivo high-resoln. dual-mode tracking. The Ag2Se@Mn QDs were fabricated by controlling the reaction of Mn2+ with the Ag2Se nanocrystals having been pretreated in 80 °C NaOH soln., with an ultrasmall size of ca. 1.8 nm, water dispersibility, high NIR fluorescence quantum yield of 13.2%, and high longitudinal relaxivity of 12.87 mM-1s-1 (almost four times that of the com. contrast agent Gd-DTPA). The ultrasmall size of the Ag2Se@Mn QDs enables them to be directly and efficiently loaded into MVs by electroporation, instantly and reliably conferring both NIR fluorescence and MR traceability on MVs. Our method for labeling MVs of different origins is universal and free of unfavorable influence on intrinsic behaviors of MVs. The complementary imaging capabilities of the Ag2Se@Mn QDs have made the long-term noninvasive whole-body high-resoln. dual-mode tracking of MVs in vivo realized, by which the dynamic biodistribution of MVs has been revealed in a real-time and in situ quant. manner. This work not only opens a new window for labeling with QDs, but also facilitates greatly the investigation and application of MVs.
- 246Gu, Y. P.; Cui, R.; Zhang, Z. L.; Xie, Z. X.; Pang, D. W. Ultrasmall Near-Infrared Ag2Se Quantum Dots with Tunable Fluorescence for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134, 79– 82, DOI: 10.1021/ja2089553246Ultrasmall Near-Infrared Ag2Se Quantum Dots with Tunable Fluorescence for in Vivo ImagingGu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Xie, Zhi-Xiong; Pang, Dai-WenJournal of the American Chemical Society (2012), 134 (1), 79-82CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A strategy is presented that involves coupling Na2SeO3 redn. with the binding of silver ions and alanine in a quasi-biosystem to obtain ultrasmall, near-IR Ag2Se quantum dots (QDs) with tunable fluorescence at 90° in aq. soln. This strategy avoids high temps., high pressures, and org. solvents so that water-dispersible sub-3 nm Ag2Se QDs can be directly obtained. The photoluminescence of the Ag2Se QDs was size-dependent over a wavelength range from 700 to 820 nm, corresponding to sizes from 1.5 ± 0.4 to 2.4 ± 0.5 nm, with good monodispersity. The Ag2Se QDs are less cytotoxic than other nanomaterials used for similar applications. Furthermore, the NIR fluorescence of the Ag2Se QDs could penetrate through the abdominal cavity of a living nude mouse and could be detected on its back side, demonstrating the potential applications of these less toxic NIR Ag2Se QDs in bioimaging.
- 247Alivisatos, A. P.; Gu, W. W.; Larabell, C. Quantum Dots as Cellular Probes. Annu. Rev. Biomed. Eng. 2005, 7, 55– 76, DOI: 10.1146/annurev.bioeng.7.060804.100432247Quantum dots as cellular probesAlivisatos, A. Paul; Gu, Weiwei; Larabell, CarolynAnnual Review of Biomedical Engineering (2005), 7 (), 55-76, 3 platesCODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews Inc.)A review. Robust and bright light emitters, semiconductor nanocrystals [quantum dots (QDs)] have been adopted as a new class of fluorescent labels. Six years after the first expts. of their uses in biol. applications, there have been dramatic improvements in understanding surface chem., biocompatibility, and targeting specificity. Many studies have shown the great potential of using quantum dots as new probes in vitro and in vivo. This review summarizes the recent advances of quantum dot usage at the cellular level, including immunolabeling, cell tracking, in situ hybridization, FRET, in vivo imaging, and other related technologies. Limitations and potential future uses of quantum dot probes are also discussed.
- 248Gao, X.; Yang, L.; Petros, J. A.; Marshall, F. F.; Simons, J. W.; Nie, S. In Vivo Molecular and Cellular Imaging with Quantum Dots. Curr. Opin. Biotechnol. 2005, 16, 63– 72, DOI: 10.1016/j.copbio.2004.11.003248In vivo molecular and cellular imaging with quantum dotsGao, Xiaohu; Yang, Lily; Petros, John A.; Marshall, Fray F.; Simons, Jonathan W.; Nie, ShumingCurrent Opinion in Biotechnology (2005), 16 (1), 63-72CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomol. and cellular imaging. In comparison with org. dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degrdn. need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of mol. targets in living cells, animal models and possibly in humans.
- 249Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science 1998, 281, 2013– 2016, DOI: 10.1126/science.281.5385.2013249Semiconductor nanocrystals as fluorescent biological labelsBruchez, Marcel, Jr.; Moronne, Mario; Gin, Peter; Weiss, Shimon; Alivisatos, A. PaulScience (Washington, D. C.) (1998), 281 (5385), 2013-2016CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Semiconductor nanocrystals were prepd. for use as fluorescent probes in biol. staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, sym. emission spectrum and are photochem. stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling expt. on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
- 250Smith, A. M.; Duan, H. W.; Mohs, A. M.; Nie, S. M. Bioconjugated Quantum Dots for in Vivo Molecular and Cellular Imaging. Adv. Drug Delivery Rev. 2008, 60, 1226– 1240, DOI: 10.1016/j.addr.2008.03.015250Bioconjugated quantum dots for in vivo molecular and cellular imagingSmith, Andrew M.; Duan, Hongwei; Mohs, Aaron M.; Nie, ShumingAdvanced Drug Delivery Reviews (2008), 60 (11), 1226-1240CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)A review. Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biol. and medicine. In comparison with org. dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small mols., QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for mol. and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicol.
- 251Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D. J.; Post, J. N.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin, T. M. Quantum Dot Ligands Provide New Insights into erbB/HER Receptor–Mediated Signal Transduction. Nat. Biotechnol. 2004, 22, 198– 203, DOI: 10.1038/nbt929251Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transductionLidke, Diane S.; Nagy, Peter; Heintzmann, Rainer; Arndt-Jovin, Donna J.; Post, Janine N.; Grecco, Hernan E.; Jares-Erijman, Elizabeth A.; Jovin, Thomas M.Nature Biotechnology (2004), 22 (2), 198-203CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. The authors have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (ii) continuous confocal laser scanning microscopy and flow cytometry. EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnol. developments.
- 252Srinivasan, C.; Lee, J.; Papadimitrakopoulos, F.; Silbart, L. K.; Zhao, M.; Burgess, D. J. Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots. Mol. Ther. 2006, 14, 192– 201, DOI: 10.1016/j.ymthe.2006.03.010252Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum DotsSrinivasan, Charudharshini; Lee, Jeunghoon; Papadimitrakopoulos, Fotios; Silbart, Lawrence K.; Zhao, Minhua; Burgess, Diane J.Molecular Therapy (2006), 14 (2), 192-201CODEN: MTOHCK; ISSN:1525-0016. (Elsevier)Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)-N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and at. force microscopy (AFM) were used to confirm the structure of QD-DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNA-reactive site of the plasmid DNA. These QD-DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ∼62%, which was comparable to the control (unconjugated) plasmid DNA.
- 253Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots. Nat. Biotechnol. 2003, 21, 41– 46, DOI: 10.1038/nbt764253Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dotsWu, Xingyong; Liu, Hongjian; Liu, Jianquan; Haley, Kari N.; Treadway, Joseph A.; Larson, J. Peter; Ge, Nianfeng; Peale, Frank; Bruchez, Marcel P.Nature Biotechnology (2003), 21 (1), 41-46CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small mols., can specifically and effectively label mol. targets at a subcellular level. Here we have used QDs linked to IgG (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable org. dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over org. dyes in multiplex target detection.
- 254Chen, C.; Peng, J.; Xia, H.; Wu, Q.; Zeng, L.; Xu, H.; Tang, H.; Zhang, Z.; Zhu, X.; Pang, D. Quantum-Dot-Based Immunofluorescent Imaging of HER2 and ER Provides New Insights into Breast Cancer Heterogeneity. Nanotechnology 2010, 21, 095101 DOI: 10.1088/0957-4484/21/9/095101254Quantum-dot-based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneityChen, Chuang; Peng, Jun; Xia, Heshun; Wu, Qiongshui; Zeng, Libo; Xu, Hao; Tang, Hongwu; Zhang, Zhiling; Zhu, Xiaobo; Pang, Daiwen; Li, YanNanotechnology (2010), 21 (9), 095101/1-095101/6CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)Breast cancer (BC) is a heterogeneous tumor, and better understanding of its heterogeneity is essential to improving treatment effect. Quantum dot (QD)-based immunofluorescent nanotechnol. (QD-IHC) for mol. pathol. has potential advantages in delineating tumor heterogeneity. This potential is explored in this paper by QD-IHC imaging of HER2 and ER. BC heterogeneity can be displayed more clearly and sensitively by QD-IHC than conventional IHC in BC tissue microarrays. Furthermore, the simultaneous imaging of ER and HER2 might help understand their interactions during the process of evolution of heterogeneous BC.
- 255Chen, C.; Xia, H. S.; Gong, Y. P.; Peng, J.; Peng, C. W.; Hu, M. B.; Zhu, X. B.; Pang, D. W.; Sun, S. R.; Li, Y. The Quantitative Detection of Total HER2 Load by Quantum Dots and the Identification of a New Subtype of Breast Cancer with Different 5-Year Prognosis. Biomaterials 2010, 31, 8818– 8825, DOI: 10.1016/j.biomaterials.2010.07.091255The quantitative detection of total HER2 load by quantum dots and the identification of a new subtype of breast cancer with different 5-year prognosisChen, Chuang; Xia, He-Shun; Gong, Yi-Ping; Peng, Jun; Peng, Chun-Wei; Hu, Ming-Bai; Zhu, Xiao-Bo; Pang, Dai-Wen; Sun, Sheng-Rong; Li, YanBiomaterials (2010), 31 (33), 8818-8825CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Accurate classification is fundamental for breast cancer (BC) personalized care. Current BC classification based on the either traditional morphol. staging or mol. signatures seems inefficient to reveal the"true"behaviors of invasive BC evolution. An appropriate approach combining the macro- and micro-pathol. information might be more useful academically as well as clin. Here the authors explore a holistic approach by integrating a key mol. prognostic indicator of BC, HER2, with quant. detn. using quantum dots (QDs)-based nanotechnol. and spectral anal., and a key macropathol. indicator, tumor size, resulting a new indicator, total HER2 load. This indicator might better reveal BC heterogeneity and new subtypes of BC with different 5-yr disease-free survival compared with current methods, which could be helpful in formulating a more personalized targeted therapy for BC. Furthermore, this mode integrating macro- and micro-pathol. indicators might help gain new insights into invasive BC biol. behaviors.
- 256Chen, C.; Liu, S. L.; Cui, R.; Huang, B. H.; Tian, Z. Q.; Jiang, P.; Pang, D. W.; Zhang, Z. L. Diffusion Behaviors of Water-Soluble CdSe/ZnS Core/Shell Quantum Dots Investigated by Single-Particle Tracking. J. Phys. Chem. C 2008, 112, 18904– 18910, DOI: 10.1021/jp807074t256Diffusion Behaviors of Water-Soluble CdSe/ZnS Core/Shell Quantum Dots Investigated by Single-Particle TrackingChen, Cheng; Liu, Shu-Lin; Cui, Ran; Huang, Bi-Hai; Tian, Zhi-Quan; Jiang, Peng; Pang, Dai-Wen; Zhang, Zhi-LingJournal of Physical Chemistry C (2008), 112 (48), 18904-18910CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)As is known to the authors all, quantum dots (QDs), the fluorescent semiconductor nanocrystals, have many excellent optical properties which make them attractive fluorescent tags in single-mol. tracking in live cells. Because the intracellular environment is so complex, this paper aimed at simulating the intracellular soln. environment in vitro and studied the influence of the soln. environment on the diffusion of water-sol. core/shell CdSe/ZnS QDs. Single-particle tracking (SPT) was applied to measure the diffusion coeffs. of two water-sol. core/shell QDs, CTAB-modified CdSe/ZnS QDs (CTAB-QDs) and octylamine-modified poly(acrylic acid)-modified CdSe/ZnS QDs (OPA-QDs). The exposure time was optimized to be 29.95 ms. Then the paper was focused on the effects of pH value, salt concn., and soln. viscosity on the diffusion coeffs. of the two water-sol. QDs. The pH value had a great influence on the diffusion coeff. of CTAB-QDs but little on that of OPA-QDs. The difference should be mainly due to the distinguishment of the charge and structure of surface ligands on the two water-sol. QDs. The diffusion coeff. of either CTAB-QDs or OPA-QDs was hardly affected by the salt concn. of the soln. Also, for both CTAB-QDs and OPA-QDs, the diffusion coeffs. decreased as the soln. viscosity increased, which obeyed the Stokes-Einstein relation. In summary, OPA-QDs have more promising applications in single-mol. tracking in live cells, as compared with CTAB-QDs. The obtained results would benefit the further applications of QDs in single-mol. tracking in live cells. This system could also serve as a model system for studying the diffusion behavior of nanoparticles.
- 257Gao, X.; Wang, T.; Wu, B.; Chen, J.; Chen, J.; Yue, Y.; Dai, N.; Chen, H.; Jiang, X. Quantum Dots for Tracking Cellular Transport of Lectin-Functionalized Nanoparticles. Biochem. Biophys. Res. Commun. 2008, 377, 35– 40, DOI: 10.1016/j.bbrc.2008.09.077257Quantum dots for tracking cellular transport of lectin-functionalized nanoparticlesGao, Xiaoling; Wang, Tao; Wu, Bingxian; Chen, Jun; Chen, Jiyao; Yue, Yang; Dai, Ning; Chen, Hongzhuan; Jiang, XinguoBiochemical and Biophysical Research Communications (2008), 377 (1), 35-40CODEN: BBRCA9; ISSN:0006-291X. (Elsevier Inc.)Successful drug delivery by functionalized nanocarriers largely depends on their efficient intracellular transport which has not yet been fully understood. We developed a new tracking technique by encapsulating quantum dots into the core of wheat germ agglutinin-conjugated nanoparticles (WGA-NP) to track cellular transport of functionalized nanocarriers. The resulting nanoparticles showed no changes in particle size, zeta potential or biobinding activity, and the loaded probe presented excellent photostability and tracking ability. Taking advantage of these properties, cellular transport profiles of WGA-NP in Caco-2 cells was demonstrated. The cellular uptake begins with binding of WGA to its receptor at the cell surface. The subsequent endocytosis happened in a cytoskeleton-dependent manner and by means of clathrin and caveolae-mediated mechanisms. After endosome creating, transport occurs to both trans-Golgi and lysosome. Our study provides new evidences for quantum dots as a cellular tracking probe of nanocarriers and helps understand intracellular transport profile of lectin-functionalized nanoparticles.
- 258Rajan, S. S.; Liu, H. Y.; Vu, T. Q. Ligand-Bound Quantum Dot Probes for Studying the Molecular Scale Dynamics of Receptor Endocytic Trafficking in Live Cells. ACS Nano 2008, 2, 1153– 1166, DOI: 10.1021/nn700399e258Ligand-Bound Quantum Dot Probes for Studying the Molecular Scale Dynamics of Receptor Endocytic Trafficking in Live CellsRajan, Sujata Sundara; Liu, Hong Yan; Vu, Tania Q.ACS Nano (2008), 2 (6), 1153-1166CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small nos. of ligand bound-receptor complexes inside live cells is currently hampered by tech. limitations. Here, the authors develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunopptn., and immuno-colocalization, the authors illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand-receptor binding and internalization (t = 15-150 min). The unique value of these probes is illustrated by new dynamic observations: (1) that endocytosis proceeds at strikingly regulated fashion, and (2) that diffusive and active forms of transport inside cells are rapid and efficient. QDs are powerful intracellular probes that can provide biologists with new capabilities and fresh insight for studying endocytic receptor signaling events, in real time, and at the resoln. of single or small nos. of receptors in live cells.
- 259Murcia, M. J.; Minner, D. E.; Mustata, G. M.; Ritchie, K.; Naumann, C. A. Design of Quantum Dot-Conjugated Lipids for Long-Term, High-Speed Tracking Experiments on Cell Surfaces. J. Am. Chem. Soc. 2008, 130, 15054– 15062, DOI: 10.1021/ja803325b259Design of Quantum Dot-Conjugated Lipids for Long-Term, High-Speed Tracking Experiments on Cell SurfacesMurcia, Michael J.; Minner, Daniel E.; Mustata, Gina-Mirela; Ritchie, Kenneth; Naumann, Christoph A.Journal of the American Chemical Society (2008), 130 (45), 15054-15062CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking expts. on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 M mixt. of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol-2000)],are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aq. soln. is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking expts. on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-mol. tracking probes in plasma membranes of several cell types. Initial tracking expts. at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-mol. imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking expts. that used larger (40 nm) Au labels, not only push the time resoln. in long-time, continuous fluorescence-based single-mol. tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor expts.
- 260Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum Dot Bioconjugates for Imaging, Labelling and Sensing. Nat. Mater. 2005, 4, 435– 446, DOI: 10.1038/nmat1390260Quantum dot bioconjugates for imaging, labelling and sensingMedintz, Igor L.; Uyeda, H. Tetsuo; Goldman, Ellen R.; Mattoussi, HediNature Materials (2005), 4 (6), 435-446CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)A review. One of the fastest moving and most exciting interfaces of nanotechnol. is the use of quantum dots (QDs) in biol. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biol. investigations, in which traditional fluorescent labels based on org. mols. fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water sol. and target them to specific biomols. has led to promising applications in cellular labeling, deep-tissue imaging, assay labeling and as efficient fluorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible bioconjugation techniques. In this review, we look at current methods for prepg. QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biol. has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.
- 261Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005, 307, 538– 544, DOI: 10.1126/science.1104274261Quantum Dots for Live Cells, in Vivo Imaging, and DiagnosticsMichalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S.Science (Washington, DC, United States) (2005), 307 (5709), 538-544CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biol. applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biol. Recent examples of their exptl. use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-IR emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-mol. level, high-resoln. cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.
- 262Bentolila, L. A.; Ebenstein, Y.; Weiss, S. Quantum Dots for in Vivo Small-Animal Imaging. J. Nucl. Med. 2009, 50, 493– 496, DOI: 10.2967/jnumed.108.053561262Quantum dots for in vivo small-animal imagingBentolila, Laurent A.; Ebenstein, Yuval; Weiss, ShimonJournal of Nuclear Medicine (2009), 50 (4), 493-496CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine)A review. Nanotechnol. is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1-10 nm) made of inorg. semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the UV to the near-IR. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clin. applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.
- 263Zrazhevskiy, P.; Sena, M.; Gao, X. Designing Multifunctional Quantum Dots for Bioimaging, Detection, and Drug Delivery. Chem. Soc. Rev. 2010, 39, 4326– 4354, DOI: 10.1039/b915139g263Designing multifunctional quantum dots for bioimaging, detection, and drug deliveryZrazhevskiy, Pavel; Sena, Mark; Gao, XiaohuChemical Society Reviews (2010), 39 (11), 4326-4354CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The emerging field of bionanotechnol. aims at revolutionizing biomedical research and clin. practice via introduction of nanoparticle-based tools, expanding capabilities of existing investigative, diagnostic, and therapeutic techniques as well as creating novel instruments and approaches for addressing challenges faced by medicine. Quantum dots (QDs), semiconductor nanoparticles with unique photo-phys. properties, have become one of the dominant classes of imaging probes as well as universal platforms for engineering of multifunctional nanodevices. Possessing versatile surface chem. and superior optical features, QDs have found initial use in a variety of in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is becoming increasingly important for successful transition of this technol. from proof-of-concept studies towards real-life clin. applications. This review outlines the major design principles and criteria, from general ones to application-specific, governing the engineering of novel QD probes satisfying the increasing demands and requirements of nanomedicine and discusses the future directions of QD-focused bionanotechnol. research.
- 264Ruan, G.; Agrawal, A.; Marcus, A. I.; Nie, S. Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J. Am. Chem. Soc. 2007, 129, 14759– 14766, DOI: 10.1021/ja074936k264Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle SheddingRuan, Gang; Agrawal, Amit; Marcus, Adam I.; Nie, ShumingJournal of the American Chemical Society (2007), 129 (47), 14759-14766CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the use of Tat peptide-conjugated quantum dots (Tat-QDs) to examine the complex behavior of nanoparticle probes in live cells, a topic that is of considerable current interest in developing advanced nanoparticle agents for mol. and cellular imaging. Dynamic confocal imaging studies indicate that the peptide-conjugated QDs are internalized by macropinocytosis, a fluid-phase endocytosis process triggered by Tat-QD binding to neg. charged cell membranes. The internalized Tat-QDs are tethered to the inner vesicle surfaces and are trapped in cytoplasmic organelles. The QD loaded vesicles are actively transported by mol. machines (such as dyneins) along microtubule tracks. The destination of this active transport is an asym. perinuclear region (outside the cell nucleus) known as the microtubule organizing center (MTOC). The authors also find that Tat-QDs strongly bind to cellular membrane structures such as filopodia and that large QD-contg. vesicles are released from the tips of filopodia by vesicle shedding. These results provide new insights into the mechanisms of Tat peptide-mediated delivery as well as toward the design of functionalized nanoparticles for mol. imaging and targeted therapy.
- 265Bruchez, M. P. Quantum Dots Find Their Stride in Single Molecule Tracking. Curr. Opin. Chem. Biol. 2011, 15, 775– 780, DOI: 10.1016/j.cbpa.2011.10.011265Quantum dots find their stride in single molecule trackingBruchez, Marcel P.Current Opinion in Chemical Biology (2011), 15 (6), 775-780CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Thirteen years after the demonstration of quantum dots as biol. imaging agents, and nine years after the initial com. introduction of bioconjugated quantum dots, the brightness and photostability of the quantum dots has enabled a range of investigations using single mol. tracking. These materials are being routinely utilized by a no. of groups to track the dynamics of single mols. in reconstituted biophys. systems and on living cells, and are esp. powerful for investigations of single mols. over long timescales with short exposure times and high pointing accuracy. New approaches are emerging where the quantum dots are used as hard-sphere' probes for intracellular compartments. Innovations in quantum dot surface modification are poised to substantially expand the utility of these materials.
- 266Tada, H.; Higuchi, H.; Wanatabe, T. M.; Ohuchi, N. In Vivo Real-Time Tracking of Single Quantum Dots Conjugated with Monoclonal Anti-HER2 Antibody in Tumors of Mice. Cancer Res. 2007, 67, 1138– 1144, DOI: 10.1158/0008-5472.CAN-06-1185266In vivo Real-time Tracking of Single Quantum Dots Conjugated with Monoclonal Anti-HER2 Antibody in Tumors of MiceTada, Hiroshi; Higuchi, Hideo; Wanatabe, Tomonobu M.; Ohuchi, NoriakiCancer Research (2007), 67 (3), 1138-1144CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)Studies with tracking of single nanoparticles are providing new insights into the interactions and processes involved in the transport of drug carriers in living mice. Here, the authors report the tracking of a single particle quantum dot (Qdot) conjugated with tumor-targeting antibody in tumors of living mice using a dorsal skinfold chamber and a high-speed confocal microscope with a high-sensitivity camera. Qdot labeled with the monoclonal anti-HER2 antibody was injected into mice with HER2-overexpressing breast cancer to analyze the mol. processes of its mechanistic delivery to the tumor. Movement of single complexes of the Qdot-antibody could be clearly obsd. at 30 frames/s inside the tumor through a dorsal skinfold chamber. The authors successfully identified six processes of delivery: initially in the circulation within a blood vessel, during extravasation, in the extracellular region, binding to HER2 on the cell membrane, moving from the cell membrane to the perinuclear region, and in the perinuclear region. The six processes were quant. analyzed to understand the rate-limiting constraints on Qdot-antibody delivery. The movement of the complexes at each stage was "stop-and-go.". The image anal. of the delivery processes of single particles in vivo provides valuable information on antibody-conjugated therapeutic nanoparticles, which will be useful in increasing therapeutic efficacy.
- 267Wells, N. P.; Lessard, G. A.; Werner, J. H. Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background Environments. Anal. Chem. 2008, 80, 9830– 9834, DOI: 10.1021/ac8021899267Confocal, Three-Dimensional Tracking of Individual Quantum Dots in High-Background EnvironmentsWells, Nathan P.; Lessard, Guillaume A.; Werner, James H.Analytical Chemistry (Washington, DC, United States) (2008), 80 (24), 9830-9834CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The authors demonstrate a custom confocal fluorescence-microscope that is capable of tracking individual quantum dots undergoing three-dimensional Brownian motion (diffusion coeff. ∼ 0.5 μm2/s) in environments with a signal-to-background ratio as low as 2:1, significantly worse than obsd. in a typical cellular environment. By utilizing a pulsed excitation source and time-correlated single photon counting, the time-resolved photon stream can be used to det. changes in the emission lifetime as a function of position and pos. identify single quantum dots via photon-pair correlations. These results indicate that this microscope will be capable of following protein and RNA transport throughout the full three-dimensional vol. of a live cell for durations up to 15 s.
- 268Cui, Z. Q.; Ren, Q.; Wei, H. P.; Chen, Z.; Deng, J. Y.; Zhang, Z. P.; Zhang, X. E. Quantum Dot-Aptamer Nanoprobes for Recognizing and Labeling Influenza A Virus Particles. Nanoscale 2011, 3, 2454– 2457, DOI: 10.1039/c1nr10218d268Quantum dot-aptamer nanoprobes for recognizing and labeling influenza A virus particlesCui, Zong-Qiang; Ren, Qian; Wei, Hong-Ping; Chen, Ze; Deng, Jiao-Yu; Zhang, Zhi-Ping; Zhang, Xian-EnNanoscale (2011), 3 (6), 2454-2457CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)The fluorescence labeling of viruses is a useful technol. for virus detection and imaging. By combining the excellent fluorescence properties of quantum dots (QDs) with the high affinity and specificity of aptamers, we constructed a QD-aptamer probe. The aptamer A22, against the hemagglutinin of influenza A virus, was linked to QDs, producing the QD-A22 probe. Fluorescence imaging and transmission electron microscopy showed that the QD-A22 probe could specifically recognize and label influenza A virus particles. This QD labeling technique provides a new strategy for labeling virus particles for virus detection and imaging.
- 269Saxton, M. J.; Jacobson, K. Single-Particle Tracking: Applications to Membrane Dynamics. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373– 399, DOI: 10.1146/annurev.biophys.26.1.373269Single-particle tracking: applications to membrane dynamicsSaxton, Michael J.; Jacobson, KenAnnual Review of Biophysics and Biomolecular Structure (1997), 26 (), 373-399CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews)Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is obsd., but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.
- 270Kusumi, A.; Sako, Y.; Yamamoto, M. Confined Lateral Diffusion of Membrane Receptors as Studied by Single Particle Tracking (Nanovid Microscopy). Effects of Calcium-Induced Differentiation in Cultured Epithelial Cells. Biophys. J. 1993, 65, 2021– 2040, DOI: 10.1016/S0006-3495(93)81253-0270Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cellsKusumi, Akihiro; Sako, Yasushi; Yamamoto, MutsuyaBiophysical Journal (1993), 65 (5), 2021-40CODEN: BIOJAU; ISSN:0006-3495.The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor mols. are labeled with 40 nm-φ colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resoln. of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor mols. obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were obsd.: (a) stationary mode, in which the microscopic diffusion coeff. is less than 4.6 × 10-12 cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coeff. between 4.6 × 10-12 and 1 × 10-9 cm2/s) are confined within a limited area, probably by the membrane-assocd. cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diam. (0.04-0.24 μm2 in area), in which receptor mols. are confined in the time scale of 3-30 s, and that the long-range diffusion obsd. by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of mols. in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from ∼60% to 8% (low- and high-calcium mediums, resp.).
- 271Wan, X. Y.; Zheng, L. L.; Gao, P. F.; Yang, X. X.; Li, C. M.; Li, Y. F.; Huang, C. Z. Real-Time Light Scattering Tracking of Gold Nanoparticles- Bioconjugated Respiratory Syncytial Virus Infecting HEp-2 Cells. Sci. Rep. 2015, 4, 4529, DOI: 10.1038/srep04529There is no corresponding record for this reference.
- 272Marjomaki, V.; Lahtinen, T.; Martikainen, M.; Koivisto, J.; Malola, S.; Salorinne, K.; Pettersson, M.; Hakkinen, H. Site-Specific Targeting of Enterovirus Capsid by Functionalized Monodisperse Gold Nanoclusters. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 1277– 1281, DOI: 10.1073/pnas.1310973111272Site-specific targeting of enterovirus capsid by functionalized monodisperse gold nanoclustersMarjomaki, Varpu; Lahtinen, Tanja; Martikainen, Mari; Koivisto, Jaakko; Malola, Sami; Salorinne, Kirsi; Pettersson, Mika; Hakkinen, HannuProceedings of the National Academy of Sciences of the United States of America (2014), 111 (4), 1277-1281CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Development of precise protocols for accurate site-specific conjugation of monodisperse inorg. nanoparticles to biol. material is one of the challenges in contemporary bionanoscience and nanomedicine. We report here a successful site-specific covalent conjugation of functionalized atomically monodisperse gold clusters with 1.5-nm metal cores to viral surfaces. Water-sol. Au102(para-mercaptobenzoic acid)44 clusters, functionalized by maleimide linkers to target cysteines of viral capsid proteins, were synthesized and conjugated to enteroviruses echovirus 1 and coxsackievirus B3. Quant. anal. of transmission electron microscopy images and the known virus structures showed high affinity and mutual ordering of the bound gold clusters on the viral surface and a clear correlation between the clusters and the targeted cysteine sites close to the viral surface. Infectivity of the viruses was not compromised by loading of several tens of gold clusters per virus. These advances allow for future investigations of the structure-function relations of enteroviruses and enterovirus-related virus-like particles, including their entry mechanisms into cells and uncoating in cellular endosomes.
- 273Wan, X. K.; Xu, W. W.; Yuan, S. F.; Gao, Y.; Zeng, X. C.; Wang, Q. M. A Near-Infrared-Emissive Alkynyl-Protected Au24 Nanocluster. Angew. Chem., Int. Ed. 2015, 54, 9683– 9686, DOI: 10.1002/anie.201503893273A Near-Infrared-Emissive Alkynyl-Protected Au24 NanoclusterWan, Xian-Kai; Xu, Wen Wu; Yuan, Shang-Fu; Gao, Yi; Zeng, Xiao-Cheng; Wang, Quan-MingAngewandte Chemie, International Edition (2015), 54 (33), 9683-9686CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)An alkynyl-protected Au nanocluster [Au24(C≡CPh)14(PPh3)4](SbF6)2 was prepd. by a direct redn. method. Single-crystal x-ray diffraction reveals that the mol. structure contains a Au22 core that is made of two Au13-centered cuboctahedra that share a square face. Two staple-like PhC≡CAuC≡CPh motifs are located around the center of the rod-like Au22 core. This Au24 nanocluster is highly emissive in the near-IR region with λmax = 925 nm and the nature of the HOMO-LUMO transition was studied by time-dependent DFT calcns.
- 274Jin, R.; Zeng, C.; Zhou, M.; Chen, Y. Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities. Chem. Rev. 2016, 116, 10346– 10413, DOI: 10.1021/acs.chemrev.5b00703274Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and OpportunitiesJin, Rongchao; Zeng, Chenjie; Zhou, Meng; Chen, YuxiangChemical Reviews (Washington, DC, United States) (2016), 116 (18), 10346-10413CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Colloidal nanoparticles are being intensely pursued in current nanoscience research. Nanochemists are often frustrated by the known fact that no two nanoparticles are the same, which precludes the deep understanding of many fundamental properties of colloidal nanoparticles in which the total structures (core plus surface) must be known. Therefore, controlling nanoparticles with at. precision and solving their total structures have long been major dreams for nanochemists. Recently, these goals are partially fulfilled in the case of gold nanoparticles, at least in the ultrasmall size regime (1-3 nm in diam., often called nanoclusters). This review summarizes the major progress in the field, including the principles that permit atomically precise synthesis, new types of at. structures, and unique phys. and chem. properties of atomically precise nanoparticles, as well as exciting opportunities for nanochemists to understand very fundamental science of colloidal nanoparticles (such as the stability, metal-ligand interfacial bonding, ligand assembly on particle surfaces, aesthetic structural patterns, periodicities, and emergence of the metallic state) and to develop a range of potential applications such as in catalysis, biomedicine, sensing, imaging, optics, and energy conversion. Although most of the research activity currently focuses on thiolate-protected gold nanoclusters, important progress also was achieved in other ligand-protected gold, silver, and bimetal (or alloy) nanoclusters. All of these types of unique nanoparticles will bring unprecedented opportunities, not only in understanding the fundamental questions of nanoparticles but also in opening up new horizons for scientific studies of nanoparticles.
- 275Zhang, L. B.; Wang, E. K. Metal Nanoclusters: New Fluorescent Probes for Sensors and Bioimaging. Nano Today 2014, 9, 132– 157, DOI: 10.1016/j.nantod.2014.02.010275Metal nanoclusters: New fluorescent probes for sensors and bioimagingZhang, Libing; Wang, ErkangNano Today (2014), 9 (1), 132-157CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.)A review. Fluorescent metal nanoclusters (NCs) as a new class of fluorophores have attracted more and more attention due to their unique electronic structures and the subsequent unusual phys. and chem. properties. The size of metal NCs approaches the Fermi wavelength of electrons, between metal atoms and nanoparticles, resulting in mol.-like properties including discrete energy levels, size-dependent fluorescence, good photostability and biocompatibility. These excellent properties make them ideal fluorescent probes for biol. application. Up to now, significant efforts have been devoted to the synthesis, property and application studies of gold and silver NCs. Recently, a growing no. of studies on copper and other metal clusters have also been reported. In this review article, we focus on summarizing recent advances in controllable synthesis strategies, chem. and optical properties, and sensing and imaging applications of metal NCs (mainly including Au, Ag, Cu, etc.). Finally, we conclude with a look at the future challenges and prospects of the future development of metal NCs.
- 276Shang, L.; Dong, S. J.; Nienhaus, G. U. Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications. Nano Today 2011, 6, 401– 418, DOI: 10.1016/j.nantod.2011.06.004276Ultra-small fluorescent metal nanoclusters: synthesis and biological applicationsShang, Li; Dong, Shaojun; Nienhaus, G. UlrichNano Today (2011), 6 (4), 401-418CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.)A review. Recent advances in nanotechnol. have given rise to a new class of fluorescent labels, fluorescent metal nanoclusters, e.g., Au and Ag. These nanoclusters are of significant interest because they provide the missing link between at. and nanoparticle behavior in metals. Composed of a few to a hundred atoms, their sizes are comparable to the Fermi wavelength of electrons, resulting in mol.-like properties including discrete electronic states and size-dependent fluorescence. Fluorescent metal nanoclusters have an attractive set of features, such as ultrasmall size, good biocompatibility and excellent photostability, making them ideal fluorescent labels for biol. applications. In this review, we summarize synthesis strategies of water-sol. fluorescent metal nanoclusters and their optical properties, highlight recent advances in their application for ultrasensitive biol. detection and fluorescent biol. imaging, and finally discuss current challenges for their potential biomedical applications.
- 277Draz, M. S.; Fang, B. A.; Li, L. J.; Chen, Z.; Wang, Y. J.; Xu, Y. H.; Yang, J.; Killeen, K.; Chen, F. F. Hybrid Nanocluster Plasmonic Resonator for Lmmunological Detection of Hepatitis B Virus. ACS Nano 2012, 6, 7634– 7643, DOI: 10.1021/nn3034056277Hybrid Nanocluster Plasmonic Resonator for Immunological Detection of Hepatitis B VirusDraz, Mohamed Shehata; Fang, Binbin Amanda; Li, Lanjuan; Chen, Zhi; Wang, Yingjie; Xu, Yuhong; Yang, Jun; Killeen, Kevin; Chen, Fanqing FrankACS Nano (2012), 6 (9), 7634-7643CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Approx. 88% of the world population lives in regions with intermediate to high incidence of Hepatitis B virus (HBV), yet current serol. and DNA-based detection methods have limited sensitivity and convenience. Here, the authors describe a preassembled plasmonic resonance nanocluster for HBV detection. The gold nanoparticle acceptors (AuNPs), with HBV surface antigen (HBsAg) epitope, and quantum dot (QD) donors with Fab antibody, are assembled into an immuno-mediated 3D-oriented complex with enhanced energy transfer and fluorescence quenching. The coherent plasmonic resonance between Au and QD nanoparticles is exploited to achieve improved donor-acceptor resonance within the nanocluster, which in the presence of HBV viral particles is disassembled in a highly specific manner. The nanocluster provides high detection specificity and sensitivity of HBV, with a sensitivity limit down to 1-100 viral particles per μL and to attomolar levels of HBsAg. This general platform could be used to establish multiplex diagnostic assays for a variety of other microbial pathogens.
- 278Shokri, E.; Hosseini, M.; Faridbod, F.; Rahaie, M. Rapid Pre-Symptomatic Recognition of Tristeza Viral RNA by a Novel Fluorescent Self-Dimerized DNA-Silver Nanocluster probe. RSC Adv. 2016, 6, 99437– 99443, DOI: 10.1039/C6RA15199J278Rapid pre-symptomatic recognition of tristeza viral RNA by a novel fluorescent self-dimerized DNA-silver nanocluster probeShokri, Ehsan; Hosseini, Morteza; Faridbod, Farnoush; Rahaie, MahdiRSC Advances (2016), 6 (101), 99437-99443CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Citrus tristeza virus (CTV), a pos.-strand RNA virus within the family of Closteroviridae, is distributed worldwide and causes one of the most economically important diseases of citrus. Since the CTV pathogen is easily spread by graft propagation and aphid vectors, continual monitoring of healthy seedlings and eradication of infected plants is essential for better disease management. In this study, a novel self-dimerized DNA-silver nanocluster probe was developed for the simple and rapid pre-symptomatic detection of CTV severe strains in biol. prepns. Insertion of a G rich loop maker sequence contg. internal complementary bases in the probe structure leads to the formation of a stable homodimer probe with a G-rich loop at the center that is more reactive than the formless probe. Interestingly, the results showed that the probe structure conversion between dimeric and non dimeric states upon DNA/RNA hybridization, produced an enhanced fluorescence signal allowing precise and sensitive detection of tristeza RNA. Based on the sensitivity test, CTV-RNA in the range of 5-210 nM, can be linearly detected with the detection limit of 2.5 nM. Finally, the results of our DNA-AgNCs based fluorometric assay were consistent with the results of the conventional RT-PCR.
- 279Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44, 8636– 8663, DOI: 10.1039/C5CS00607D279Metal nanoclusters: novel probes for diagnostic and therapeutic applicationsTao, Yu; Li, Mingqiang; Ren, Jinsong; Qu, XiaogangChemical Society Reviews (2015), 44 (23), 8636-8663CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Metal nanoclusters, composed of several to a few hundred metal atoms, have received worldwide attention due to their extraordinary phys. and chem. characteristics. Recently, great efforts have been devoted to the exploration of the potential diagnostic and therapeutic applications of metal nanoclusters. Here we focus on the recent advances and new horizons in this area, and introduce the rising progress on the use of metal nanoclusters for biol. anal., biol. imaging, therapeutic applications, DNA assembly and logic gate construction, enzyme mimic catalysis, as well as thermometers and pH meters. Furthermore, the future challenges in the construction of biofunctional metal nanoclusters for diagnostic and therapeutic applications are also discussed. We expect that the rapidly growing interest in metal nanocluster-based theranostic applications will certainly not only fuel the excitement and stimulate research in this highly active field, but also inspire broader concerns across various disciplines.
- 280Basle, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17, 213– 227, DOI: 10.1016/j.chembiol.2010.02.008280Protein Chemical Modification on Endogenous Amino AcidsBasle, Emmanuel; Joubert, Nicolas; Pucheault, MathieuChemistry & Biology (Cambridge, MA, United States) (2010), 17 (3), 213-227CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Chem. modification of protein is an arduous but fruitful task. Many chem. methods have been developed for such purpose by carefully balancing reactivity and selectivity. Now both chemists and biologists have in hand an arsenal of tools from which they can select a relevant reaction to tackle their problems. This review focuses on the various chem. transformations available for selective modification of proteins. It also provides a brief overview of some of their main applications, including detection of protein interactions, prepn. of bioconjugates, and protein microarrays.
- 281Hong, Z. Y.; Zhang, Z. L.; Tang, B.; Ao, J.; Wang, C.; Yu, C.; Pang, D. W. Equipping Inner Central Components of Influenza A Virus with Quantum Dots. Anal. Chem. 2018, 90, 14020– 14028, DOI: 10.1021/acs.analchem.8b03995281Equipping Inner Central Components of Influenza A Virus with Quantum DotsHong, Zheng-Yuan; Zhang, Zhi-Ling; Tang, Bo; Ao, Jian; Wang, Chuan; Yu, Cong; Pang, Dai-WenAnalytical Chemistry (Washington, DC, United States) (2018), 90 (23), 14020-14028CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Influenza A virus (IAV), a risk to public health, is enveloped and contains viral ribonucleoprotein (vRNP) complexes, where vRNP complexes are central to every aspect of the IAV life cycle. Labeling both the vRNP complexes and viral envelope with quantum dots (QDs) is conducive to achieving global long-term tracking of a single IAV infecting host cell, which has potential to provide valuable information for revealing mechanisms of IAV infection. However, even though some strategies for labeling of the viral envelope with QDs have been developed, there are few strategies for coupling of QDs to the vRNP complexes inside IAV so far. Herein, we devised a convenient electroporation-based strategy, coupled with antibody binding, to transfer green QDs-labeled nucleoprotein antibodies (GQDs-NPAb) into H1N1 and achieved the labeling of vRNP complexes with QDs [H1N1(GQDs)]. Under the optimal condition of 20 nM GQDs-NPAb and a single pulse with 20 ms duration and 750 V/cm pulse intensity, the actual efficiency of labeling is ca. 34% and H1N1(GQDs) can retain 93% infectivity. Then, dual labeling of H1N1 was realized by labeling the envelope of H1N1(GQDs) with red QDs (RQDs) via a mild and efficient hydrazine-aldehyde-based strategy. At the optimal RQDs concn. of 5 nM, the actual efficiency of dual labeling can reach to 11% and the dual-labeled H1N1 can retain 93% infectivity. Because of the similar components and structure of different IAV subtypes, this dual-labeling strategy is applicable to other subtypes of IAV, e.g., H9N2.
- 282Huisgen, R. 1.3-Dipolare Cycloadditionen Rückschau und Ausblick. Angew. Chem. 1963, 75, 604– 637, DOI: 10.1002/ange.196307513042821,3-Dipolar cycloadditionsHuisgen, RolfAngewandte Chemie (1963), 75 (13), 604-37CODEN: ANCEAD; ISSN:0044-8249.cf. CA 56, 7096c. A review with 211 references.
- 283Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. 2001, 40, 2004– 2021, DOI: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5283Click chemistry: diverse chemical function from a few good reactionsKolb, Hartmuth C.; Finn, M. G.; Sharpless, K. BarryAngewandte Chemie, International Edition (2001), 40 (11), 2004-2021CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)Review with > 88 refs. Examn. of nature's favorite mols. reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds - surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon-heteroatom bonds. Even the 35 or so building blocks from which these crucial mols. are made each contain, at most, six contiguous C-C bonds, except for the three arom. amino acids. Taking a cue from nature's approach, the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compds. and combinatorial libraries through heteroatom links (C-X-C), an approach called "click chem." is addressed. Click chem. is at once defined, enabled, and constrained by a handful of nearly perfect "spring-loaded" reactions. The stringent criteria for a process to earn click chem. status are described along with examples of the mol. frameworks that are easily made using this spartan, but powerful, synthetic strategy.
- 284Nwe, K.; Brechbiel, M. W. Growing Applications of ″Click Chemistry″ for Bioconjugation in Contemporary Biomedical Research. Cancer Biother.Radiopharm. 2009, 24, 289– 302, DOI: 10.1089/cbr.2008.0626284Growing Applications of "Click Chemistry" for Bioconjugation in Contemporary Biomedical ResearchNwe, Kido; Brechbiel, Martin W.Cancer Biotherapy & Radiopharmaceuticals (2009), 24 (3), 289-302CODEN: CBRAFJ; ISSN:1084-9785. (Mary Ann Liebert, Inc.)A review. This update summarizes the growing application of "click" chem. in diverse areas such as bioconjugation, drug discovery, materials science, and radiochem. This update also discusses click chem. reactions that proceed rapidly with high selectivity, specificity, and yield. Two important characteristics make click chem. so attractive for assembling compds., reagents, and biomols. for preclin. and clin. applications. First, click reactions are bio-orthogonal; neither the reactants nor their product's functional groups interact with functionalized biomols. Second, the reactions proceed with ease under mild nontoxic conditions, such as at room temp. and, usually, in water. The copper-catalyzed Huisgen cycloaddn., azide-alkyne [3 + 2] dipolar cycloaddn., Staudinger ligation, and azide-phosphine ligation each possess these unique qualities. These reactions can be used to modify one cellular component while leaving others unharmed or untouched. Click chem. has found increasing applications in all aspects of drug discovery in medicinal chem., such as for generating lead compds. through combinatorial methods. Bioconjugation via click chem. is rigorously employed in proteomics and nucleic research. In radiochem., selective radiolabeling of biomols. in cells and living organisms for imaging and therapy has been realized by this technol. Bifunctional chelating agents for several radionuclides useful for positron emission tomog. and single-photon emission computed tomog. imaging have also been prepd. by using click chem. This review concludes that click chem. is not the perfect conjugation and assembly technol. for all applications, but provides a powerful, attractive alternative to conventional chem. This chem. has proven itself to be superior in satisfying many criteria (e.g., biocompatibility, selectivity, yield, stereospecificity, and so forth); thus, one can expect it will consequently become a more routine strategy in the near future for a wide range of applications.
- 285Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry. J. Am. Chem. Soc. 2008, 130, 11486– 11493, DOI: 10.1021/ja803086r285Second-Generation Difluorinated Cyclooctynes for Copper-Free Click ChemistryCodelli, Julian A.; Baskin, Jeremy M.; Agard, Nicholas J.; Bertozzi, Carolyn R.Journal of the American Chemical Society (2008), 130 (34), 11486-11493CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The 1,3-dipolar cycloaddn. of azides and activated alkynes has been used for site-selective labeling of biomols. in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3+2] cycloaddn., this method, often termed "click chem.", is currently incompatible with living systems because of the toxicity of the metal. The authors recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here the authors report a novel class of DIFO reagents for copper-free click chem. that are considerably more synthetically tractable. The new analogs maintained the same elevated rates of [3+2] cycloaddn. as the parent compd. and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chem. in the hands of biologists.
- 286Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Copper-Free Click Chemistry for Dynamic in Vivo Imaging. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16793– 16797, DOI: 10.1073/pnas.0707090104286Copper-free click chemistry for dynamic in vivo imagingBaskin, Jeremy M.; Prescher, Jennifer A.; Laughlin, Scott T.; Agard, Nicholas J.; Chang, Pamela V.; Miller, Isaac A.; Lo, Anderson; Codelli, Julian A.; Bertozzi, Carolyn R.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (43), 16793-16797CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomols. such as glycans and lipids. Here, the authors report a Cu-free variant of click chem. that can label these biomols. rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The crit. reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3+2] dipolar cycloaddn. with azides installed metabolically into biomols. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, the authors studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
- 287Hoyle, C. E.; Bowman, C. N. Thiol-Ene Click Chemistry. Angew. Chem., Int. Ed. 2010, 49, 1540– 1573, DOI: 10.1002/anie.200903924287Thiol-Ene Click ChemistryHoyle, Charles E.; Bowman, Christopher N.Angewandte Chemie, International Edition (2010), 49 (9), 1540-1573CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Following Sharpless' visionary characterization of several idealized reactions as click reactions, the materials science and synthetic chem. communities have pursued numerous routes toward the identification and implementation of these click reactions. Herein, the authors review the radical-mediated thiol-ene reaction as one such click reaction. This reaction has all the desirable features of a click reaction, being highly efficient, simple to execute with no side products and proceeding rapidly to high yield. Further, the thiol-ene reaction is most frequently photoinitiated, particularly for photopolymns. resulting in highly uniform polymer networks, promoting unique capabilities related to spatial and temporal control of the click reaction. The reaction mechanism and its implementation in various synthetic methodologies, biofunctionalization, surface and polymer modification, and polymn. are all reviewed.
- 288Nikic, I.; Plass, T.; Schraidt, O.; Szymanski, J.; Briggs, J. A.; Schultz, C.; Lemke, E. A. Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy. Angew. Chem., Int. Ed. 2014, 53, 2245– 2249, DOI: 10.1002/anie.201309847288Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution MicroscopyNikic, Ivana; Plass, Tilman; Schraidt, Oliver; Szymanski, Jedrzej; Briggs, John A. G.; Schultz, Carsten; Lemke, Edward A.Angewandte Chemie, International Edition (2014), 53 (8), 2245-2249CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The growing demands of advanced fluorescence and super-resoln. microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chem. handles into proteins in living cells. Here the authors present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized org. dyes by the inverse-electron-demand Diels-Alder cycloaddn. (SPIEDAC). Furthermore, the authors fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which the authors termed selectivity enhanced (se) SPIEDAC. SeSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. The authors demonstrate the strength of the authors' method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resoln. microscopy.
- 289Salic, A.; Mitchison, T. J. A Chemical Method for Fast and Sensitive Detection of DNA Synthesis in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2415– 2420, DOI: 10.1073/pnas.0712168105289A chemical method for fast and sensitive detection of DNA synthesis in vivoSalic, Adrian; Mitchison, Timothy J.Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (7), 2415-2420CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddn. reaction ("click" chem.). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount prepns. of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.
- 290Jao, C. Y.; Salic, A. Exploring RNA Transcription and Turnover in Vivo by Using Click Chemistry. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15779– 15784, DOI: 10.1073/pnas.0808480105290Exploring RNA transcription and turnover in vivo by using click chemistryJao, Cindy Y.; Salic, AdrianProceedings of the National Academy of Sciences of the United States of America (2008), 105 (41), 15779-15784CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We describe a chem. method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on av. once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddn. reaction (often referred to as "click" chem.) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.
- 291Jewett, J. C.; Bertozzi, C. R. Cu-Free Click Cycloaddition Reactions in Chemical Biology. Chem. Soc. Rev. 2010, 39, 1272– 1279, DOI: 10.1039/b901970g291Cu-free click cycloaddition reactions in chemical biologyJewett, John C.; Bertozzi, Carolyn R.Chemical Society Reviews (2010), 39 (4), 1272-1279CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Bioorthogonal chem. reactions are paving the way for new innovations in biol. These reactions possess extreme selectivity and biocompatibility, such that their participating reagents can form covalent bonds within richly functionalized biol. systems-in some cases, living organisms. This tutorial review summarizes the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddn. reactions. As we get a better idea of the powers and limitations of each bioorthogonal reaction pair, we can start using them in concert to study increasingly complex interactions in biol. settings. While the field of bioorthogonal chem. has expanded rapidly, there remains a pressing need for new reagents with fewer side reactions and increased efficiencies. Where will these new reactions come from Using history as a guide, they are found in the least likely of places, buried in a piece of purely academic scholarship that is being written up at this very moment.
- 292Rubino, F. A.; Oum, Y. H.; Rajaram, L.; Chu, Y.; Carrico, I. S. Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry. J. Visualized Exp. 2012, e4246 DOI: 10.3791/4246292Chemoselective modification of viral surfaces via bioorthogonal click chemistryRubino, Frederick A.; Oum, Yoon Hyeun; Rajaram, Lakshmi; Chu, Yanjie; Carrico, Isaac S.Journal of Visualized Experiments (2012), (66), E4246/1-E4246/7CODEN: JVEOA4; ISSN:1940-087X. (Journal of Visualized Experiments)The modification of virus particles has received a significant amt. of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development. Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus prodn., infectivity and cellular transduction. Using chemoselective click chem., we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible. The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chem. to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically or anal., as it allows for chemoselective ligation of targeting mols., dyes or other mols. of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness, (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access. In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purifn. of the azide-modified virus particles, an alkyne probe contg. the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE anal. is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only. In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein - strain-promoted azide-alkyne cycloaddn. (SPAAC) and copper-catalyzed azide-alkyne cycloaddn. (CuAAC) under deoxygenated atm.
- 293Oum, Y. H.; Desai, T. M.; Marin, M.; Melikyan, G. B. Click Labeling of Unnatural Sugars Metabolically Incorporated into Viral Envelope Glycoproteins Enables Visualization of Single Particle Fusion. J. Virol. Methods 2016, 233, 62– 71, DOI: 10.1016/j.jviromet.2016.02.016293Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusionOum, Yoon Hyeun; Desai, Tanay M.; Marin, Mariana; Melikyan, Gregory B.Journal of Virological Methods (2016), 233 (), 62-71CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with org. fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells.
- 294Huang, L. L.; Lu, G. H.; Hao, J.; Wang, H. Z.; Yin, D. L.; Xie, H. Y. Enveloped Virus Labeling via Both Intrinsic Biosynthesis and Metabolic Incorporation of Phospholipids in Host Cells. Anal. Chem. 2013, 85, 5263– 5270, DOI: 10.1021/ac4008144294Enveloped Virus Labeling via Both Intrinsic Biosynthesis and Metabolic Incorporation of Phospholipids in Host CellsHuang, Li-Li; Lu, Gui-Hong; Hao, Jian; Wang, Hanzhong; Yin, Du-Lin; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2013), 85 (10), 5263-5270CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)An alternative method for labeling fully replicative enveloped viruses was developed, in which both the biosynthesis and metabolic incorporation of phospholipids in host cells were simultaneously utilized to introduce an azide group to the envelope of the vaccinia virus by taking advantage of the host-derived lipid membrane formation mechanism. Such an azide group could be subsequently used to fluorescently label the envelope of the virus via a bioorthogonal reaction. Furthermore, simultaneous dual-labeling of the virus through the virus replication was realized skillfully by coupling this envelope labeling strategy with "replication-intercalation labeling" of viral nucleic acid. For the first time, it is by natural propagation of the virus in its host cells in the presence of fluorophores that simultaneous dual-labeling of living viruses can be mildly realized with high efficiency in facile and mild conditions.
- 295Hou, W.; Li, Y.; Kang, W.; Wang, X.; Wu, X.; Wang, S.; Liu, F. Real-Time Analysis of Quantum Dot Labeled Single Porcine Epidemic Diarrhea Virus Moving along the Microtubules Using Single Particle Tracking. Sci. Rep. 2019, 9, 1307, DOI: 10.1038/s41598-018-37789-9295Real-time analysis of quantum dot labeled single porcine epidemic diarrhea virus moving along the microtubules using single particle trackingHou Wei; Li Yangyang; Kang Wenjie; Wang Xin; Wang Shouyu; Liu Fei; Wu Xuping; Wang ShouyuScientific reports (2019), 9 (1), 1307 ISSN:.In order to study the infection mechanism of porcine epidemic diarrhea virus (PEDV), which causes porcine epidemic diarrhea, a highly contagious enteric disease, we combined quantum dot labeled method, which could hold intact infectivity of the labeled viruses to the largest extent, with the single particle tracking technique to dynamically and globally visualize the transport behaviors of PEDVs in live Vero cells. Our results were the first time to uncover the dynamic characteristics of PEDVs moving along the microtubules in the host cells. It is found that PEDVs kept restricted motion mode with a relatively stable speed in the cell membrane region; while performed a slow-fast-slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule organizing center region. In addition, the return movements of small amount of PEDVs were also observed in the live cells. Collectively, our work is crucial for understanding the movement mechanisms of PEDV in the live cells, and the proposed work also provided important references for further analysis and study on the infection mechanism of PEDVs.
- 296Lin, S.; Yan, H.; Li, L.; Yang, M.; Peng, B.; Chen, S.; Li, W.; Chen, P. R. Site-Specific Engineering of Chemical Functionalities on the Surface of Live Hepatitis D Virus. Angew. Chem., Int. Ed. 2013, 52, 13970– 13974, DOI: 10.1002/anie.201305787296Site-specific engineering of chemical functionalities on the surface of live hepatitis D virusLin, Shixian; Yan, Huan; Li, Lin; Yang, Maiyun; Peng, Bo; Chen, She; Li, Wenhui; Chen, Peng R.Angewandte Chemie, International Edition (2013), 52 (52), 13970-13974CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The hepatitis D virus (HDV) assembly process was engineered to accommodate pyrrolysine (Pyl)-genetic code expansion within viable human hepatocytes, which allowed the genetic and site-specific incorporation of Pyl analogs carrying various functional handles into HDV surface envelope proteins with high prodn. yield. Five recently developed Pyl analogs were used: PenK and ACPK contain an alkyne and an azide group, resp., which can participate in the CuAAC reaction for protein click chem. labeling; BCN bears a cyclooctyne moiety capable of participating in the inverse electron-demand Diels-Alder reaction and the 1,3-dipolar cycloaddn. on proteins; DiZPK possesses a photo-reactive diazirine for protein photocrosslinking; and ONBK carries a photolytically removable O-nitrobenzyloxycarbonyl group. These 5 Pyl analogs can be recognized by wild-type or mutant PylRS and were incorporated into residues located at Pre S1, Pre S2, or S domains on the HBV L protein. These HBV envelope proteins were assembled with pregenerated HDV RNA and delta antigen into infectious HDV in human Huh-7 cells and successfully labeled using the Pyl analogs.
- 297Huang, L. L.; Liu, K.; Zhang, Q.; Xu, J.; Zhao, D.; Zhu, H.; Xie, H. Y. Integrating Two Efficient and Specific Bioorthogonal Ligation Reactions with Natural Metabolic Incorporation in One Cell for Virus Dual Labeling. Anal. Chem. 2017, 89, 11620– 11627, DOI: 10.1021/acs.analchem.7b03043297Integrating Two Efficient and Specific Bioorthogonal Ligation Reactions with Natural Metabolic Incorporation in One Cell for Virus Dual LabelingHuang, Li-Li; Liu, Kejiang; Zhang, Qianmei; Xu, Jin; Zhao, Dongxu; Zhu, Houshun; Xie, Hai-YanAnalytical Chemistry (Washington, DC, United States) (2017), 89 (21), 11620-11627CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Though techniques in bioorthogonal chem. and metabolic incorporation have been developed over the past decade, it remains difficult to integrate different bioorthogonal reactions or metabolic incorporations into one system. The protein and DNA metabolic incorporations were combined with two bioorthogonal reactions in one cell to develop a facile and universal method for virus dual labeling. Azide and vinyl groups were introduced into the proteins or genomes of viruses, resp., through the intrinsic biosynthesis of biomols., which were subsequently fluorescently labeled via copper-free click chem. or alkene-tetrazine ligation reactions during natural propagation process in host cells. Both the envelope viruses and the capsid viruses could be labeled, and the dual labeling efficiency was >80%. The labeled progeny virions were structurally intact and fully infectious, and their fluorescence was strong enough to track single virions.
- 298Wang, I. H.; Suomalainen, M.; Andriasyan, V.; Kilcher, S.; Mercer, J.; Neef, A.; Luedtke, N. W.; Greber, U. F. Tracking Viral Genomes in Host Cells at Single-Molecule Resolution. Cell Host Microbe 2013, 14, 468– 480, DOI: 10.1016/j.chom.2013.09.004298Tracking Viral Genomes in Host Cells at Single-Molecule ResolutionWang, I.-Hsuan; Suomalainen, Maarit; Andriasyan, Vardan; Kilcher, Samuel; Mercer, Jason; Neef, Anne; Luedtke, Nathan W.; Greber, Urs F.Cell Host & Microbe (2013), 14 (4), 468-480CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Viral DNA trafficking in cells has large impacts on physiol. and disease development. Current methods lack the resoln. and accuracy to visualize and quantify viral DNA trafficking at single-mol. resoln. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresoln. microscopy and copper(I)-catalyzed azide-alkyne cycloaddn. (click) reactions allowed visualization of infection at single vDNA resoln. within mammalian cells. Anal. of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.
- 299Green, N. M. Avidin and Streptavidin. Methods Enzymol; Elsevier: 1990; Vol. 184, pp 51– 67.There is no corresponding record for this reference.
- 300Zhang, F.; Zheng, Z.; Liu, S. L.; Lu, W.; Zhang, Z.; Zhang, C.; Zhou, P.; Zhang, Y.; Long, G.; He, Z. Self-Biotinylation and Site-Specific Double Labeling of Baculovirus Using Quantum Dots for Single-Virus in-Situ Tracking. Biomaterials 2013, 34, 7506– 7518, DOI: 10.1016/j.biomaterials.2013.06.030300Self-biotinylation and site-specific double labeling of baculovirus using quantum dots for single-virus in-situ trackingZhang, Fuxian; Zheng, Zhenhua; Liu, Shu-Lin; Lu, Wen; Zhang, Zhenfeng; Zhang, Cuiling; Zhou, Peng; Zhang, Yuan; Long, Gang; He, Zhike; Pang, Dai-Wen; Hu, Qinxue; Wang, HanzhongBiomaterials (2013), 34 (30), 7506-7518CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Single-virus labeling and tracking represent a powerful tool to study virus-cell interactions. Using baculovirus as a model, here the authors developed a biochem. method for labeling both the viral envelope and the viral capsid of a virus. Viral envelope of the baculovirus AcMNPV was self-biotinylated and site-specifically conjugated with quantum dots (QDs) following one-step binding reaction, while the viral nucleocapsid was site-specifically labeled with green fluorescent protein (GFP) during viral replication. The established procedure of labeling did not affect viral infectivity, showing that the double-labeled virus retained functional structure and could be tracked for viral localization and movement in the host cells. The double-labeled virus also demonstrated the potential to be used for in-situ and real-time visualizing the internalization of a single viral particle into the host cells. Furthermore, the disassembly processes of the viral envelope and the viral nucleocapsid could be monitored for a long period of time (up to 2 h). Using the established method, several interaction details between the labeled baculoviruses and the host cells have been revealed. Given its advantages in high efficiency, high specificity, convenience and the maintenance of viral infectivity, the established approach provides a promising means for elucidating virus-cell interactions.
- 301Liu, J.; Xu, M.; Tang, B.; Hu, L.; Deng, F.; Wang, H.; Pang, D. W.; Hu, Z.; Wang, M.; Zhou, Y. Single-Particle Tracking Reveals the Sequential Entry Process of the Bunyavirus Severe Fever with Thrombocytopenia Syndrome Virus. Small 2019, 15, e1803788 DOI: 10.1002/smll.201803788There is no corresponding record for this reference.
- 302Liu, J.; Yu, C.; Gui, J. F.; Pang, D. W.; Zhang, Q. Y. Real-Time Dissecting the Entry and Intracellular Dynamics of Single Reovirus Particle. Front. Microbiol. 2018, 9, 2797, DOI: 10.3389/fmicb.2018.02797302Real-Time Dissecting the Entry and Intracellular Dynamics of Single Reovirus ParticleLiu Jia; Gui Jian-Fang; Zhang Qi-Ya; Yu Cong; Pang Dai-WenFrontiers in microbiology (2018), 9 (), 2797 ISSN:1664-302X.Reoviruses are non-enveloped viruses with wide host range, can cause serious infections in animals, plants and microorganism, e.g., aquareovirus, which is capable of causing serious haemorrhagic in aquatic animals. To date, the entry process of aquareovirus infection remains obscure. Real-time single-virus tracking are effective tools for exploring the details in viral infection process, which are crucial for understanding the pathogenic mechanism. Here, we used quantum dots-based single particle tracking technology combined with biochemical assays and ultrastructural observation to reveal unobservable infection steps and map dynamic interactions between a reovirus, Scophthalmus maximus reovirus (SMReV), and its host cell in real time. The results showed that the single membrane-bound reovirus particle can enter into the cell within several seconds through nascent clathrin-caoted pits, and most of the particles could internalize into cytoplasm within 30 min post-infection. The specific inhibitors analysis also showed that entry of SMREV depended on clathrin-mediated endocytosis rather than cavolin-mediated endocytosis. The motion analysis of internalized single particle indicated that the reovirus initially experienced slow and directed motion in the actin-enriched cell periphery, while it underwent relatively faster and directed movement toward the cell interior, suggesting that transport of SMReV was dependent on the cytoskeleton. Further, dual-labeling of virus and cytoskeleton and inhibitor analysis both demonstrated that transport of internalized SMReV was firstly dependent on actin filaments at the cell periphery, and then on microtubules toward the cell interior. Then visualization of SMReV trafficking in the endosomes revealed that the internalized reovirus particles were sorted from early endosomes to late endosomes, then part of them were delivered to lysosome. This study for the first time revealed the entry pathway, intracellular dynamic and the infection fate of fish reovirus in host cell in real time and in situ, which provided new insights into the infection mechanism of non-enveloped viruses.
- 303Dixit, S. K.; Goicochea, N. L.; Daniel, M. C.; Murali, A.; Bronstein, L.; De, M.; Stein, B.; Rotello, V. M.; Kao, C. C.; Dragnea, B. Quantum Dot Encapsulation in Viral Capsids. Nano Lett. 2006, 6, 1993– 1999, DOI: 10.1021/nl061165u303Quantum Dot Encapsulation in Viral CapsidsDixit, Suraj K.; Goicochea, Nancy L.; Daniel, Marie-Christine; Murali, Ayaluru; Bronstein, Lyudmila; De, Mrinmoy; Stein, Barry; Rotello, Vincent M.; Kao, C. Cheng; Dragnea, BogdanNano Letters (2006), 6 (9), 1993-1999CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Incorporation of CdSe/ZnS semiconductor quantum dots (QDs) into viral particles provides a new paradigm for the design of intracellular microscopic probes and vectors. Several strategies for the incorporation of QDs into viral capsids were explored; those functionalized with poly(ethylene glycol) (PEG) can be self-assembled into viral particles with minimal release of photoreaction products and enhanced stability against prolonged irradn.
- 304Huang, B. H.; Lin, Y.; Zhang, Z. L.; Zhuan, F.; Liu, A. A.; Xie, M.; Tian, Z. Q.; Zhang, Z.; Wang, H.; Pang, D. W. Surface Labeling of Enveloped Viruses Assisted by Host Cells. ACS Chem. Biol. 2012, 7, 683– 688, DOI: 10.1021/cb2001878304Surface Labeling of Enveloped Viruses Assisted by Host CellsHuang, Bi-Hai; Lin, Yi; Zhang, Zhi-Ling; Zhuan, Fangfang; Liu, An-An; Xie, Min; Tian, Zhi-Quan; Zhang, Zhenfeng; Wang, Hanzhong; Pang, Dai-WenACS Chemical Biology (2012), 7 (4), 683-688CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Labeling of virus opens new pathways for the understanding of viruses themselves and facilitates the utilization of viruses in modern biol., medicine, and materials. Based on the characteristic that viruses hijack their host cellular machineries to survive and reproduce themselves, a host-cell-assisted strategy is proposed to label enveloped viruses. By simply feeding Vero cells with com. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotin-Cap-PE), the authors obtained biotinylated Vero cells whose membrane systems were modified with biotin. Subsequently, pseudorabies viruses (PrV) were cultivated in the biotinylated Vero cells, and the PrV progenies were spontaneously labeled with Biotin-Cap-PE during viral natural assembly process. Since the viral natural assembly process was employed for the labeling, potential threats of genetic engineering and difficulties in keeping viral natural bioactivity were avoided. Importantly, this labeling strategy for enveloped virus greatly reduces the tech. complexity and allows researchers from different backgrounds to apply it for their specified demands.
- 305You, J. O.; Liu, Y. S.; Liu, Y. C.; Joo, K. I.; Peng, C. A. Incorporation of Quantum Dots on Virus in Polycationic Solution. Int. J. Nanomedicine 2006, 1, 59– 64, DOI: 10.2147/nano.2006.1.1.59305Incorporation of quantum dots on virus in polycationic solutionYou, Jin-Oh; Liu, Yu-San; Liu, Yu-Chuan; Joo, Kye-Il; Peng, Ching-AnInternational Journal of Nanomedicine (2006), 1 (1), 59-64CODEN: IJNNHQ; ISSN:1176-9114. (Dove Medical Press (NZ) Ltd.)Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biol. imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both neg. charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examg. the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 h, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.
- 306Chen, Y. H.; Wang, C. H.; Chang, C. W.; Peng, C. A. In Situ Formation of Viruses Tagged with Quantum Dots. Integr. Biol. 2010, 2, 258– 264, DOI: 10.1039/b926852a306In situ formation of viruses tagged with quantum dotsChen, Yu-Hao; Wang, Chung-Hao; Chang, Chia-Wei; Peng, Ching-AnIntegrative Biology (2010), 2 (5-6), 258-264CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)Quantum dots (QDs) have great potential for applications in bio-related fields, due to their high photoluminescence, photochem. stability and size-dependent emission. QDs used for the construction of QD-virus hybrids can be harnessed as an imaging probe to reveal viral infection pathways and screen antiviral agents. In the study, human embryonic kidney (HEK) 293T cells were transfected with three plasmids, pSIN-EGFP, pMDG, and p8.91, to produce lentiviruses which can make infected cells express enhanced green fluorescent protein (EGFP). The QDs employed were CdSe-ZnS semiconductor nanocrystals emitting red fluorescence. The QD-virus hybrids, constructed as lentiviruses, were budding from the membrane surface of HEK 293T producer cells on which QDs encapsulated with alkylated chitosan (Chitosan-QDs) were pre-adsorbed via electrostatic attraction force. Such in situ formation of QD-virus hybrids was confirmed by TEM micrographs indicating the lentivirus was capped with chitosan-modified QDs. To further illustrate the effectiveness (i.e., infectivity and photoluminescence) of the constructed QD-virus hybrids, NIH 3T3 cells were infected with the in situ fabricated QD-virus hybrids. Our results showed QDs were indeed entering NIH 3T3 cells along with lentiviruses as hybrids. Moreover, photoluminescence and infectivity of QD-virus hybrids remained intact, as compared to QDs and lentivirus alone. The unique approach of constructing QD-virus hybrids taking advantage of the viral budding process offers a feasible tool to create enveloped virus incorporated with nanomaterials for the study of fundamental and applied virol.
- 307Li, F.; Zhang, Z. P.; Peng, J.; Cui, Z. Q.; Pang, D. W.; Li, K.; Wei, H. P.; Zhou, Y. F.; Wen, J. K.; Zhang, X. E. Imaging Viral Behavior in Mammalian Cells with Self-Assembled Capsid-Quantum-Dot Hybrid Particles. Small 2009, 5, 718– 726, DOI: 10.1002/smll.200801303307Imaging viral behavior in mammalian cells with self-assembled capsid-quantum-dot hybrid particlesLi, Feng; Zhang, Zhi-Ping; Peng, Jun; Cui, Zong-Qiang; Pang, Dai-Wen; Li, Ke; Wei, Hong-Ping; Zhou, Ya-Feng; Wen, Ji-Kai; Zhang, Xian-EnSmall (2009), 5 (6), 718-726CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long-term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD-contg. virus-like particles (VLPs) of simian virus 40 (SV40), termed SVLP-QDs, are constructed by in vitro self-assembly of the major capsid protein of SV40. SVLP-QDs show homogeneity in size (≈ 24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP-QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.
- 308Johnson, H. E.; Haugh, J. M. Quantitative Analysis of Phosphoinositide 3-Kinase (PI3K) Signaling Using Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr. Protoc. Cell Biol. 2013, 61, 14.14.1– 14.14.24, DOI: 10.1002/0471143030.cb1414s61There is no corresponding record for this reference.
- 309Chen, I.; Ting, A. Y. Site-Specific Labeling of Proteins with Small Molecules in Live Cells. Curr. Opin. Biotechnol. 2005, 16, 35– 40, DOI: 10.1016/j.copbio.2004.12.003309Site-specific labeling of proteins with small molecules in live cellsChen, Irwin; Ting, Alice Y.Current Opinion in Biotechnology (2005), 16 (1), 35-40CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)A review. The principal bottleneck for the utilization of small-mol. probes in live cells is the shortage of methodologies for targeting them with very high specificity to biol. mols. or compartments of interest. Recently developed methods for labeling proteins with small-mol. probes in cells employ special protein or peptide handles that recruit small-mol. ligands, harness enzymes to catalyze small-mol. conjugation or hijack the cell's protein translation machinery.
- 310Gong, Y. K.; Pan, L. F. Recent Advances in Bioorthogonal Reactions for Site-Specific Protein Labeling and Engineering. Tetrahedron Lett. 2015, 56, 2123– 2132, DOI: 10.1016/j.tetlet.2015.03.065310Recent advances in bioorthogonal reactions for site-specific protein labeling and engineeringGong, Yukang; Pan, LifengTetrahedron Letters (2015), 56 (17), 2123-2132CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)A review. In the past two decades, with the rapid development of chem. biol., tremendous small-mol. based toolkits were created by org. chemists, and were widely used to study and manipulate proteins to dissect their complicated biol. functions. This review summarizes some recent progresses of bioorthogonal reactions for site-specific protein labeling and engineering, and highlights the powers of using these methods to study the biol. functions of some proteins.
- 311Zhang, G.; Zheng, S. Q.; Liu, H. P.; Chen, P. R. Illuminating Biological Processes through Site-Specific Protein Labeling. Chem. Soc. Rev. 2015, 44, 3405– 3417, DOI: 10.1039/C4CS00393D311Illuminating biological processes through site-specific protein labelingZhang, Gong; Zheng, Siqi; Liu, Haiping; Chen, Peng R.Chemical Society Reviews (2015), 44 (11), 3405-3417CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biol. setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biol. problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biol. processes.
- 312Jing, C.; Cornish, V. W. Chemical Tags for Labeling Proteins inside Living Cells. Acc. Chem. Res. 2011, 44, 784– 792, DOI: 10.1021/ar200099f312Chemical Tags for Labeling Proteins Inside Living CellsJing, Chaoran; Cornish, Virginia W.Accounts of Chemical Research (2011), 44 (9), 784-792CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, the challenge of understanding how macromol. machines, signaling pathways, and other biol. networks operate in the complex environment of the living cell must now be faced. The fluorescent proteins (FPs) revolutionized the ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biol., they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chem. methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chem. tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chem. tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an org. fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chem. tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, the authors focus on the development and application of chem. tags for studying protein function within living cells. Thus, in the authors' overview of different chem. tagging strategies and technologies, the authors emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. The authors highlight recent applications in which the chem. tags have enabled sophisticated biophys. measurements that would be difficult or even impossible with FPs. Finally, the authors conclude by looking forward to (i) the development of high-photon-output chem. tags compatible with living cells to enable high-resoln. imaging, (ii) the realization of the potential of the chem. tags to significantly reduce tag size, and (iii) the exploitation of the modular chem. tag label to go beyond fluorescent imaging.
- 313Klein, T.; Loschberger, A.; Proppert, S.; Wolter, S.; van de Linde, S.; Sauer, M. Live-Cell dSTORM with SNAP-Tag Fusion Proteins. Nat. Methods 2011, 8, 7– 9, DOI: 10.1038/nmeth0111-7b313Live-cell dSTORM with SNAP-tag fusion proteinsKlein, Teresa; Loeschberger, Anna; Proppert, Sven; Wolter, Steve; van de Linde, Sebastian; Sauer, MarkusNature Methods (2011), 8 (1), 7-9CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)In the Sept. 2010 issue of Nature Methods, live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins was demonstrated using a trimethoprim chem. tag (TMP tag) for genetic encoding with photo stable std. fluorophores. In line with this, this study reported that live-cell dSTORM of core histone H2B proteins in different eukaryotic cell lines is possible using com. available SNAP tags as well. Protein-specific labeling was based on the irreversible reaction of human O6-alkylguanine-DNA alkyltransferase with the rhodamine O6-benzylguanine derivs. SNAP-Cell 505 (rhodamine green) and SNAP-Cell TMR-Star (tetramethylrhodamine) (New England BioLabs). Data generally suggest that dSTORM used in combination with std. chem. tags and conventional synthetic org. fluorophores is a simple method for live-cell super-resoln. imaging with high spatiotemporal resoln. that can be advantageously combined with photoactivatable fluorescent proteins for multicolor applications. Notably, the highly reversible and reliable photoswitching process of rhodamine and oxazine fluorophores in the cellular environment enables reversible photoswitching of most com. org. fluorophores in living cells without any additives.
- 314Chen, Z.; Cornish, V. W.; Min, W. Chemical Tags: Inspiration for Advanced Imaging Techniques. Curr. Opin. Chem. Biol. 2013, 17, 637– 643, DOI: 10.1016/j.cbpa.2013.05.018314Chemical tags: inspiration for advanced imaging techniquesChen, Zhixing; Cornish, Virginia W.; Min, WeiCurrent Opinion in Chemical Biology (2013), 17 (4), 637-643CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)This review summarizes recent applications of chem. tags in conjunction with advanced bio-imaging techniques including single-mol. fluorescence, spatiotemporally resolved ensemble microscopy techniques, and imaging modalities beyond fluorescence. The authors aim to illustrate the unique advantages of chem. tags in facilitating contemporary microscopy to address biol. problems that are difficult or near impossible to approach otherwise. The authors hope the authors' review will inspire more innovative applications enabled by the mingling of these two growing fields.
- 315Wombacher, R.; Cornish, V. W. Chemical Tags: Applications in Live Cell Fluorescence Imaging. J. Biophotonics 2011, 4, 391– 402, DOI: 10.1002/jbio.201100018315Chemical tags: Applications in live cell fluorescence imagingWombacher, Richard; Cornish, Virginia W.Journal of Biophotonics (2011), 4 (6), 391-402CODEN: JBOIBX; ISSN:1864-063X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biol. processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chem. biol. techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chem. tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected expts. in which chem. tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
- 316Specht, E. A.; Braselmann, E.; Palmer, A. E. A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging. Annu. Rev. Physiol. 2017, 79, 93– 117, DOI: 10.1146/annurev-physiol-022516-034055316A Critical and Comparative Review of Fluorescent Tools for Live-Cell ImagingSpecht, Elizabeth A.; Braselmann, Esther; Palmer, Amy E.Annual Review of Physiology (2017), 79 (), 93-117CODEN: ARPHAD; ISSN:0066-4278. (Annual Reviews)Fluorescent tools have revolutionized our ability to probe biol. dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-mol. dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small mols., and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell anal. and esp. live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.
- 317Eckhardt, M.; Anders, M.; Muranyi, W.; Heilemann, M.; Krijnse-Locker, J.; Muller, B. A SNAP-Tagged Derivative of HIV-1--A Versatile Tool to Study Virus-Cell Interactions. PLoS One 2011, 6, e22007 DOI: 10.1371/journal.pone.0022007317A SNAP-tagged derivative of HIV-1 - a versatile tool to study virus-cell interactionsEckhardt, Manon; Anders, Maria; Muranyi, Walter; Heilemann, Mike; Krijnse-Locker, Jacomine; Mueller, BarbaraPLoS One (2011), 6 (7), e22007CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Fluorescently labeled human immunodeficiency virus (HIV) derivs., combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochem. properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate mols. in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV deriv. HIVSNAP, which carries the SNAP-tag as an addnl. domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the anal. of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.
- 318Hanne, J.; Gottfert, F.; Schimer, J.; Anders-Osswein, M.; Konvalinka, J.; Engelhardt, J.; Muller, B.; Hell, S. W.; Krausslich, H. G. Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation. ACS Nano 2016, 10, 8215– 8222, DOI: 10.1021/acsnano.6b03850318Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic MaturationHanne, Janina; Goettfert, Fabian; Schimer, Jiri; Anders-Oesswein, Maria; Konvalinka, Jan; Engelhardt, Johann; Mueller, Barbara; Hell, Stefan W.; Kraeusslich, Hans-GeorgACS Nano (2016), 10 (9), 8215-8222CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomog. provided structures of immature and mature HIV-1 with a diam. of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, the authors employed super-resoln. STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resoln. microscopy. This study shows the rearrangement of subviral structures in a super-resoln. light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biol. process by an optical switch.
- 319Sun, X.; Zhang, A.; Baker, B.; Sun, L.; Howard, A.; Buswell, J.; Maurel, D.; Masharina, A.; Johnsson, K.; Noren, C. J. Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging. ChemBioChem 2011, 12, 2217– 2226, DOI: 10.1002/cbic.201100173319Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence ImagingSun, Xiaoli; Zhang, Aihua; Baker, Brenda; Sun, Luo; Howard, Angela; Buswell, John; Maurel, Damien; Masharina, Anastasiya; Johnsson, Kai; Noren, Christopher J.; Xu, Ming-Qun; Correa, Ivan R.ChemBioChem (2011), 12 (14), 2217-2226CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The ability to specifically attach chem. probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of exptl. settings. However, a potential drawback of detection using chem. probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report the authors present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivs. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. The authors describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addn., the authors have characterized a fast-labeling variant of SNAP-tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
- 320Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Labeling of Fusion Proteins with Synthetic Fluorophores in Live Cells. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9955– 9959, DOI: 10.1073/pnas.0401923101320Labeling of fusion proteins with synthetic fluorophores in live cellsKeppler, Antje; Pick, Horst; Arrivoli, Claudio; Vogel, Horst; Johnsson, KaiProceedings of the National Academy of Sciences of the United States of America (2004), 101 (27), 9955-9959CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A general approach for the sequential labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor anal. of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.
- 321Lavis, L. D. Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines. Annu. Rev. Biochem. 2017, 86, 825– 843, DOI: 10.1146/annurev-biochem-061516-044839321Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and RhodaminesLavis, Luke D.Annual Review of Biochemistry (2017), 86 (), 825-843CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)Small-mol. fluorophores, such as fluorescein and rhodamine derivs., are crit. tools in modern biochem. and biol. research. The field of chem. dyes is old; colored mols. were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochem. and biol. The application of modern chem., biochem., mol. genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This crit. review focuses on an important example of chem. biol.-the melding of old and new chem. knowledge-leading to useful mols. for advanced biochem. and biol. expts.
- 322Ross-Thriepland, D.; Mankouri, J.; Harris, M. Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes. J. Virol. 2015, 89, 3123– 3135, DOI: 10.1128/JVI.02995-14322Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexesRoss-Thriepland, Douglas; Mankouri, Jamel; Harris, MarkJournal of Virology (2015), 89 (6), 3123-3135CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold redn. in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.
- 323Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.; Kindermann, M.; Beaufils, F.; Johnsson, K. An Engineered Protein Tag for Multiprotein Labeling in Living Cells. Chem. Biol. 2008, 15, 128– 136, DOI: 10.1016/j.chembiol.2008.01.007323An Engineered Protein Tag for Multiprotein Labeling in Living CellsGautier, Arnaud; Juillerat, Alexandre; Heinis, Christian; Correa, Ivan Reis; Kindermann, Maik; Beaufils, Florent; Johnsson, KaiChemistry & Biology (Cambridge, MA, United States) (2008), 15 (2), 128-136CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Summary: The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivs. bearing a chem. probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivs. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different mol. probes in living cells. We furthermore show simultaneous pulse-chase expts. to visualize different generations of two different proteins in one sample.
- 324Liu, A. A.; Zhang, Z.; Sun, E. Z.; Zheng, Z.; Zhang, Z. L.; Hu, Q.; Wang, H.; Pang, D. W. Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy. ACS Nano 2016, 10, 1147– 1155, DOI: 10.1021/acsnano.5b06438324Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific Halo Tag Labeling StrategyLiu, An-An; Zhang, Zhenfeng; Sun, En-Ze; Zheng, Zhenhua; Zhang, Zhi-Ling; Hu, Qinxue; Wang, Hanzhong; Pang, Dai-WenACS Nano (2016), 10 (1), 1147-1155CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Real-time, long-term, single-particle tracking (SPR) provides an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, the authors established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technol. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT expts. without further modification. Thus, virus infectivity, which is crit. for the visualization and anal. of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.
- 325Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3, 373– 382, DOI: 10.1021/cb800025k325HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein AnalysisLos, Georgyi V.; Encell, Lance P.; McDougall, Mark G.; Hartzell, Danette D.; Karassina, Natasha; Zimprich, Chad; Wood, Monika G.; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.ACS Chemical Biology (2008), 3 (6), 373-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in soln., in living cells, or in chem. fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful mols., such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiol. conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple mol. processes assocd. with NF-κB-mediated cellular physiol., including imaging of subcellular protein translocation and capture of protein-protein and protein-DNA complexes.
- 326Chen, Z.; Jing, C.; Gallagher, S. S.; Sheetz, M. P.; Cornish, V. W. Second-Generation Covalent TMP-Tag for Live Cell Imaging. J. Am. Chem. Soc. 2012, 134, 13692– 13699, DOI: 10.1021/ja303374p326Second-Generation Covalent TMP-Tag for Live Cell ImagingChen, Zhixing; Jing, Chaoran; Gallagher, Sarah S.; Sheetz, Michael P.; Cornish, Virginia W.Journal of the American Chemical Society (2012), 134 (33), 13692-13699CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with org. fluorophores that have improved brightness and other specialized properties. Recently, the authors successfully rendered the their TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here the authors report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, the authors designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chem. synthesis for routine prodn. of a variety of A-TMP-probe v2.0 labels. The authors then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, the authors demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited no. of chem. tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and org. chem. as tools not only for chem. tag engineering but also more broadly for synthetic biol.
- 327Gallagher, S. S.; Jing, C.; Peterka, D. S.; Konate, M.; Wombacher, R.; Kaufman, L. J.; Yuste, R.; Cornish, V. W. A Trimethoprim-Based Chemical Tag for Live Cell Two-Photon Imaging. ChemBioChem 2010, 11, 782– 784, DOI: 10.1002/cbic.200900731327A Trimethoprim-Based Chemical Tag for Live Cell Two-Photon ImagingGallagher, Sarah S.; Jing, Chaoran; Peterka, Darcy S.; Konate, Mariam; Wombacher, Richard; Kaufman, Laura J.; Yuste, Rafael; Cornish, Virginia W.ChemBioChem (2010), 11 (6), 782-784CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)TMP-BC575 is a viable tool for imaging proteins in live cells by using two-photon microscopy. This two-photon fluorophore expands the TMP-tag tool kit, adding to the value of this modular-labeling technol. A protein of interest can be tagged with Escherichia coli dihydrofolate reductase (eDHFR), and different labels can then be swapped in, allowing the protein to be readily analyzed by multiple techniques. While cell permeability and lipid partitioning appear to be tag dependent, these expts. suggest BC575 might also be compatible with other chem. tags. Given its broad excitation maxima and distinct emission wavelength, TMP-BC575 offers an alternative to other fluorophores for multicolor two-photon imaging with enhanced green fluorescent protein (EGFP).
- 328Wombacher, R.; Heidbreder, M.; van de Linde, S.; Sheetz, M. P.; Heilemann, M.; Cornish, V. W.; Sauer, M. Live-Cell Super-Resolution Imaging with Trimethoprim Conjugates. Nat. Methods 2010, 7, 717– 719, DOI: 10.1038/nmeth.1489328Live-cell super-resolution imaging with trimethoprim conjugatesWombacher, Richard; Heidbreder, Meike; van de Linde, Sebastian; Sheetz, Michael P.; Heilemann, Mike; Cornish, Virginia W.; Sauer, MarkusNature Methods (2010), 7 (9), 717-719CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The spatiotemporal resoln. of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chem. tag that allows proteins to be labeled with an org. fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at ∼ 20 nm resoln.
- 329Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W. In Vivo Protein Labeling with Trimethoprim Conjugates: A Flexible Chemical Tag. Nat. Methods 2005, 2, 255– 257, DOI: 10.1038/nmeth749329In vivo protein labeling with trimethoprim conjugates: a flexible chemical tagMiller, Lawrence W.; Cai, Yunfei; Sheetz, Michael P.; Cornish, Virginia W.Nature Methods (2005), 2 (4), 255-257CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The introduction of green fluorescent protein and its variants (GFPs) has allowed protein anal. at the level of the cell. Now, chem. methods are needed to label proteins in vivo with a wider variety of functionalities so that mechanistic questions about protein function in the complex cellular environment can be addressed. Here we demonstrate that trimethoprim derivs. can be used to selectively tag Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins in wild-type mammalian cells with minimal background and fast kinetics.
- 330Rudner, L.; Nydegger, S.; Coren, L. V.; Nagashima, K.; Thali, M.; Ott, D. E. Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling. J. Virol. 2005, 79, 4055– 4065, DOI: 10.1128/JVI.79.7.4055-4065.2005330Dynamic fluorescent imaging of Human immunodeficiency virus type 1 Gag in live cells by biarsenical labelingRudner, Lynnie; Nydegger, Sascha; Coren, Lori V.; Nagashima, Kunio; Thali, Markus; Ott, David E.Journal of Virology (2005), 79 (7), 4055-4065CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Human immunodeficiency virus type 1 (HIV-1) Gag is the primary structural protein of the virus and is sufficient for particle formation. We utilized the recently developed biarsenical-labeling method to dynamically observe HIV-1 Gag within live cells by adding a tetracysteine tag (C-C-P-G-C-C) to the C terminus of Gag in both Pr55Gag expression and full-length proviral constructs. Membrane-permeable biarsenical compds. FlAsH and ReAsH covalently bond to this tetracysteine sequence and specifically fluoresce, effectively labeling Gag in the cell. Biarsenical labeling readily and specifically detected a tetracysteine-tagged HIV-1 Gag protein (Gag-TC) in HeLa, Mel JuSo, and Jurkat T cells by deconvolution fluorescence microscopy. Gag-TC was localized primarily at or near the plasma membrane in all cell types examd. Fluorescent two-color anal. of Gag-TC in HeLa cells revealed that nascent Gag was present mostly at the plasma membrane in distinct regions. Intracellular imaging of a Gag-TC myristoylation mutant obsd. a diffuse signal throughout the cell, consistent with the role of myristoylation in Gag localization to the plasma membrane. In contrast, mutation of the L-domain core sequence did not appreciably alter the localization of Gag, suggesting that the PTAP L domain functions at the site of budding rather than as a targeting signal. Taken together, our results show that Gag concs. in specific plasma membrane areas rapidly after translation and demonstrate the utility of biarsenical labeling for visualizing the dynamic localization of Gag.
- 331Das, S. C.; Panda, D.; Nayak, D.; Pattnaik, A. K. Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells. J. Virol. 2009, 83, 2611– 2622, DOI: 10.1128/JVI.01668-08331Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged M protein allows dynamic imaging of M protein and virus uncoating in infected cellsDas, Subash C.; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K.Journal of Virology (2009), 83 (6), 2611-2622CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addn. to wild-type (wt.) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt. VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent assocn. of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 h. Using dually fluorescent VSV, we detd. that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approx. 28 min.
- 332Li, Y.; Lu, X.; Li, J.; Berube, N.; Giest, K. L.; Liu, Q.; Anderson, D. H.; Zhou, Y. Genetically Engineered, Biarsenically Labeled Influenza Virus Allows Visualization of Viral NS1 Protein in Living Cells. J. Virol. 2010, 84, 7204– 7213, DOI: 10.1128/JVI.00203-10332Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cellsLi, Yang; Lu, Xinya; Li, Junwei; Berube, Nathalie; Giest, Kerri-Lane; Liu, Qiang; Anderson, Deborah H.; Zhou, YanJournal of Virology (2010), 84 (14), 7204-7213CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.
- 333Martin, B. R.; Giepmans, B. N.; Adams, S. R.; Tsien, R. Y. Mammalian Cell-Based Optimization of the Biarsenical-Binding Tetracysteine Motif for Improved Fluorescence and Affinity. Nat. Biotechnol. 2005, 23, 1308– 1314, DOI: 10.1038/nbt1136333Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinityMartin, Brent R.; Giepmans, Ben N. G.; Adams, Stephen R.; Tsien, Roger Y.Nature Biotechnology (2005), 23 (10), 1308-1314CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dil. proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concns. of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.
- 334Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications. J. Am. Chem. Soc. 2002, 124, 6063– 6076, DOI: 10.1021/ja017687n334New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applicationsAdams, Stephen R.; Campbell, Robert E.; Gross, Larry A.; Martin, Brent R.; Walkup, Grant K.; Yao, Yong; Llopis, Juan; Tsien, Roger Y.Journal of the American Chemical Society (2002), 124 (21), 6063-6076CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein deriv. with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissocn. consts. (∼10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed α-helix. Many analogs of FlAsH have been synthesized, including ReAsH, a resorufin deriv. excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatog., fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
- 335Zheng, L. L.; Li, C. M.; Zhen, S. J.; Li, Y. F.; Huang, C. Z. His-Tag Based in Situ Labelling of Progeny Viruses for Real-Time Single Virus Tracking in Living Cells. Nanoscale 2016, 8, 18635– 18639, DOI: 10.1039/C6NR05806J335His-tag based in situ labelling of progeny viruses for real-time single virus tracking in living cellsZheng, Lin Ling; Li, Chun Mei; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng ZhiNanoscale (2016), 8 (44), 18635-18639CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)Tracking virus infection events in live cells is useful for understanding the mechanism of virus infection, and fluorescent labeling is a crit. step. Herein a noninvasive strategy for labeling viruses with His-tags was developed by in situ modifying the cell surface proteins with polypeptides contg. His-tags during progeny virus assembly. The His-tagged viruses were further conjugated with Ni2+-nitrilotriacetate complex modified quantum dots, and retained their infectivity for real-time single virus tracking in living cells.
- 336Guignet, E. G.; Hovius, R.; Vogel, H. Reversible Site-Selective Labeling of Membrane Proteins in Live Cells. Nat. Biotechnol. 2004, 22, 440– 444, DOI: 10.1038/nbt954336Reversible site-selective labeling of membrane proteins in live cellsGuignet, Emmanuel G.; Hovius, Ruud; Vogel, HorstNature Biotechnology (2004), 22 (4), 440-444CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Chem. and biol. labeling is fundamental for the elucidation of the function of proteins within biochem. cellular networks. In particular, fluorescent probes allow detection of mol. interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resoln. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small mol. probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each contg. a polyhistidine sequence. We investigated the ionotropic 5HT3 serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.
- 337Chen, I.; Howarth, M.; Lin, W.; Ting, A. Y. Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes Using Biotin Ligase. Nat. Methods 2005, 2, 99– 104, DOI: 10.1038/nmeth735337Site-specific labeling of cell surface proteins with biophysical probes using biotin ligaseChen, Irwin; Howarth, Mark; Lin, Weiying; Ting, Alice Y.Nature Methods (2005), 2 (2), 99-104CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophys. probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized mols. We demonstrate this two-stage protein labeling methodol. on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.
- 338Howarth, M.; Ting, A. Y. Imaging Proteins in Live Mammalian Cells with Biotin Ligase and Monovalent Streptavidin. Nat. Protoc. 2008, 3, 534– 545, DOI: 10.1038/nprot.2008.20338Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidinHowarth, Mark; Ting, Alice Y.Nature Protocols (2008), 3 (3), 534-545CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a simple and efficient way to label specific cell surface proteins with biophys. probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the crosslinking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.
- 339Uttamapinant, C.; White, K. A.; Baruah, H.; Thompson, S.; Fernandez-Suarez, M.; Puthenveetil, S.; Ting, A. Y. A Fluorophore Ligase for Site-Specific Protein Labeling inside Living Cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 10914– 10919, DOI: 10.1073/pnas.0914067107339A fluorophore ligase for site-specific protein labeling inside living cellsUttamapinant, Chayasith; White, Katharine A.; Baruah, Hemanta; Thompson, Samuel; Fernandez-Subrez, Marta; Puthenveetil, Sujiet; Ting, Alice Y.Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (24), 10914-10919, S10914/1-S10914/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Biol. microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LpIA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LpIA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.
- 340Fernandez-Suarez, M.; Baruah, H.; Martinez-Hernandez, L.; Xie, K. T.; Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Redirecting Lipoic Acid Ligase for Cell Surface Protein Labeling with Small-Molecule Probes. Nat. Biotechnol. 2007, 25, 1483– 1487, DOI: 10.1038/nbt1355340Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probesFernandez-Suarez, Marta; Baruah, Hemanta; Martinez-Hernandez, Laura; Xie, Kathleen T.; Baskin, Jeremy M.; Bertozzi, Carolyn R.; Ting, Alice Y.Nature Biotechnology (2007), 25 (12), 1483-1487CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissoc. from proteins after internalization. Here, we report technol. for covalent, specific tagging of cellular proteins with chem. probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodol. should provide general access to biochem. and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.
- 341Schumacher, D.; Helma, J.; Mann, F. A.; Pichler, G.; Natale, F.; Krause, E.; Cardoso, M. C.; Hackenberger, C. P.; Leonhardt, H. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase. Angew. Chem., Int. Ed. 2015, 54, 13787– 13791, DOI: 10.1002/anie.201505456341Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine LigaseSchumacher, Dominik; Helma, Jonas; Mann, Florian A.; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M. Cristina; Hackenberger, Christian P. R.; Leonhardt, HeinrichAngewandte Chemie, International Edition (2015), 54 (46), 13787-13791CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A novel chemoenzymic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivs. as small bioorthogonal handles to proteins contg. a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochem., cell biol., and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.
- 342Schumacher, D.; Lemke, O.; Helma, J.; Gerszonowicz, L.; Waller, V.; Stoschek, T.; Durkin, P. M.; Budisa, N.; Leonhardt, H.; Keller, B. G. Broad Substrate Tolerance of Tubulin Tyrosine Ligase Enables One-Step Site-Specific Enzymatic Protein Labeling. Chem. Sci. 2017, 8, 3471– 3478, DOI: 10.1039/C7SC00574A342Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labelingSchumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M.; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G.; Hackenberger, Christian P. R.Chemical Science (2017), 8 (5), 3471-3478CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking expts. and all-atom mol. dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.
- 343Zhou, Z.; Cironi, P.; Lin, A. J.; Xu, Y.; Hrvatin, S.; Golan, D. E.; Silver, P. A.; Walsh, C. T.; Yin, J. Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases. ACS Chem. Biol. 2007, 2, 337– 346, DOI: 10.1021/cb700054k343Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl TransferasesZhou, Zhe; Cironi, Pablo; Lin, Alison J.; Xu, Yangqing; Hrvatin, Sinisa; Golan, David E.; Silver, Pamela A.; Walsh, Christopher T.; Yin, JunACS Chemical Biology (2007), 2 (5), 337-346CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), resp. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small mols. of diverse structures to the target proteins with high efficiency and specificity.
- 344Yin, J.; Straight, P. D.; McLoughlin, S. M.; Zhou, Z.; Lin, A. J.; Golan, D. E.; Kelleher, N. L.; Kolter, R.; Walsh, C. T. Genetically Encoded Short Peptide Tag for Versatile Protein Labeling by Sfp Phosphopantetheinyl Transferase. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 15815– 15820, DOI: 10.1073/pnas.0507705102344Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferaseYin, Jun; Straight, Paul D.; McLoughlin, Shaun M.; Zhou, Zhe; Lin, Alison J.; Golan, David E.; Kelleher, Neil L.; Kolter, Roberto; Walsh, Christopher T.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (44), 15815-15820CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)An 11-residue peptide with the sequence DSLEFIASKLA was identified from a genomic library of Bacillus subtilis by phage display as an efficient substrate for Sfp phosphopantetheinyltransferase-catalyzed protein labeling by small mol.-CoA conjugates. The authors name this peptide the "ybbR tag," because part of its sequence is derived from the ybbR ORF in the B. subtilis genome. The site of Sfp-catalyzed ybbR tag labeling was mapped to the first Ser residue, and the ybbR tag was found to have a strong tendency for adopting an α-helical conformation in soln. Here the authors demonstrate that the ybbR tag can be fused to the N or C termini of target proteins or inserted in a flexible loop in the middle of a target protein for site-specific protein labeling by Sfp. The short size of the ybbR tag and its compatibility with various target proteins, the broad substrate specificity of Sfp for labeling the ybbR tag with small-mol. probes of diverse structures, and the high specificity and efficiency of the labeling reaction make Sfp-catalyzed ybbR tag labeling an attractive tool for expanding protein structural and functional diversities by posttranslational modification.
- 345Crivat, G.; Taraska, J. W. Imaging Proteins inside Cells with Fluorescent Tags. Trends Biotechnol. 2012, 30, 8– 16, DOI: 10.1016/j.tibtech.2011.08.002345Imaging proteins inside cells with fluorescent tagsCrivat, Georgeta; Taraska, Justin W.Trends in Biotechnology (2012), 30 (1), 8-16CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. Watching biol. mols. provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four std. genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.
- 346Hoehnel, S.; Lutolf, M. P. Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag Technology. Bioconjugate Chem. 2015, 26, 1678– 1686, DOI: 10.1021/acs.bioconjchem.5b00268346Capturing Cell-Cell Interactions via SNAP-tag and CLIP-tag TechnologyHoehnel, S.; Lutolf, M. P.Bioconjugate Chemistry (2015), 26 (8), 1678-1686CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Juxtacrine or contact-dependent signaling is a major form of cell communication in multicellular organisms. The involved cell-cell and cell-extracellular-matrix (ECM) interactions are crucial for the organization and maintenance of tissue architecture and function. However, because cell-cell contacts are relatively weak, it is difficult to isolate interacting cells in their native state to study, for example, how specific cell types interact with others (e.g., stem cells with niche cells) or where they locate within tissues to execute specific tasks. To achieve this, the authors propose artificial in situ cell-to-cell linking systems that are based on SNAP-tag and CLIP-tag, engineered mutants of the human O6-alkylguanine-DNA alkyltransferase. SNAP-tag can be used to efficiently and covalently tether cells to poly(ethylene glycol) (PEG)-based hydrogel surfaces that have been functionalized with the SNAP-tag substrate benzylguanine (BG). Furthermore, using PEG-based spherical microgels as an artificial cell model, the authors provide proof-of-principle for inducing clustering that mimics cell-cell pairing.
- 347Liss, V.; Barlag, B.; Nietschke, M.; Hensel, M. Self-Labelling Enzymes as Universal Tags for Fluorescence Microscopy, Super-Resolution Microscopy and Rectron Microscopy. Sci. Rep. 2016, 5, 17740, DOI: 10.1038/srep17740There is no corresponding record for this reference.
- 348Jing, C.; Cornish, V. W. A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell Imaging. ACS Chem. Biol. 2013, 8, 1704– 1712, DOI: 10.1021/cb300657r348A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell ImagingJing, Chaoran; Cornish, Virginia W.ACS Chemical Biology (2013), 8 (8), 1704-1712CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Developed to complement the use of fluorescent proteins in live cell imaging, chem. tags enjoy the benefit of modular incorporation of org. fluorophores, opening the possibility of high photon output and special photophys. properties. However, the theor. challenge in using chem. tags as opposed to fluorescent proteins for high-resoln. imaging is background noise from unbound and/or nonspecifically bound ligand-fluorophore. The authors envisioned the authors could overcome this limit by engineering fluorogenic trimethoprim-based chem. tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR)-tagged protein displaces the quencher. Thus, the authors began by building a nonfluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here the authors take the final step and render the covalent TMP-tag fluorogenic. In brief, the authors designed a trimeric TMP-fluorophore-quencher mol. (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. The authors present the in vitro expts. showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only the authors' initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins but also less abundant, more dynamic cytoplasmic proteins. These results suggest that the fluorogenic TMP-tag can significantly impact high-resoln. live cell imaging and further establish the potential of proximity-induced reactivity and org. chem. more broadly as part of the growing toolbox for synthetic biol. and cell engineering.
- 349Lai, Y. T.; Chang, Y. Y.; Hu, L.; Yang, Y.; Chao, A.; Du, Z. Y.; Tanner, J. A.; Chye, M. L.; Qian, C.; Ng, K. M. Rapid Labeling of Intracellular His-Tagged Proteins in Living Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 2948– 2953, DOI: 10.1073/pnas.1419598112349Rapid labeling of intracellular His-tagged proteins in living cellsLai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A.; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, HongzheProceedings of the National Academy of Sciences of the United States of America (2015), 112 (10), 2948-2953CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Small mol.-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purifn., there is significant interest in fluorescent Ni2+-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.
- 350Stephanopoulos, N.; Francis, M. B. Choosing an Effective Protein Bioconjugation Strategy. Nat. Chem. Biol. 2011, 7, 876– 884, DOI: 10.1038/nchembio.720350Choosing an effective protein bioconjugation strategyStephanopoulos, Nicholas; Francis, Matthew B.Nature Chemical Biology (2011), 7 (12), 876-884CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. The collection of chem. techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biol. understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate prodn., and a no. of techniques can be used to label a single protein in a complex biol. mixt. This review examines the way in which exptl. circumstances influence one's selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.
- 351Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7583– 7588, DOI: 10.1073/pnas.0503125102351Targeting quantum dots to surface proteins in living cells with biotin ligaseHowarth, Mark; Takao, Keizo; Hayashi, Yasunori; Ting, Alice Y.Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (21), 7583-7588CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.
- 352Cohen, J. D.; Zou, P.; Ting, A. Y. Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone Formation. ChemBioChem 2012, 13, 888– 894, DOI: 10.1002/cbic.201100764352Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone FormationCohen, Justin D.; Zou, Peng; Ting, Alice Y.ChemBioChem (2012), 13 (6), 888-894CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for Alexa Fluor 568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, the authors labeled the low-d. lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-mol. imaging and tracking over time.
- 353Lotze, J.; Reinhardt, U.; Seitz, O.; Beck-Sickinger, A. G. Peptide-Tags for Site-Specific Protein Labelling in Vitro and in Vivo. Mol. BioSyst. 2016, 12, 1731– 1745, DOI: 10.1039/C6MB00023A353Peptide-tags for site-specific protein labelling in vitro and in vivoLotze, Jonathan; Reinhardt, Ulrike; Seitz, Oliver; Beck-Sickinger, Annette G.Molecular BioSystems (2016), 12 (6), 1731-1745CODEN: MBOIBW; ISSN:1742-2051. (Royal Society of Chemistry)Peptide-tag based labeling can be achieved by (i) enzymes (ii) recognition of metal ions or small mols. and (iii) peptide-peptide interactions and enables site-specific protein visualization to investigate protein localization and trafficking.
- 354Sunbul, M.; Yen, M.; Zou, Y.; Yin, J. Enzyme Catalyzed Site-Specific Protein Labeling and Cell Imaging with Quantum Dots. Chem. Commun. 2008, 5927– 5929, DOI: 10.1039/b812162a354Enzyme catalyzed site-specific protein labeling and cell imaging with quantum dotsSunbul, Murat; Yen, Michelle; Zou, Yekui; Yin, JunChemical Communications (Cambridge, United Kingdom) (2008), (45), 5927-5929CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)We have developed an efficient method for one-step covalent labeling of cell surface proteins with quantum dots based on enzyme catalyzed site-specific modification of short peptide tags.
- 355Zhang, Y.; Ke, X.; Zheng, Z.; Zhang, C.; Zhang, Z.; Zhang, F.; Hu, Q.; He, Z.; Wang, H. Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus Infection. ACS Nano 2013, 7, 3896– 3904, DOI: 10.1021/nn305189n355Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus InfectionZhang, Yuan; Ke, Xianliang; Zheng, Zhenhua; Zhang, Cuiling; Zhang, Zhenfeng; Zhang, Fuxian; Hu, Qinxue; He, Zhike; Wang, HanzhongACS Nano (2013), 7 (5), 3896-3904CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Utilization of quantum dots (QDs) for single virus tracking has attracted growing interest. Through modification of viral surface proteins, viruses can be labeled with various functionalized QDs and used for tracking the routes of viral infections. However, incorporation of QDs on the viral surface may affect the efficiency of viral entry and alter virus-cell interactions. Here, we describe that QDs can be encapsulated into the capsid of vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus (PTLV) in living cells without modification of the viral surface. QDs conjugated with modified genomic RNAs (gRNAs), which contain a packaging signal (Psi) sequence for viral genome encapsulation, can be packaged into virions together with the gRNAs. QD-contg. PTLV demonstrated similar entry efficiency as the wild-type PTLV. After infection, QD signals entered the Rab5+ endosome and then moved to the microtubule organizing center of the infected cells in a microtubule-dependent manner. Findings in this study are consistent with previously reported infection routes of VSV and VSV-G pseudotyped lentivirus, indicating that our established QD packaging approach can be used for enveloped virus labeling and tracking.
- 356Molenaar, C.; Marras, S. A.; Slats, J. C.; Truffert, J. C.; Lemaitre, M.; Raap, A. K.; Dirks, R. W.; Tanke, H. J. Linear 2’ O-Methyl RNA Probes for the Visualization of RNA in Living Cells. Nucleic Acids Res. 2001, 29, E89– 9, DOI: 10.1093/nar/29.17.e89356Linear 2' O-methyl RNA probes for the visualization of RNA in living cellsMolenaar, C.; Marras, S. A.; Slats, J. C. M.; Truffert, J.-C.; Lemaitre, M.; Raap, A. K.; Dirks, R. W.; Tanke, H. J.Nucleic Acids Research (2001), 29 (17), e89/1-e89/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)U1snRNA, U3snRNA, 28S rRNA, poly(A) RNA and a specific mRNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Me oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic rRNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of mol. beacons, which due to their structure should theor. fluoresce only upon hybridization. No improvements were achieved however with the mol. beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.
- 357Ma, Y.; Mao, G.; Huang, W.; Wu, G.; Yin, W.; Ji, X.; Deng, Z.; Cai, Z.; Zhang, X. E.; He, Z. Quantum Dot Nanobeacons for Single RNA Labeling and Imaging. J. Am. Chem. Soc. 2019, 141, 13454– 13458, DOI: 10.1021/jacs.9b04659357Quantum Dot Nanobeacons for Single RNA Labeling and ImagingMa, Yingxin; Mao, Guobin; Huang, Weiren; Wu, Guoqiang; Yin, Wen; Ji, Xinghu; Deng, Zishi; Cai, Zhiming; Zhang, Xian-En; He, Zhike; Cui, ZongqiangJournal of the American Chemical Society (2019), 141 (34), 13454-13458CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Detection and imaging RNAs in live cells is in high demand. Methodol. for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate co-modified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Addnl., QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being esp. meaningful for tracking of individual RNAs in live cells.
- 358Ortega-Arroyo, J.; Kukura, P. Interferometric Scattering Microscopy (iSCAT): New Frontiers in Ultrafast and Ultrasensitive Optical Microscopy. Phys. Chem. Chem. Phys. 2012, 14, 15625– 15636, DOI: 10.1039/c2cp41013c358Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopyOrtega-Arroyo, Jaime; Kukura, PhilippPhysical Chemistry Chemical Physics (2012), 14 (45), 15625-15636CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)A review. Optical microscopes have for centuries been window to the microscopic world. The advent of single-mol. optics over the past few decades has ushered in a new era in optical imaging, partly because it has enabled the observation of motion and more recently structure on the nanoscopic scale through the development of super-resoln. techniques. The large majority of these studies have relied on the efficient detection of fluorescence as the basis of single-mol. sensitivity. Despite the many advantages of using single emitters as light sources, the intensity and duration of their emission impose fundamental limits on the imaging speed and precision for tracking studies. Here, the potential of a novel imaging technique based on interferometric scattering (iSCAT) that pushes both the sensitivity and time resoln. far beyond what is currently achievable by single-emitter-based approaches are discussed. The authors present recent results that demonstrate single-mol. sensitivity and imaging speeds on the microsecond timescale.
- 359Kukura, P.; Ewers, H.; Muller, C.; Renn, A.; Helenius, A.; Sandoghdar, V. High-Speed Nanoscopic Tracking of the Position and Orientation of a Single Virus. Nat. Methods 2009, 6, 923– 927, DOI: 10.1038/nmeth.1395359High-speed nanoscopic tracking of the position and orientation of a single virusKukura, Philipp; Ewers, Helge; Mueller, Christian; Renn, Alois; Helenius, Ari; Sandoghdar, VahidNature Methods (2009), 6 (12), 923-927CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the mol. dynamics of early virus-host couplings, which are important for cell infection. Here, we present a colocalization methodol. that combines scattering interferometry and single-mol. fluorescence microscopy to visualize both position and orientation of single quantum dot-labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resoln., we obsd. sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions sepd. by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biol. nano-objects.
- 360Renz, M. Fluorescence Microscopy-A Historical and Technical Perspective. Cytometry, Part A 2013, 83, 767– 779, DOI: 10.1002/cyto.a.22295360Fluorescence microscopy-a historical and technical perspectiveRenz MalteCytometry. Part A : the journal of the International Society for Analytical Cytology (2013), 83 (9), 767-79 ISSN:.For a little more than a century, fluorescence microscopy has been an essential source of major discoveries in cell biology. Recent developments improved both visualization and quantification by fluorescence microscopy imaging and established a methodology of fluorescence microscopy. By outlining basic principles and their historical development, I seek to provide insight into and understanding of the ever-growing tools of fluorescence microscopy. Thereby, this synopsis may help the interested researcher to choose a fluorescence microscopic method capable of addressing a specific scientific question.
- 361Lorenz, K. S.; Salama, P.; Dunn, K. W.; Delp, E. J. Digital Correction of Motion Artefacts in Microscopy Image Sequences Collected from Living Animals Using Rigid and Nonrigid Registration. J. Microsc. 2012, 245, 148– 160, DOI: 10.1111/j.1365-2818.2011.03557.x361Digital correction of motion artefacts in microscopy image sequences collected from living animals using rigid and nonrigid registrationLorenz K S; Salama P; Dunn K W; Delp E JJournal of microscopy (2012), 245 (2), 148-60 ISSN:.Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artefacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and nonrigid components. The rigid registration component corrects global image translations, whereas the nonrigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung and salivary gland of living rodents.
- 362Combs, C. A.; Shroff, H. Fluorescence Microscopy: A Concise Guide to Current Imaging Methods. Curr. Protoc. Neurosci. 2017, 79, 2.1.1– 2.1.25, DOI: 10.1002/cpns.29There is no corresponding record for this reference.
- 363Masters, B. R. Fluorescence Microscopy: from Principles to Biological Applications. J. Biomed. Opt. 2014, 19, 049901 DOI: 10.1117/1.JBO.19.4.049901There is no corresponding record for this reference.
- 364Ma, Y. Y.; Wang, X.; Liu, H.; Wei, L.; Xiao, L. H. Recent Advances in Optical Microscopic Methods for Single-Particle Tracking in Biological Samples. Anal. Bioanal. Chem. 2019, 411, 4445– 4463, DOI: 10.1007/s00216-019-01638-z364Recent advances in optical microscopic methods for single-particle tracking in biological samplesMa, Yuanyuan; Wang, Xiao; Liu, Hua; Wei, Lin; Xiao, LehuiAnalytical and Bioanalytical Chemistry (2019), 411 (19), 4445-4463CODEN: ABCNBP; ISSN:1618-2642. (Springer)A review. With the rapid development of optical microscopic techniques, explorations on the chem. and biol. properties of target objects in biol. samples at single-mol./particle level have received great attention recently. In the past decades, various powerful techniques have been developed for single-particle tracking (SPT) in biol. samples. In this review, we summarize the commonly used optical microscopic methods for SPT, such as total internal reflection fluorescence microscopy (TIRFM), super-resoln. fluorescence microscopy (SRM), dark-field optical microscopy (DFM), total internal reflection scattering microscopy (TIRSM), and differential interference contrast microscopy (DICM). We then discuss the image processing and data anal. methods, including particle localization, trajectory reconstruction, and diffusion behavior anal. The application of SPT on the cell membrane, within the cell, and the cellular invading process of viruses are introduced. Finally, the challenges and prospects of optical microscopic technologies for SPT are delineated. [Figure not available: see fulltext.].
- 365Deschout, H.; Cella Zanacchi, F.; Mlodzianoski, M.; Diaspro, A.; Bewersdorf, J.; Hess, S. T.; Braeckmans, K. Precisely and Accurately Localizing Single Emitters in Fluorescence Microscopy. Nat. Methods 2014, 11, 253– 266, DOI: 10.1038/nmeth.2843365Precisely and accurately localizing single emitters in fluorescence microscopyDeschout, Hendrik; Zanacchi, Francesca Cella; Mlodzianoski, Michael; Diaspro, Alberto; Bewersdorf, Joerg; Hess, Samuel T.; Braeckmans, KevinNature Methods (2014), 11 (3), 253-266CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Methods based on single-mol. localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of mols. and nanoparticles and contributed to the recent revolution in super-resoln. localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing no. of researchers using single-particle tracking and super-resoln. localization microscopy.
- 366Lichtman, J. W.; Conchello, J. A. Fluorescence Microscopy. Nat. Methods 2005, 2, 910– 919, DOI: 10.1038/nmeth817366Fluorescence microscopyLichtman, Jeff W.; Conchello, Jose-AngelNature Methods (2005), 2 (12), 910-919CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Although fluorescence microscopy permeates all of cell and mol. biol., most biologists have little experience with the underlying photophys. phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Addnl., fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.
- 367Beier, H. T.; Ibey, B. L. Experimental Comparison of the High-Speed Imaging Performance of an EM-CCD and sCMOS Camera in a Dynamic Live-Cell Imaging Test Case. PLoS One 2014, 9, e84614 DOI: 10.1371/journal.pone.0084614367Experimental comparison of the high-speed imaging performance of an EM-CCD and sCMOS camera in a dynamic live-cell imaging test caseBeier, Hope T.; Ibey, Bennett L.PLoS One (2014), 9 (1), e84614/1-e84614/6, 6 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The study of living cells may require advanced imaging techniques to track weak and rapidly changing signals. Fundamental to this need is the recent advancement in camera technol. Two camera types, specifically sCMOS and EM-CCD, promise both high signal-to-noise and high speed (>100 fps), leaving researchers with a crit. decision when detg. the best technol. for their application. In this article, we compare two cameras using a live-cell imaging test case in which small changes in cellular fluorescence must be rapidly detected with high spatial resoln. The EM-CCD maintained an advantage of being able to acquire discernible images with a lower no. of photons due to its EM-enhancement. However, if high-resoln. images at speeds approaching or exceeding 1000 fps are desired, the flexibility of the full-frame imaging capabilities of sCMOS is superior.
- 368Long, F.; Zeng, S.; Huang, Z. L. Localization-Based Super-Resolution Microscopy with an sCMOS Camera Part II: Experimental Methodology for Comparing sCMOS with EMCCD Cameras. Opt. Express 2012, 20, 17741– 17759, DOI: 10.1364/OE.20.017741368Localization-based super-resolution microscopy with an sCMOS camera part II: experimental methodology for comparing sCMOS with EMCCD camerasLong Fan; Zeng Shaoqun; Huang Zhen-LiOptics express (2012), 20 (16), 17741-59 ISSN:.Nowadays, there is a hot debate among industry and academic researchers that whether the newly developed scientific-grade Complementary Metal Oxide Semiconductor (sCMOS) cameras could become the image sensors of choice in localization-based super-resolution microscopy. To help researchers find answers to this question, here we reported an experimental methodology for quantitatively comparing the performance of low-light cameras in single molecule detection (characterized via image SNR) and localization (via localization accuracy). We found that a newly launched sCMOS camera can present superior imaging performance than a popular Electron Multiplying Charge Coupled Device (EMCCD) camera in a signal range (15-12000 photon/pixel) more than enough for typical localization-based super-resolution microscopy.
- 369Fullerton, S. A Guide to Choosing and Using Scientific Imaging Cameras. Laser Focus World 2014, 50, 37– 40There is no corresponding record for this reference.
- 370Ivanchenko, S.; Godinez, W. J.; Lampe, M.; Krausslich, H. G.; Eils, R.; Rohr, K.; Brauchle, C.; Muller, B.; Lamb, D. C. Dynamics of HIV-1 Assembly and Release. PLoS Pathog. 2009, 5, e1000652 DOI: 10.1371/journal.ppat.1000652370Dynamics of HIV-1 assembly and releaseIvanchenko Sergey; Godinez William J; Lampe Marko; Krausslich Hans-Georg; Eils Roland; Rohr Karl; Brauchle Christoph; Muller Barbara; Lamb Don CPLoS pathogens (2009), 5 (11), e1000652 ISSN:.Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of approximately 5 x 10(-3) s(-1), corresponding to 8-9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at approximately 1,500+/-700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.
- 371Axelrod, D. Cell-Substrate Contacts Illuminated by Total Internal Reflection Fluorescence. J. Cell Biol. 1981, 89, 141– 145, DOI: 10.1083/jcb.89.1.141371Cell-substrate contacts illuminated by total internal reflection fluorescenceAxelrod DThe Journal of cell biology (1981), 89 (1), 141-5 ISSN:0021-9525.A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent alpha-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membranes topography, and visualization of reversible bound fluorescent ligands at membrane receptors.
- 372Axelrod, D. Total Internal Reflection Fluorescence Microscopy in Cell Biology. Traffic 2001, 2, 764– 774, DOI: 10.1034/j.1600-0854.2001.21104.x372Total internal reflection fluorescence microscopy in cell biologyAxelrod, DanielTraffic (Copenhagen, Denmark) (2001), 2 (11), 764-774CODEN: TRAFFA; ISSN:1398-9219. (Munksgaard International Publishers Ltd.)A review. Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochem. kinetics and single biomol. dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the phys. basis for the technique and the various ways to implement total internal reflection fluorescence in a std. fluorescence microscope.
- 373Reichert, W. M.; Truskey, G. A. Total Internal Reflection Fluorescence (TIRF) Microscopy. I. Modelling Cell Contact Region Fluorescence. J. Cell Sci. 1990, 96, 219– 230373Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescenceReichert W M; Truskey G AJournal of cell science (1990), 96 ( Pt 2) (), 219-30 ISSN:0021-9533.Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.
- 374Johnson, C. Spectroscopy and Dynamics of Single Molecules: Methods and Applications; Elsevier: 2019.There is no corresponding record for this reference.
- 375Tokunaga, M.; Kitamura, K.; Saito, K.; Iwane, A. H.; Yanagida, T. Single Molecule Imaging of Fluorophores and Enzymatic Reactions Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy. Biochem. Biophys. Res. Commun. 1997, 235, 47– 53, DOI: 10.1006/bbrc.1997.6732375Single molecule imaging of fluorophores and enzymic reactions achieved by objective-type total internal reflection fluorescence microscopyTokunaga, Makio; Kitamura, Kazuo; Saito, Kiwamu; Iwane, Atsuko Hikikoshi; Yanagida, ToshioBiochemical and Biophysical Research Communications (1997), 235 (1), 47-53CODEN: BBRCA9; ISSN:0006-291X. (Academic)Imaging of single fluorescence mols. has been achieved in a relatively simple manner using objective-type total internal reflection fluorescence microscopy (TIRFM). Switching from epi-fluorescence microscopy to objective-type TIRFM was achieved by translation of a single mirror in the system. Clear images of single mols. of an orange fluorescent dye, Cy3, were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective (PlanApo, 100 ×, Na 1.4) with ordinary coverslips and immersion oil. This method allowed visualization of single mols. under scanning probe microscopes. Taking advantage of the technique of single mol. imaging, individual ATP turnovers have been visualized with a fluorescent ATP analog, Cy3-ATP, using a simple exptl. strategy. Clear on/off signals were obtained that correspond to the assocn. and dissocn. of single Cy3-ATP/ADP mols. with a single myosin head mol. This method will allow a variety of single-mol. assays of biomol. functions to be performed using fluorescently labeled substrates, ligands, messengers, and biol. active mols. Thus, the present technique provides a simple yet powerful and universal tool for researchers to probe the events of single mols.
- 376Parhamifar, L.; Moghimi, S. M. Total Internal Reflection Fluorescence (TIRF) Microscopy for Real-Time Imaging of Nanoparticle-Cell Plasma Membrane Interaction. Methods Mol. Biol. 2012, 906, 473– 482, DOI: 10.1007/978-1-61779-953-2_38376Total internal reflection fluorescence (TIRF) microscopy for real-time imaging of nanoparticle-cell plasma membrane interactionParhamifar, Ladan; Moghimi, S. MoeinMethods in Molecular Biology (New York, NY, United States) (2012), 906 (Nanoparticles in Biology and Medicine), 473-482CODEN: MMBIED; ISSN:1064-3745. (Springer)Nanoparticulate systems are widely used for site-specific drug and gene delivery as well as for medical imaging. The mode of nanoparticle-cell interaction may have a significant effect on the pathway of nanoparticle internalization and subsequent intracellular trafficking. Total internal reflection fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane.
- 377Fish, K. N. Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr. Protoc. Cytom. 2009, 12, 12– 18, DOI: 10.1002/0471142956.cy1218s50There is no corresponding record for this reference.
- 378Mattheyses, A. L.; Simon, S. M.; Rappoport, J. Z. Imaging with Total Internal Reflection Fluorescence Microscopy for the Cell Biologist. J. Cell Sci. 2010, 123, 3621– 3628, DOI: 10.1242/jcs.056218378Imaging with total internal reflection fluorescence microscopy for the cell biologistMattheyses, Alexa L.; Simon, Sanford M.; Rappoport, Joshua Z.Journal of Cell Science (2010), 123 (21), 3621-3628CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)A review. Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biol. applications, and is particularly well suited to anal. of the localization and dynamics of mols. and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ∼100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid expsoure times. In this Commentary, we discuss the applications of TIRF to the study of cell biol., the phys. basis of TIRF, exptl. setup and troubleshooting.
- 379Johnson, D. S.; Jaiswal, J. K.; Simon, S. Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events. Curr. Protoc. Cytom. 2012, 12, 12– 29, DOI: 10.1002/0471142956.cy1229s61There is no corresponding record for this reference.
- 380Ewers, H.; Schelhaas, M. Analysis of Virus Entry and Cellular Membrane Dynamics by Single Particle Tracking. Methods Enzymol. 2012, 506, 63– 80, DOI: 10.1016/B978-0-12-391856-7.00028-7380Analysis of virus entry and cellular membrane dynamics by single particle trackingEwers, Helge; Schelhaas, MarioMethods in Enzymology (2012), 506 (Imaging and Spectroscopic Analysis of Living Cells), 63-80CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)A review. Viruses have evolved to mimic cellular ligands in order to gain access to their host cells for replication. Since viruses are simple in structure, they rely on host cells for all their transportation needs. Following single virus particles during the initial phase of infection, i.e., virus entry into target cells, can reveal crucial information on the mechanism of pathogen infections and likewise cellular transport and membrane dynamics. Here, we give an overview on how to fluorescently label virus particles for live cell microscopy, and on how virus entry can be analyzed by single particle tracking expts. Highlighted are strategies, on how to chem. introduce fluorophores into virions, and on how to ext. quant. information from live cell data.
- 381Tokunaga, M.; Imamoto, N.; Sakata-Sogawa, K. Highly Inclined Thin Illumination Enables Clear Single-Molecule Imaging in Cells. Nat. Methods 2008, 5, 159– 161, DOI: 10.1038/nmeth1171381Highly inclined thin illumination enables clear single-molecule imaging in cellsTokunaga, Makio; Imamoto, Naoko; Sakata-Sogawa, KumikoNature Methods (2008), 5 (2), 159-161CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe a simple illumination method of fluorescence microscopy for mol. imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-mol. images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify mol. dynamics, interactions and kinetics in cells for mol. systems biol.
- 382Schmidt, F. I.; Kuhn, P.; Robinson, T.; Mercer, J.; Dittrich, P. S. Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times. Biophys. J. 2013, 105, 420– 431, DOI: 10.1016/j.bpj.2013.06.016382Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag TimesSchmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.Biophysical Journal (2013), 105 (2), 420-431CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Recent studies revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. The authors developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which the authors analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concns. of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addn., EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiol. pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further studies into a potential VACV viroporin are justified. The authors' findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.
- 383Naredi-Rainer, N.; Prescher, J.; Hartschuh, A.; Lamb, D. C. Confocal Microscopy. Fluorescence Microscopy 2013, 1075, 175– 213, DOI: 10.1002/9783527671595.ch5There is no corresponding record for this reference.
- 384Minsky, M. Memoir on Inventing the Confocal Scanning Microscope. Scanning 1988, 10, 128– 138, DOI: 10.1002/sca.4950100403There is no corresponding record for this reference.
- 385Graf, R.; Rietdorf, J.; Zimmermann, T. Live Cell Spinning Disk Microscopy. Adv. Biochem. Eng./Biotechnol. 2005, 95, 57– 75, DOI: 10.1007/b102210385Live cell spinning disk microscopyGraf Ralph; Rietdorf Jens; Zimmermann TimoAdvances in biochemical engineering/biotechnology (2005), 95 (), 57-75 ISSN:0724-6145.In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. Confocal laser scanning microscopy is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi-beam scanning is much faster. Spinning disk confocal microscopes are therefore very suited for fast in vivo imaging. The principles of spinning disk microscopy will be explained in this chapter and a thorough comparison of the performance of single beam and multi-beam scanning systems is made and illustrated with an example of in vivo imaging in Dictyostelium discoideum.
- 386Egger, M. D.; Petran, M. New Reflected-Light Microscope for Viewing Unstained Brain and Ganglion Cells. Science 1967, 157, 305– 307, DOI: 10.1126/science.157.3786.305386New reflected-light microscope for viewing unstained brain and ganglion cellsEgger M D; Petran MScience (New York, N.Y.) (1967), 157 (3786), 305-7 ISSN:0036-8075.We have designed and constructed a new type of reflected-light microscope to form images including only light reflected near the plane of the object. This selectivity of image formation is based on a mechanical flying-spot technique. Objects difficult or impossible to see with earlier microscopes, such as unstained cells and cell processes in brains of living salamanders and in excised dorsal root ganglia of frogs, have been observed routinely with this microscope.
- 387Andersson, S. B. Tracking a Single Fluorescent Molecule with a Confocal Microscope. Appl. Phys. B: Lasers Opt. 2005, 80, 809– 816, DOI: 10.1007/s00340-005-1801-x387Tracking a single fluorescent molecule with a confocal microscopeAndersson, S. B.Applied Physics B: Lasers and Optics (2005), 80 (7), 809-816CODEN: APBOEM; ISSN:0946-2171. (Springer GmbH)We consider the problem of tracking a single fluorescent mol. in both two and three dimensions using a confocal laser scanning microscope. An est. of the position of the mol. is generated from the measured fluorescence signal through the use of parameter estn. theory. This est. is used in a nonlinear controller designed both to track the position of the mol. and to provide good measurements for use in the estn. algorithm. The performance of the approach is investigated through numerical simulation for mols. undergoing diffusion and directed transport and the capabilities of the controller relative to exptl. limitations are discussed.
- 388Han, J. J.; Kiss, C.; Bradbury, A. R. M.; Werner, J. H. Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three Dimensions. ACS Nano 2012, 6, 8922– 8932, DOI: 10.1021/nn302912j388Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three DimensionsHan, Jason J.; Kiss, Csaba; Bradbury, Andrew R. M.; Werner, James H.ACS Nano (2012), 6 (10), 8922-8932CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The authors demonstrate following individual fluorescent protein constructs and individual org. dyes as they diffuse in 3-D in soln. at rates up to 1 μm2/s over distances of several micrometers in X, Y, and Z. The authors' 3-D tracking method is essentially a stage scanning confocal microscope that uses a unique spatial filter geometry and active feedback 200 times/s to follow fast 3-D motion. Here the authors detail simulations used to find optimal feedback parameters for following individual fluorescent proteins in 3-D and show that a wide range of parameters are capable of following individual proteins diffusing at 1 μm2/s rates. In addn., exptl. through 3-D single-mol. tracking of a protein oligomer series (monomer, dimer, and tetramer) of the fluorescent protein Azami Green, the protein oligomerization state can be detd. The authors also perform time-resolved spectroscopy (photon pair correlation measurements) during the measured 3-D trajectories. The photon pair correlation measurements show clear fluorescence photon antibunching, demonstrating that the trajectories are of single fluorescent mols. The rates of single-mol. diffusive motion the authors follow (∼1 μm2/s) are comparable to or faster than many intracellular transport processes.
- 389Krzic, U.; Gunther, S.; Saunders, T. E.; Streichan, S. J.; Hufnagel, L. Multiview Light-Sheet Microscope for Rapid in toto Imaging. Nat. Methods 2012, 9, 730– 733, DOI: 10.1038/nmeth.2064389Multiview light-sheet microscope for rapid in toto imagingKrzic, Uros; Gunther, Stefan; Saunders, Timothy E.; Streichan, Sebastian J.; Hufnagel, LarsNature Methods (2012), 9 (7_part1), 730-733CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biol. specimens with subcellular resoln. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.
- 390Vettenburg, T.; Dalgarno, H. I. C.; Nylk, J.; Coll-Llado, C.; Ferrier, D. E. K.; Cizmar, T.; Gunn-Moore, F. J.; Dholakia, K. Light-Sheet Microscopy Using An Airy Beam. Nat. Methods 2014, 11, 541– 544, DOI: 10.1038/nmeth.2922390Light-sheet microscopy using an Airy beamVettenburg, Tom; Dalgarno, Heather I. C.; Nylk, Jonathan; Coll-Llado, Clara; Ferrier, David E. K.; Cizmar, Tomas; Gunn-Moore, Frank J.; Dholakia, KishanNature Methods (2014), 11 (5), 541-544CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resoln. We show that the Airy beam innately yields high contrast and resoln. up to a tenfold larger FOV. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beam's characteristic asym. excitation pattern results in all fluorescence contributing pos. to the contrast, enabling a step change for light-sheet microscopy.
- 391Pitrone, P. G.; Schindelin, J.; Stuyvenberg, L.; Preibisch, S.; Weber, M.; Eliceiri, K. W.; Huisken, J.; Tomancak, P. OpenSPIM: An Open-Access Light-Sheet Microscopy Platform. Nat. Methods 2013, 10, 598– 599, DOI: 10.1038/nmeth.2507391OpenSPIM: an open-access light-sheet microscopy platformPitrone, Peter G.; Schindelin, Johannes; Stuyvenberg, Luke; Preibisch, Stephan; Weber, Michael; Eliceiri, Kevin W.; Huisken, Jan; Tomancak, PavelNature Methods (2013), 10 (7), 598-599CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)An open hardware and software platform for constructing a customizable microscope for selective-plane illumination microscopy (SPIM) is presented.
- 392Li, Y.; Hu, Y.; Cang, H. Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living Cells. J. Phys. Chem. B 2013, 117, 15503– 15511, DOI: 10.1021/jp405380g392Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living CellsLi, Yu; Hu, Ying; Cang, HuJournal of Physical Chemistry B (2013), 117 (49), 15503-15511CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Single particle tracking is a powerful tool to study single mol. dynamics in living biol. samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single mols. on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF mols. on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temp., the av. diffusion coeff. of EGF on A549 cells was measured to be 0.13 μm2/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coeffs. and an increase of the av. diffusion coeff. at room temp. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole lifetime of a particle from binding till photobleaching or internalization.
- 393Keller, P. J.; Schmidt, A. D.; Wittbrodt, J.; Stelzer, E. H. Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy. Science 2008, 322, 1065– 1069, DOI: 10.1126/science.1162493393Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet MicroscopyKeller, Philipp J.; Schmidt, Annette D.; Wittbrodt, Joachim; Stelzer, Ernst H. K.Science (Washington, DC, United States) (2008), 322 (5904), 1065-1069CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A long-standing goal of biol. is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 h of development. Multiview in vivo imaging at 1.5 billion voxels per min provides "digital embryos," i.e., comprehensive databases of cell positions, divisions, and migratory tracks. Our anal. of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
- 394Ritter, J. G.; Veith, R.; Veenendaal, A.; Siebrasse, J. P.; Kubitscheck, U. Light Sheet Microscopy for Single Molecule Tracking in Living Tissue. PLoS One 2010, 5, e11639 DOI: 10.1371/journal.pone.0011639There is no corresponding record for this reference.
- 395Wan, Y.; McDole, K.; Keller, P. J. Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes. Annu. Rev. Cell Dev. Biol. 2019, 35, 655– 681, DOI: 10.1146/annurev-cellbio-100818-125311395Light-Sheet Microscopy and Its Potential for Understanding Developmental ProcessesWan, Yinan; McDole, Katie; Keller, Philipp J.Annual Review of Cell and Developmental Biology (2019), 35 (), 655-681CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews)The ability to visualize and quant. measure dynamic biol. processes in vivo and at high spatiotemporal resoln. is of fundamental importance to exptl. investigations in developmental biol. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resoln. while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biol. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-mol. to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data anal. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technol., and identify exciting opportunities for further advances.
- 396Hillman, E. M. C.; Voleti, V.; Li, W.; Yu, H. Light-Sheet Microscopy in Neuroscience. Annu. Rev. Neurosci. 2019, 42, 295– 313, DOI: 10.1146/annurev-neuro-070918-050357396Light-Sheet Microscopy in NeuroscienceHillman, Elizabeth M. C.; Voleti, Venkatakaushik; Li, Wenze; Yu, HangAnnual Review of Neuroscience (2019), 42 (), 295-313CODEN: ARNSD5; ISSN:0147-006X. (Annual Reviews)Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.
- 397Stelzer, E. H. K. Light-Sheet Fluorescence Microscopy for Quantitative Biology. Nat. Methods 2015, 12, 23– 26, DOI: 10.1038/nmeth.3219397Light-sheet fluorescence microscopy for quantitative biologyStelzer, Ernst H. K.Nature Methods (2015), 12 (1), 23-26CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)In light sheet-based fluorescence microscopy (LSFM), optical sectioning in the excitation process minimizes fluorophore bleaching and phototoxic effects. Because biol. specimens survive long-term three-dimensional imaging at high spatiotemporal resoln., LSFM has become the tool of choice in developmental biol.
- 398Bosse, J. B.; Hogue, I. B.; Feric, M.; Thiberge, S. Y.; Sodeik, B.; Brangwynne, C. P.; Enquist, L. W. Remodeling Nuclear Architecture Allows Efficient Transport of Herpesvirus Capsids by Diffusion. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E5725– E5733, DOI: 10.1073/pnas.1513876112398Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusionBosse, Jens B.; Hogue, Ian B.; Feric, Marina; Thiberge, Stephan Y.; Sodeik, Beate; Brangwynne, Clifford P.; Enquist, Lynn W.Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (42), E5725-E5733CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The nuclear chromatin structure confines the movement of large macromol. complexes to interchromatin corrals. Herpesvirus capsids of approx. 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resoln. Here, the authors use a rotating, oblique light sheet, which they dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resoln. They do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.
- 399Hoyer, P.; de Medeiros, G.; Balazs, B.; Norlin, N.; Besir, C.; Hanne, J.; Krausslich, H. G.; Engelhardt, J.; Sahl, S. J.; Hell, S. W. Breaking the Diffraction Limit of Light-Sheet Fluorescence Microscopy by RESOLFT. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 3442– 3446, DOI: 10.1073/pnas.1522292113399Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFTHoyer, Patrick; de Medeiros, Gustavo; Balazs, Balint; Norlin, Nils; Besir, Christina; Hanne, Janina; Kraeusslich, Hans-Georg; Engelhardt, Johann; Sahl, Steffen J.; Hell, Stefan W.; Hufnagel, LarsProceedings of the National Academy of Sciences of the United States of America (2016), 113 (13), 3442-3446CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent mol. state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biol. specimens with low light dose and axial resoln. far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.
- 400Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793– 795, DOI: 10.1038/nmeth929400Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)Rust, Michael J.; Bates, Mark; Zhuang, XiaoweiNature Methods (2006), 3 (10), 793-796CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors have developed a high-resoln. fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be detd. with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. The authors demonstrated an imaging resoln. of 20 nm. This technique can, in principle, reach mol.-scale resoln.
- 401Malkusch, S.; Muranyi, W.; Muller, B.; Krausslich, H. G.; Heilemann, M. Single-Molecule Coordinate-Based Analysis of the Morphology of HIV-1 Assembly Sites with Near-Molecular Spatial Resolution. Histochem. Cell Biol. 2013, 139, 173– 179, DOI: 10.1007/s00418-012-1014-4401Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolutionMalkusch, Sebastian; Muranyi, Walter; Mueller, Barbara; Kraeusslich, Hans-Georg; Heilemann, MikeHistochemistry and Cell Biology (2013), 139 (1), 173-179CODEN: HCBIFP; ISSN:0948-6143. (Springer)We apply single-mol. super-resoln. microscopy and coordinate-based cluster anal. to ext. information on the distribution and on the morphol. and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly obsd. was in accordance with previous electron microscopy analyses revealing ∼140 nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag mols. in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster anal. and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to ext. information on the properties of the different Gag clusters.
- 402Lehmann, M.; Rocha, S.; Mangeat, B.; Blanchet, F.; Uji, I. H.; Hofkens, J.; Piguet, V. Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction. PLoS Pathog. 2011, 7, e1002456 DOI: 10.1371/journal.ppat.1002456402Quantitative multicolor super-resolution microscopy reveals tetherin HIV-1 interactionLehmann, Martin; Rocha, Susana; Mangeat, Bastien; Blanchet, Fabien; Uji-i, Hiroshi; Hofkens, Johan; Piguet, VincentPLoS Pathogens (2011), 7 (12), e1002456CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resoln. microscopy techniques achieve nanometer resoln. of fluorescently labeled mols. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resoln. anal. of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, resp. Multicolor super-resoln. microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters contg. on av. 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochem. and super-resoln. anal. revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resoln. microscopy anal. of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions.
- 403Inamdar, K.; Floderer, C.; Favard, C.; Muriaux, D. Monitoring HIV-1 Assembly in Living Cells: Insights from Dynamic and Single Molecule Microscopy. Viruses 2019, 11, 72, DOI: 10.3390/v11010072403Monitoring HIV-1 assembly in living cells: insights from dynamic and single molecule microscopyInamdar, Kaushik; Floderer, Charlotte; Favard, Cyril; Muriaux, DelphineViruses (2019), 11 (1), 72CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)The HIV-1 assembly process is a multi-complex mechanism that takes place at the host cell plasma membrane. It requires a spatio-temporal coordination of events to end up with a full mature and infectious virus. The mol. mechanisms of HIV-1 assembly have been extensively studied during the past decades, in order to dissect the resp. roles of the structural and non-structural viral proteins of the viral RNA genome and of some host cell factors. Nevertheless, the time course of HIV-1 assembly was obsd. in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resoln., in addn. to improved fluorescent tags for proteins, now permits study of HIV-1 assembly at the single mol. level within living cells. In this review, after a short description of these new approaches, we will discuss how HIV-1 assembly at the cell plasma membrane has been revisited using advanced super resoln. microscopy techniques and how it can bridge the study of viral assembly from the single mol. to the entire host cell.
- 404Bleck, M.; Itano, M. S.; Johnson, D. S.; Thomas, V. K.; North, A. J.; Bieniasz, P. D.; Simon, S. M. Temporal and Spatial Organization of ESCRT Protein Recruitment During HIV-1 Budding. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 12211– 12216, DOI: 10.1073/pnas.1321655111404Temporal and spatial organization of ESCRT protein recruitment during HIV-1 buddingBleck, Marina; Itano, Michelle S.; Johnson, Daniel S.; Thomas, V. Kaye; North, Alison J.; Bieniasz, Paul D.; Simon, Sanford M.Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (33), 12211-12216CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the host ESCRT (endosomal sorting complex required for transport) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we obsd. that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins like Chmp4b (charged multivesicular body protein 4b) and Vps4A (vacuolar protein sorting-assocd. protein 4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresoln. imaging, we obsd. that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.
- 405Prescher, J.; Baumgartel, V.; Ivanchenko, S.; Torrano, A. A.; Brauchle, C.; Muller, B.; Lamb, D. C. Super-Resolution Imaging of ESCRT-Proteins at HIV-1 Assembly Sites. PLoS Pathog. 2015, 11, e1004677 DOI: 10.1371/journal.ppat.1004677405Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sitesPrescher, Jens; Baumgaertel, Viola; Ivanchenko, Sergey; Torrano, Adriano A.; Braeuchle, Christoph; Mueller, Barbara; Lamb, Don C.PLoS Pathogens (2015), 11 (2), e1004677CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane assocd. lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be obsd., the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resoln. fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an av. size (full-width at half-max.) between 45 and 60 nm or a diam. (detd. using a Ripley's L-function anal.) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual mols. surrounding the central clusters was often obsd., which we attribute to ALIX mols. incorporated into the nascent HIV-1 Gag shell. Expts. performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.
- 406Shao, L.; Kner, P.; Rego, E. H.; Gustafsson, M. G. Super-Resolution 3D Microscopy of Live Whole Cells Using Structured Illumination. Nat. Methods 2011, 8, 1044– 1046, DOI: 10.1038/nmeth.1734406Super-resolution 3D microscopy of live whole cells using structured illuminationShao, Lin; Kner, Peter; Rego, E. Hesper; Gustafsson, Mats G. L.Nature Methods (2011), 8 (12), 1044-1046CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resoln. of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per vol. for >50 time points with 120-nm lateral and 360-nm axial resoln. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.
- 407Jones, S. A.; Shim, S. H.; He, J.; Zhuang, X. Fast, Three-Dimensional Super-Resolution Imaging of Live Cells. Nat. Methods 2011, 8, 499– 508, DOI: 10.1038/nmeth.1605407Fast, three-dimensional super-resolution imaging of live cellsJones, Sara A.; Shim, Sang-Hee; He, Jiang; Zhuang, XiaoweiNature Methods (2011), 8 (6), 499-505CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We report super-resoln. fluorescence imaging of live cells with high spatiotemporal resoln. using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resoln. images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolns. of ∼25 nm and temporal resolns. as fast as 0.5 s. We also demonstrated live-cell 3D super-resoln. imaging. We obtained 3D spatial resoln. of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolns. as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resoln. imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
- 408Nair, D.; Hosy, E.; Petersen, J. D.; Constals, A.; Giannone, G.; Choquet, D.; Sibarita, J. B. Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95. J. Neurosci. 2013, 33, 13204– 13224, DOI: 10.1523/JNEUROSCI.2381-12.2013408Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95Nair, Deepak; Hosy, Eric; Petersen, Jennifer D.; Constals, Audrey; Giannone, Gregory; Choquet, Daniel; Sibarita, Jean-BaptisteJournal of Neuroscience (2013), 33 (32), 13204-13224CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resoln. light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concd. inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them. Nanodomains are dynamic in their shape and position within synapses and can form or disappear within minutes, although they are mostly stable for up to 1 h. AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95, which are generally of larger size than AMPAR nanoclusters. PSD95 expression level regulates AMPAR nanodomain size and compactness in parallel to miniature EPSC amplitude. Monte Carlo simulations further indicate the impact of AMPAR concn. in clusters on the efficacy of synaptic transmission. The observation that AMPARs are highly concd. in nanodomains, instead of diffusively distributed in the PSD as generally thought, has important consequences on our understanding of excitatory neurotransmission. Furthermore, our results indicate that glutamatergic synaptic transmission is controlled by the nanometer-scale regulation of the size of these highly concd. nanodomains.
- 409Izeddin, I.; Recamier, V.; Bosanac, L.; Cisse, I. I.; Boudarene, L.; Dugast-Darzacq, C.; Proux, F.; Benichou, O.; Voituriez, R.; Bensaude, O. Single-Molecule Tracking in Live Cells Reveals Distinct Target-Search Strategies of Transcription Factors in the Nucleus. eLife 2014, 3, e02230 DOI: 10.7554/eLife.02230409Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleusIzeddin, Ignacio; Recamier, Vincent; Bosanac, Lana; Cisse, Ibrahim I.; Boudarene, Lydia; Dugast-Darzacq, Claire; Proux, Florence; Benichou, Olivier; Voituriez, Raphael; Bensaude, Olivier; Dahan, Maxime; Darzacq, XaviereLife (2014), 3 (), e02230/1-e02230/27, 27 pp.CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-mol. tracking assay to det. TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the pos. transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc mol. is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.
- 410Yang, L.; Dun, A. R.; Martin, K. J.; Qiu, Z.; Dunn, A.; Lord, G. J.; Lu, W.; Duncan, R. R.; Rickman, C. Secretory Vesicles Are Preferentially Targeted to Areas of Low Molecular SNARE Density. PLoS One 2012, 7, e49514 DOI: 10.1371/journal.pone.0049514410Secretory vesicles are preferentially targeted to areas of low molecular SNARE densityYang, Lei; Dun, Alison R.; Martin, Kirsty J.; Qiu, Zhen; Dunn, Andrew; Lord, Gabriel J.; Lu, Weiping; Duncan, Rory R.; Rickman, ColinPLoS One (2012), 7 (11), e49514CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (sol. N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters contg. many mols., thus providing a concd. reservoir to promote membrane fusion. However, biophys. expts. suggest that a small no. of SNAREs are sufficient to drive a single fusion event. Here we show, using mol. imaging, that the majority of tSNARE mols. are spatially sepd. from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random mol. distribution and limiting the max. no. of mols. encountered by secretory vesicles. Together our results provide a new model for the mol. mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual mol. machines.
- 411Juette, M. F.; Gould, T. J.; Lessard, M. D.; Mlodzianoski, M. J.; Nagpure, B. S.; Bennett, B. T.; Hess, S. T.; Bewersdorf, J. Three-Dimensional Sub-100 nm Resolution Fluorescence Microscopy of Thick Samples. Nat. Methods 2008, 5, 527– 529, DOI: 10.1038/nmeth.1211411Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samplesJuette, Manuel F.; Gould, Travis J.; Lessard, Mark D.; Mlodzianoski, Michael J.; Nagpure, Bhupendra S.; Bennett, Brian T.; Hess, Samuel T.; Bewersdorf, JoergNature Methods (2008), 5 (6), 527-529CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The ability to image thick vols. with invariant high axial and lateral resoln. is a challenge for existing super-resoln. fluorescence microscopy techniques. The combination of a double-plane detection scheme with fluorescence photoactivation microscopy (FPALM) allows three-dimensional sub-diffraction resoln. imaging of samples as thick as whole cells. Imaging vols. as thick as whole cells at three-dimensional (3D) super-resoln. is required to reveal unknown features of cellular organization. The authors report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resoln. over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resoln. without compromising speed or sensitivity.
- 412Pavani, S. R.; Thompson, M. A.; Biteen, J. S.; Lord, S. J.; Liu, N.; Twieg, R. J.; Piestun, R.; Moerner, W. E. Three-Dimensional, Single-Molecule Fluorescence Imaging Beyond the Diffraction Limit by Using a Double-Helix Point Spread Function. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 2995– 2999, DOI: 10.1073/pnas.0900245106412Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread functionPavani, Sri Rama Prasanna; Thompson, Michael A.; Biteen, Julie S.; Lord, Samuel J.; Liu, Na; Twieg, Robert J.; Piestun, Rafael; Moerner, W. E.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (9), 2995-2999CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We demonstrate single-mol. fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent mols. in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 μm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresoln. imaging of high concns. of mols. in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resoln. beyond the Rayleigh diffraction limit.
- 413Thompson, M. A.; Lew, M. D.; Badieirostami, M.; Moerner, W. E. Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function. Nano Lett. 2010, 10, 211– 218, DOI: 10.1021/nl903295p413Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread FunctionThompson, Michael A.; Lew, Matthew D.; Badieirostami, Majid; Moerner, W. E.Nano Letters (2010), 10 (1), 211-218CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a wide-field fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). The authors describe in detail how the localization precision quant. depends upon the no. of photons detected and the z position of the nanoscale emitter, thereby showing a ∼10 nm localization capability along x, y, and z in the limit of weak emitters. Exptl. measurements are compared to Fisher information calcns. of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aq. soln. and a quantum dot-labeled structure inside a living cell in three dimensions.
- 414Yu, B.; Yu, J.; Li, W.; Cao, B.; Li, H.; Chen, D.; Niu, H. Nanoscale Three-Dimensional Single Particle Tracking by Light-Sheet-Based Double-Helix Point Spread Function Microscopy. Appl. Opt. 2016, 55, 449– 453, DOI: 10.1364/AO.55.000449414Nanoscale three-dimensional single particle tracking by light-sheet-based double-helix point spread function microscopyYu, Bin; Yu, Jie; Li, Weihai; Cao, Bo; Li, Heng; Chen, Danni; Niu, HanbenApplied Optics (2016), 55 (3), 449-453CODEN: APOPAI; ISSN:1559-128X. (Optical Society of America)The double-helix point spread function (DH-PSF) microscopy has become an essential tool for nanoscale three-dimensional (3D) localization and tracking of single mols. in living cells. However, its localization precision is limited by fluorescent contrast in thick samples because the signal-to-noise ratio of the system is low due to the inherent low transfer function efficiency and background fluorescence. Here we combine DH-PSF microscopy with light-sheet illumination to eliminate out-of-focus background fluorescence for high-precision 3D single particle tracking. To demonstrate the capability of the method, we obtain the single fluorescent bead image with light-sheet illumination, with three-dimensional localization accuracy better than that of epi-illumination. We also show that the single fluorescent beads in agarose soln. can be tracked, which demonstrates the possibility of our method for the study of dynamic processes in complex biol. specimens.
- 415Holtzer, L.; Meckel, T.; Schmidt, T. Nanometric Three-Dimensional Tracking of Individual Quantum Dots in Cells. Appl. Phys. Lett. 2007, 90, 053902 DOI: 10.1063/1.2437066415Nanometric three-dimensional tracking of individual quantum dots in cellsHoltzer, Laurent; Meckel, Tobias; Schmidt, ThomasApplied Physics Letters (2007), 90 (5), 053902/1-053902/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)Wide-field single-mol. fluorescence microscopy has become an established tool for the study of dynamic biol. processes which occur in the plane of a cellular membrane. In the current study we have extended this technique to the three-dimensional anal. of mol. mobility. Introduction of a cylindrical lens into the emission path of a microscope produced some astigmatism which was used to obtain the full three-dimensional position information. The localization accuracy of fluorescent objects was calcd. theor. and subsequently confirmed by simulations and by expts. For further validation individual quantum dots were followed when passively diffusing and actively transported within life cells.
- 416Huang, B.; Wang, W.; Bates, M.; Zhuang, X. Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy. Science 2008, 319, 810– 813, DOI: 10.1126/science.1153529416Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction MicroscopyHuang, Bo; Wang, Wenqin; Bates, Mark; Zhuang, XiaoweiScience (Washington, DC, United States) (2008), 319 (5864), 810-813CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resoln., achieving a near-mol. resoln. of 20 to 30 nm in the two lateral dimensions. Three-dimensional (3D) nanoscale-resoln. imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to det. both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resoln. of 20 to 30 nm in the lateral dimensions and 50 to 60 nm in the axial dimension. This development allowed us to resolve the 3D morphol. of nanoscopic cellular structures.
- 417Lange, S.; Katayama, Y.; Schmid, M.; Burkacky, O.; Brauchle, C.; Lamb, D. C.; Jansen, R. P. Simultaneous Transport of Different Localized mRNA Species Revealed by Live-Cell Imaging. Traffic 2008, 9, 1256– 1267, DOI: 10.1111/j.1600-0854.2008.00763.x417Simultaneous transport of different localized mRNA species revealed by live-cell imagingLange, Susanne; Katayama, Yoshihiko; Schmid, Maria; Burkacky, Ondrej; Braeuchle, Christoph; Lamb, Don C.; Jansen, Ralf-PeterTraffic (Oxford, United Kingdom) (2008), 9 (8), 1256-1267CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)Intracellular mRNA localization is a common mechanism to achieve asym. distributions of proteins. Previous studies have revealed that in a no. of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into sep. or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, the authors analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, the authors have tracked the transport of two different localized mRNA species in real time. Their observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form sep. particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.
- 418Wells, N. P.; Lessard, G. A.; Goodwin, P. M.; Phipps, M. E.; Cutler, P. J.; Lidke, D. S.; Wilson, B. S.; Werner, J. H. Time-Resolved Three-Dimensional Molecular Tracking in Live Cells. Nano Lett. 2010, 10, 4732– 4737, DOI: 10.1021/nl103247v418Time-Resolved Three-Dimensional Molecular Tracking in Live CellsWells, Nathan P.; Lessard, Guillaume A.; Goodwin, Peter M.; Phipps, Mary E.; Cutler, Patrick J.; Lidke, Diane S.; Wilson, Bridget S.; Werner, James H.Nano Letters (2010), 10 (11), 4732-4737CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The authors report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses 4 overlapping confocal vol. elements and active feedback once every 5 ms to follow 3-dimensional mol. motion. This method has substantial advantages over three-dimensional mol. tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the mols. being tracked. In particular, the authors show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biol. relevant transport rates. To demonstrate the power of these methods for exploring the spatiotemporal dynamics of live cells, the authors follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale features of the cell surface topol. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (∼950 nm/s) vesicular transit through the cell.
- 419Cang, H.; Xu, C. S.; Montiel, D.; Yang, H. Guiding a Confocal Microscope by Single Fluorescent Nanoparticles. Opt. Lett. 2007, 32, 2729– 2731, DOI: 10.1364/OL.32.002729419Guiding a confocal microscope by single fluorescent nanoparticlesCang Hu; Xu C Shan; Montiel Daniel; Yang HawOptics letters (2007), 32 (18), 2729-31 ISSN:0146-9592.Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.
- 420Balzarotti, F.; Eilers, Y.; Gwosch, K. C.; Gynna, A. H.; Westphal, V.; Stefani, F. D.; Elf, J.; Hell, S. W. Nanometer Resolution Imaging and Tracking of Fluorescent Molecules with Minimal Photon Fluxes. Science 2017, 355, 606– 612, DOI: 10.1126/science.aak9913420Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxesBalzarotti, Francisco; Eilers, Yvan; Gwosch, Klaus C.; Gynna, Arvid H.; Westphal, Volker; Stefani, Fernando D.; Elf, Johan; Hell, Stefan W.Science (Washington, DC, United States) (2017), 355 (6325), 606-612CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity min. of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our expts., 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresoln. microscopy, MINFLUX attained ∼1-nm precision, resolving mols. only 6 nm apart. MINFLUX tracking of single fluorescent proteins increased the temporal resoln. and the no. of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromols. in living cells and beyond.
- 421Enderlein, J. Tracking of Fluorescent Molecules Diffusing within Membranes. Appl. Phys. B: Lasers Opt. 2000, 71, 773– 777, DOI: 10.1007/s003400000409421Tracking of fluorescent molecules diffusing within membranesEnderlein, J.Applied Physics B: Lasers and Optics (2000), 71 (5), 773-777CODEN: APBOEM; ISSN:0946-2171. (Springer-Verlag)A new method is proposed for tracking fluorescing single mols. diffusing within a two-dimensional membrane. It is based on a confocal microscopy setup with a constantly rotating laser focus, which follows the position of the mol. The optimization and efficiency of the method are theor. studied for a broad range of exptl. realistic conditions. The proposed method allows for a longtime observation of diffusing mols. while allowing the application of fast spectroscopic techniques such as fluorescence decay time detn. or fluorescence anisotropy measurements.
- 422Kis-Petikova, K.; Gratton, E. Distance Measurement by Circular Scanning of the Excitation Beam in the Two-Photon Microscope. Microsc. Res. Tech. 2004, 63, 34– 49, DOI: 10.1002/jemt.10417422Distance measurement by circular scanning of the excitation beam in the two-photon microscopeKis-Petikova Katarina; Gratton EnricoMicroscopy research and technique (2004), 63 (1), 34-49 ISSN:1059-910X.We developed a method to measure relative distances with nanometer accuracy of fluorescent particles of different color in a two-photon scanning fluorescence microscope, with two-channel photon counting detection. The method can be used in the 10-500 nm range, for distances below the resolution limit of standard far field microscopy. The proposed technique is more efficient than the methods using raster scanning. To achieve maximum sensitivity in the radial direction, the excitation beam is moved periodically in a circular orbit with a radius of the size of the point spread function. The phase and the modulation of the periodic fluorescence signal, calculated by fast Fourier transform, gives the phase and the radial distance of the particle from the center of scanning. The coordinates of particles are recovered simultaneously in the two channels and the relative distance is calculated in real time. Particles can be tracked by moving the center of scanning to the recovered position, while measuring the distance from the second particle. Intensity data are saved and fitted later by a model accounting for light leakage between the channels. The total number of detected photons limited the accuracy of the position and distance measurement. Experiments demonstrating the advantages of the method were performed on fluorescent spheres and single dye molecules immobilized on quartz surface.
- 423Lessard, G. A.; Goodwin, P. M.; Werner, J. H. Three-Dimensional Tracking of Individual Quantum Dots. Appl. Phys. Lett. 2007, 91, 224106, DOI: 10.1063/1.2819074423Three-dimensional tracking of individual quantum dotsLessard, Guillaume A.; Goodwin, Peter M.; Werner, James H.Applied Physics Letters (2007), 91 (22), 224106/1-224106/3CODEN: APPLAB; ISSN:0003-6951. (American Institute of Physics)The authors describe an instrument that extends the state of the art in a single-mol. tracking technol., allowing extended observations of single fluorophores and fluorescently labeled proteins as they undergo directed and diffusive transport in three dimensions. The authors demonstrate three-dimensional tracking of individual quantum dots undergoing diffusion for durations of over a second at velocities comparable to those of intracellular signaling processes.
- 424Verdeny-Vilanova, I.; Wehnekamp, F.; Mohan, N.; Alvarez, A. S.; Borbely, J. S.; Otterstrom, J. J.; Lamb, D. C.; Lakadamyali, M. 3D Motion of Vesicles along Microtubules Helps Them to Circumvent Obstacles in Cells. J. Cell Sci. 2017, 130, 1904– 1916, DOI: 10.1242/jcs.2011784243D motion of vesicles along microtubules helps them to circumvent obstacles in cellsVerdeny-Vilanova, Ione; Wehnekamp, Fabian; Mohan, Nitin; Alvarez, AngelSandoval; Borbely, Joseph Steven; Otterstrom, Jason John; Lamb, Don C.; Lakadamyali, MelikeJournal of Cell Science (2017), 130 (11), 1904-1916CODEN: JNCSAI; ISSN:1477-9137. (Company of Biologists Ltd.)Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the sepn. of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them.
- 425Wehnekamp, F.; Plucinska, G.; Thong, R.; Misgeld, T.; Lamb, D. C. Nanoresolution Real-Time 3D Orbital Tracking for Studying Mitochondrial Trafficking in Vertebrate Axons in Vivo. eLife 2019, 8, e46059 DOI: 10.7554/eLife.46059425Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivoWehnekamp, Fabian; Plucinska, Gabriela; Thong, Rachel; Misgeld, Thomas; Lamb, Don C.eLife (2019), 8 (), e46059/1-e46059/22CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resoln. By combining various tech. improvements, we tracked individual mitochondria with unprecedented spatiotemporal resoln. over distances of >100 μm. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resoln. in a living organism offers a new approach to elucidating their function in its complete physiol. context.
- 426Katayama, Y.; Burkacky, O.; Meyer, M.; Brauchle, C.; Gratton, E.; Lamb, D. C. Real-Time Nanomicroscopy via Three-Dimensional Single-Particle Tracking. ChemPhysChem 2009, 10, 2458– 2464, DOI: 10.1002/cphc.200900436426Real-Time Nanomicroscopy via Three-Dimensional Single-Particle TrackingKatayama, Yoshihiko; Burkacky, Ondrej; Meyer, Martin; Braeuchle, Christoph; Gratton, Enrico; Lamb, Don C.ChemPhysChem (2009), 10 (14), 2458-2464CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resoln. in the lateral dimensions and 50 nm in the axial dimension at a temporal resoln. of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resoln.
- 427Hellriegel, C.; Gratton, E. Real-Time Multi-Parameter Spectroscopy and Localization in Three-Dimensional Single-Particle Tracking. J. R. Soc., Interface 2009, 6, S3– S14, DOI: 10.1098/rsif.2008.0313.focus427Real-time multi-parameter spectroscopy and localization in three-dimensional single-particle trackingHellriegel, Christian; Gratton, EnricoJournal of the Royal Society, Interface (2009), 6 (Suppl. 1), S3-S14CODEN: JRSICU; ISSN:1742-5689. (Royal Society)Tracking of single particles in optical microscopy has been employed in studies ranging from material sciences to biophysics down to the level of single mols. The technique intrinsically circumvents ensemble averaging and may therefore reveal directly mechanistic details of the involved dynamic processes. Such processes range from translational and rotational motion to spectral dynamics. We distinguish between conventional a posteriori tracking of objects (e.g. from the sequences of images) and the exptl. more refined 'on-the-fly' tracking technique. In this technique, the observation vol. of the microscope is kept centered with respect to the moving object via a feedback algorithm. This approach brings a series of advantages in comparison with the tracking from images, ranging from a superior spatio-temporal resoln. (2-50 nm and 1-32 ms) to the capability of inferring addnl. data (e.g. fluorescence lifetime, emission spectrum, polarization, intensity dynamics) from an object as it moves over several microns in three dimensions. In this contribution, we describe the principle of the tracking technique as implemented on a two-photon laser scanning microscope and illustrate its capabilities with exptl. data, from particles labeled with different dyes moving in a liq. to the characterization of small fluorescently labeled protein assemblies in living cells.
- 428Liu, Z.; Lavis, L. D.; Betzig, E. Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level. Mol. Cell 2015, 58, 644– 659, DOI: 10.1016/j.molcel.2015.02.033428Imaging Live-Cell Dynamics and Structure at the Single-Molecule LevelLiu, Zhe; Lavis, Luke D.; Betzig, EricMolecular Cell (2015), 58 (4), 644-659CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)Observation of mol. processes inside living cells is fundamental to a quant. understanding of how biol. systems function. Specifically, decoding the complex behavior of single mols. enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble expts. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new expts. to investigate the robustness and stochasticity of diverse mol. mechanisms with high spatiotemporal resoln. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-mol. level with minimal perturbations to the specimen.
- 429Saxton, M. J. Single-Particle Tracking: Connecting the Dots. Nat. Methods 2008, 5, 671– 672, DOI: 10.1038/nmeth0808-671429Single-particle tracking: connecting the dotsSaxton, Michael J.Nature Methods (2008), 5 (8), 671-672CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review commentary on the accompanying articles by (ibid. A. Serge et al., 687-694 and K. Jaqaman et al., 695-702). Algorithms for analyzing single-particle tracking images to obtain the paths of individual particles are challenged by high-d. data. Improvements in algorithms help to overcome these limitations.
- 430Carter, B. C.; Shubeita, G. T.; Gross, S. P. Tracking Single Particles: A User-Friendly Quantitative Evaluation. Phys. Biol. 2005, 2, 60– 72, DOI: 10.1088/1478-3967/2/1/008430Tracking single particles: a user-friendly quantitative evaluationCarter Brian C; Shubeita George T; Gross Steven PPhysical biology (2005), 2 (1), 60-72 ISSN:.As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.
- 431Thompson, R. E.; Larson, D. R.; Webb, W. W. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophys. J. 2002, 82, 2775– 2783, DOI: 10.1016/S0006-3495(02)75618-X431Precise nanometer localization analysis for individual fluorescent probesThompson, Russell E.; Larson, Daniel R.; Webb, Watt W.Biophysical Journal (2002), 82 (5), 2775-2783CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Calcn. of the centroid of the images of individual fluorescent particles and mols. allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resoln. The factors that limit the precision of these techniques are examd. and a simple equation derived that describes the precision of localization over a wide range of conditions. In addn., a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an exptl. app. and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the no. of photons in the spot for the shot noise limited case and as the inverse of the no. of photons for the background noise limited case. The optimal image magnification depends on the expected no. of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the std. deviation of the point spread function.
- 432Small, A.; Stahlheber, S. Fluorophore Localization Algorithms for Super-Resolution Microscopy. Nat. Methods 2014, 11, 267– 279, DOI: 10.1038/nmeth.2844432Fluorophore localization algorithms for super-resolution microscopySmall, Alex; Stahlheber, ShaneNature Methods (2014), 11 (3), 267-279CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review. Super-resoln. localization microscopy methods provide powerful new capabilities for probing biol. at the nanometer scale via fluorescence. These methods rely on two key innovations: switchable fluorophores (which blink on and off and can be sequentially imaged) and powerful localization algorithms (which est. the positions of the fluorophores in the images). These techniques have spurred a flurry of innovation in algorithm development over the last several years. In this Review, we survey the fundamental issues for single-fluorophore fitting routines, localization algorithms based on principles other than fitting, three-dimensional imaging, dipole imaging and techniques for estg. fluorophore positions from images of multiple activated fluorophores. We offer practical advice for users and adopters of algorithms, and we identify areas for further development.
- 433Ernst, D.; Kohler, J. How the Number of Fitting Points for the Slope of the Mean-Square Displacement Influences the Experimentally Determined Particle Size Distribution from Single-Particle Tracking. Phys. Chem. Chem. Phys. 2013, 15, 3429– 3432, DOI: 10.1039/c3cp44391d433How the number of fitting points for the slope of the mean-square displacement influences the experimentally determined particle size distribution from single-particle trackingErnst, Dominique; Koehler, JuergenPhysical Chemistry Chemical Physics (2013), 15 (10), 3429-3432CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The size distribution of nanoparticles can be detd. by single-particle tracking. This yields the mean-squared displacement (MSD) as a function of the lag time, and for normal diffusion the slope of this curve is directly related to the diffusion coeff. or via the Stokes-Einstein relation to the particle size. Here we demonstrate how the exptl. detd. size distributions are affected by the no. of fitting points used to det. the slope of the MSD curve.
- 434Born, M.; Wolf, E. Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light; Elsevier: 2013.There is no corresponding record for this reference.
- 435Liu, S. L.; Li, J.; Zhang, Z. L.; Wang, Z. G.; Tian, Z. Q.; Wang, G. P.; Pang, D. W. Fast and High-Accuracy Localization for Three-Dimensional Single-Particle Tracking. Sci. Rep. 2013, 3, 2462, DOI: 10.1038/srep02462435Fast and high-accuracy localization for three-dimensional single-particle trackingLiu Shu-Lin; Li Jicun; Zhang Zhi-Ling; Wang Zhi-Gang; Tian Zhi-Quan; Wang Guo-Ping; Pang Dai-WenScientific reports (2013), 3 (), 2462 ISSN:.We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization. Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. The precision is almost the same as that of the iterative nonlinear least-squares 3D Gaussian fitting method, but with two orders of magnitude higher computation speed. This approach can reduce considerably the time and costs for processing the large volume data of 3D images for 3D single-particle tracking, which is especially suited for 3D high-precision single-particle tracking, 3D single-molecule imaging and even new microscopy techniques.
- 436Qu, X. H.; Wu, D.; Mets, L.; Scherer, N. F. Nanometer-Localized Multiple Single-Molecule Fluorescence Microscopy. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11298– 11303, DOI: 10.1073/pnas.0402155101436Nanometer-localized multiple single-molecule fluorescence microscopyQu, Xiaohui; Wu, David; Mets, Laurens; Scherer, Norbert F.Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (31), 11298-11303CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Fitting the image of a single mol. to the point spread function of an optical system greatly improves the precision with which single mols. can be located. Centroid localization with nanometer precision has been achieved when a sufficient no. of photons are collected. However, if multiple single mols. reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-mol. (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single mols. within a diffraction-limited area. NALMS microscopy will greatly facilitate single-mol. study of biol. systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resoln. is demonstrated.
- 437Hess, S. T.; Girirajan, T. P.; Mason, M. D. Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy. Biophys. J. 2006, 91, 4258– 4272, DOI: 10.1529/biophysj.106.091116437Ultra-high resolution imaging by fluorescence photoactivation localization microscopyHess, Samuel T.; Girirajan, Thanu P. K.; Mason, Michael D.Biophysical Journal (2006), 91 (11), 4258-4272CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Biol. structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resoln. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large nos. of fluorescent mols. on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small nos. of them at a time, at low excitation intensity. To control the no. of visible fluorophores in the field of view and ensure that optically active mols. are sepd. by much more than the width of the point spread function, photoactivatable fluorescent mols. are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable mols., the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive mols. are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the mols. are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small no. of mols. are visible at a given time, their positions can be detd. precisely; with only ∼100 detected photons per mol., the localization precision can be as much as 10-fold better than the resoln., depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are obsd. by FPALM of PA-GFP on glass. FPALM images are compared with images of the same mols. by wide-field fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with at. force microscopy and show that the full width at half-max. of features ∼86±4 nm is significantly better than the expected diffraction-limited optical resoln. The no. of fluorescent mols. and their brightness distribution have also been detd. using FPALM. This new method suggests a means to address a significant no. of biol. questions that had previously been limited by microscope resoln.
- 438Parthasarathy, R. Rapid, Accurate Particle Tracking by Calculation of Radial Symmetry Centers. Nat. Methods 2012, 9, 724– 726, DOI: 10.1038/nmeth.2071438Rapid, accurate particle tracking by calculation of radial symmetry centersParthasarathy, RaghuveerNature Methods (2012), 9 (7_part3), 724-726CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)I introduce an algorithm for subpixel localization of imaged objects based on an analytic, non-iterative calcn. of the best-fit radial symmetry center. This approach yields tracking accuracies that are near theor. limits, similarly to Gaussian fitting, but with orders-of-magnitude faster execution time, lower sensitivity to nearby particles and applicability to any radially sym. intensity distribution. I demonstrate the method with several types of data, including super-resoln. microscopy images.
- 439Sage, D.; Neumann, F. R.; Hediger, F.; Gasser, S. M.; Unser, M. Automatic Tracking of Individual Fluorescence Particles: Application to the Study of Chromosome Dynamics. IEEE Trans. Image Process. 2005, 14, 1372– 1383, DOI: 10.1109/TIP.2005.852787439Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamicsSage Daniel; Neumann Franck R; Hediger Florence; Gasser Susan M; Unser MichaelIEEE transactions on image processing : a publication of the IEEE Signal Processing Society (2005), 14 (9), 1372-83 ISSN:1057-7149.We present a new, robust, computational procedure for tracking fluorescent markers in time-lapse microscopy. The algorithm is optimized for finding the time-trajectory of single particles in very noisy dynamic (two- or three-dimensional) image sequences. It proceeds in three steps. First, the images are aligned to compensate for the movement of the biological structure under investigation. Second, the particle's signature is enhanced by applying a Mexican hat filter, which we show to be the optimal detector of a Gaussian-like spot in 1/omega2 noise. Finally, the optimal trajectory of the particle is extracted by applying a dynamic programming optimization procedure. We have used this software, which is implemented as a Java plug-in for the public-domain ImageJ software, to track the movement of chromosomal loci within nuclei of budding yeast cells. Besides reducing trajectory analysis time by several 100-fold, we achieve high reproducibility and accuracy of tracking. The application of the method to yeast chromatin dynamics reveals different classes of constraints on mobility of telomeres, reflecting differences in nuclear envelope association. The generic nature of the software allows application to a variety of similar biological imaging tasks that require the extraction and quantitation of a moving particle's trajectory.
- 440Godinez, W. J.; Lampe, M.; Worz, S.; Muller, B.; Eils, R.; Rohr, K. Deterministic and Probabilistic Approaches for Tracking Virus Particles in Time-Lapse Fluorescence Microscopy Image Sequences. Med. Image Anal. 2009, 13, 325– 342, DOI: 10.1016/j.media.2008.12.004440Deterministic and probabilistic approaches for tracking virus particles in time-lapse fluorescence microscopy image sequencesGodinez W J; Lampe M; Worz S; Muller B; Eils R; Rohr KMedical image analysis (2009), 13 (2), 325-42 ISSN:.Modern developments in time-lapse fluorescence microscopy enable the observation of a variety of processes exhibited by viruses. The dynamic nature of these processes requires the tracking of viruses over time to explore spatial-temporal relationships. In this work, we developed deterministic and probabilistic approaches for multiple virus tracking in multi-channel fluorescence microscopy images. The deterministic approaches follow a traditional two-step paradigm comprising particle localization based on either the spot-enhancing filter or 2D Gaussian fitting, as well as motion correspondence based on a global nearest neighbor scheme. Our probabilistic approaches are based on particle filters. We describe approaches based on a mixture of particle filters and based on independent particle filters. For the latter, we have developed a penalization strategy that prevents the problem of filter coalescence (merging) in cases where objects lie in close proximity. A quantitative comparison based on synthetic image sequences is carried out to evaluate the performance of our approaches. In total, eight different tracking approaches have been evaluated. We have also applied these approaches to real microscopy images of HIV-1 particles and have compared the tracking results with ground truth obtained from manual tracking. It turns out that the probabilistic approaches based on independent particle filters are superior to the deterministic schemes as well as to the approaches based on a mixture of particle filters.
- 441Chenouard, N.; Smal, I.; de Chaumont, F.; Maska, M.; Sbalzarini, I. F.; Gong, Y.; Cardinale, J.; Carthel, C.; Coraluppi, S.; Winter, M. Objective Comparison of Particle Tracking Methods. Nat. Methods 2014, 11, 281– 289, DOI: 10.1038/nmeth.2808441Objective comparison of particle tracking methodsChenouard, Nicolas; Smal, Ihor; de Chaumont, Fabrice; Maska, Martin; Sbalzarini, Ivo F.; Gong, Yuanhao; Cardinale, Janick; Carthel, Craig; Coraluppi, Stefano; Winter, Mark; Cohen, Andrew R.; Godinez, William J.; Rohr, Karl; Kalaidzidis, Yannis; Liang, Liang; Duncan, James; Shen, Hongying; Xu, Yingke; Magnusson, Klas E. G.; Jalden, Joakim; Blau, Helen M.; Paul-Gilloteaux, Perrine; Roudot, Philippe; Kervrann, Charles; Waharte, Francois; Tinevez, Jean-Yves; Shorte, Spencer L.; Willemse, Joost; Celler, Katherine; van Wezel, Gilles P.; Dan, Han-Wei; Tsai, Yuh-Show; Ortiz de Solorzano, Carlos; Olivo-Marin, Jean-Christophe; Meijering, ErikNature Methods (2014), 11 (3), 281-289CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Particle tracking is of key importance for quant. anal. of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large nos. of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.
- 442Reid, D. An Algorithm for Tracking Multiple Targets. IEEE Trans. Autom. Control 1979, 24, 843– 854, DOI: 10.1109/TAC.1979.1102177There is no corresponding record for this reference.
- 443Veenman, C. J.; Reinders, M. J.; Backer, E. Resolving Motion Correspondence for Densely Moving Points. IEEE T. Pattern Anal. 2001, 23, 54– 72, DOI: 10.1109/34.899946There is no corresponding record for this reference.
- 444Jiang, S.; Zhou, X.; Kirchhausen, T.; Wong, S. T. Tracking Molecular Particles in Live Cells Using Fuzzy Rule-Based System. Cytometry, Part A 2007, 71, 576– 584, DOI: 10.1002/cyto.a.20411444Tracking molecular particles in live cells using fuzzy rule-based systemJiang Shan; Zhou Xiaobo; Kirchhausen Tom; Wong Stephen T CCytometry. Part A : the journal of the International Society for Analytical Cytology (2007), 71 (8), 576-84 ISSN:1552-4922.Recent development of detection techniques of molecular particles in live cells has stimulated interest in developing the new powerful techniques to track the molecular particles in live cells. One special type of cellular microscopy images is about the formation and transportation of clathrin-coated pits and vesicles. Clathrin-coated pits are very important in studying the behavior of proteins and lipids in live cells. To answer the question, whether there exist "hot spots" for the formation of Clathrin-coated pits or the pits and arrays formed randomly on the plasma membrane, it is necessary to track many hundreds of individual pits dynamically in live-cell microscope movies to capture and monitor how pits and vesicles were formed. Therefore, a motion correspondence algorithm based on fuzzy rule-based system is proposed to resolve the problem of ambiguous association encountered in these dynamic, live-cell images of clathrin assemblies. Results show that this method can accurately track most of the particles in the high volume images.
- 445Shafique, K.; Shah, M. A Noniterative Greedy Algorithm for Multiframe Point Correspondence. IEEE Trans. Pattern Anal. Mach. Intell. 2005, 27, 51– 65, DOI: 10.1109/TPAMI.2005.1445A noniterative greedy algorithm for multiframe point correspondenceShafique Khurram; Shah MubarakIEEE transactions on pattern analysis and machine intelligence (2005), 27 (1), 51-65 ISSN:0162-8828.This paper presents a framework for finding point correspondences in monocular image sequences over multiple frames. The general problem of multiframe point correspondence is NP-hard for three or more frames. A polynomial time algorithm for a restriction of this problem is presented and is used as the basis of the proposed greedy algorithm for the general problem. The greedy nature of the proposed algorithm allows it to be used in real-time systems for tracking and surveillance, etc. In addition, the proposed algorithm deals with the problems of occlusion, missed detections, and false positives by using a single noniterative greedy optimization scheme and, hence, reduces the complexity of the overall algorithm as compared to most existing approaches where multiple heuristics are used for the same purpose. While most greedy algorithms for point tracking do not allow for entry and exit of the points from the scene, this is not a limitation for the proposed algorithm. Experiments with real and synthetic data over a wide range of scenarios and system parameters are presented to validate the claims about the performance of the proposed algorithm.
- 446Jaqaman, K.; Loerke, D.; Mettlen, M.; Kuwata, H.; Grinstein, S.; Schmid, S. L.; Danuser, G. Robust Single-Particle Tracking in Live-Cell Time-Lapse Sequences. Nat. Methods 2008, 5, 695– 702, DOI: 10.1038/nmeth.1237446Robust single-particle tracking in live-cell time-lapse sequencesJaqaman, Khuloud; Loerke, Dinah; Mettlen, Marcel; Kuwata, Hirotaka; Grinstein, Sergio; Schmid, Sandra L.; Danuser, GaudenzNature Methods (2008), 5 (8), 695-702CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here the authors present a tracking algorithm that addresses the principal challenges of SPT, namely high particle d., particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose soln. identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, the authors show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.
- 447Saxton, M. J. Single-Particle Tracking: The Distribution of Diffusion Coefficients. Biophys. J. 1997, 72, 1744– 1753, DOI: 10.1016/S0006-3495(97)78820-9447Single-particle tracking: the distribution of diffusion coefficientsSaxton, Michael J.Biophysical Journal (1997), 72 (4), 1744-1753CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)In single-particle tracking expts., the diffusion coeff. D may be measured from the trajectory of an individual particle in the cell membrane. The statistical distribution of single-trajectory diffusion coeffs. is examd. by Monte Carlo calcns. The width of this distribution may be useful as a measure of the heterogeneity of the membrane and as a test of models of hindered diffusion in the membrane. For some models, the distribution of the short-range diffusion coeff. is much narrower than the obsd. distribution for proteins diffusing in cell membranes. To aid in the anal. of single-particle tracking measurements, the distribution of D is examd. for various definitions of D and for various trajectory lengths.
- 448Dahan, M. From Analog to Digital: Exploring Cell Dynamics with Single Quantum Dots. Histochem. Cell Biol. 2006, 125, 451– 456, DOI: 10.1007/s00418-005-0105-x448From analog to digital: exploring cell dynamics with single quantum dotsDahan, MaximeHistochemistry and Cell Biology (2006), 125 (5), 451-456CODEN: HCBIFP; ISSN:0948-6143. (Springer)Semiconductor quantum dots (QDs) have emerged as new fluorescent probes for biol. When combined with ultrasensitive optical techniques, they allow motions of individual biomols. to be tracked in live cells with high signal-to-noise and over unprecedented durations. Single QD imaging readily offers a powerful tool to investigate the organization in cell membranes. Altogether QDs will contribute to more advanced biol. imaging and enable new studies on the dynamics of cellular processes.
- 449Dupont, A.; Gorelashvili, M.; Schuller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D. Three-Dimensional Single-Particle Tracking in Live Cells: News from the Third Dimension. New J. Phys. 2013, 15, 075008 DOI: 10.1088/1367-2630/15/7/075008449Three-dimensional single-particle tracking in live cells: news from the third dimensionDupont, A.; Gorelashvili, M.; Schueller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D.New Journal of Physics (2013), 15 (July), 075008CODEN: NJOPFM; ISSN:1367-2630. (IOP Publishing Ltd.)Single-particle tracking (SPT) is of growing importance in the biophys. community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its tech. simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort. Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) anal. introduced previously. The 3D trajectories and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells' cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coeff. values derived from the 2D anal. were therefore biased.
- 450Bannai, H.; Lévi, S.; Schweizer, C.; Dahan, M.; Triller, A. Imaging the Lateral Diffusion of Membrane Molecules with Quantum Dots. Nat. Protoc. 2006, 1, 2628– 2634, DOI: 10.1038/nprot.2006.429450Imaging the lateral diffusion of membrane molecules with quantum dotsBannai, Hiroko; Levi, Sabine; Schweizer, Claude; Dahan, Maxime; Triller, AntoineNature Protocols (2006), 1 (6), 2628-2634CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol describes a sensitive approach to tracking the motion of membrane mols. such as lipids and proteins with mol. resoln. in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane mols. of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and org. dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The exptl. procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.
- 451Saxton, M. J. Single-Particle Tracking: Effects of Corrals. Biophys. J. 1995, 69, 389– 398, DOI: 10.1016/S0006-3495(95)79911-8451Single-particle tracking: effects of corralsSaxton, Michael J.Biophysical Journal (1995), 69 (2), 389-95CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Structural proteins of the membrane skeleton are thought to form "corrals" at the membrane surface, and these corrals may restrict lateral diffusion of membrane proteins. Recent exptl. developments in single-particle tracking and laser trapping make it possible to examine the corral model in detail. Techniques to interpret these expts. are presented. First, escape times for a diffusing particle in a corral are obtained from Monte Carlo calcns. and anal. solns. for various corral sizes, shapes, and escape probabilities, and reduced to a common curve. Second, the identification of corrals in tracking expts. is considered. The simplest way to identify corrals is by sight. If the walls are impermeable enough, a trajectory fills the corral before the diffusing particle escapes. The fraction of distinct sites visited before escape is calcd. for corrals of various sizes, shapes, and escape probabilities, and reduced to a common curve. This fraction is also a measure of the probability that the diffusing species will react with another species in the corral before escaping. Finally, the effect of the sampling interval on the measurement of the short-range diffusion coeff. is examd.
- 452Rock, R. S. Myosin VI Is a Processive Motor with a Large Step Size. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 13655– 13659, DOI: 10.1073/pnas.191512398452Myosin VI is a processive motor with a large step sizeRock, Ronald S.; Rice, Sarah E.; Wells, Amber L.; Purcell, Thomas J.; Spudich, James A.; Sweeney, H. LeeProceedings of the National Academy of Sciences of the United States of America (2001), 98 (24), 13655-13659CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Myosin VI is a mol. motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mech. advances before it releases from actin. Here we show that myosin VI is also processive by using single mol. motility and optical trapping expts. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.
- 453Ma, S.; Chisholm, R. L. Cytoplasmic Dynein-Associated Structures Move Bidirectionally in Vivo. J. Cell Sci. 2002, 115, 1453– 1460453Cytoplasmic dynein-associated structures move bidirectionally in vivoMa, Shuo; Chisholm, Rex L.Journal of Cell Science (2002), 115 (7), 1453-1460CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubule-based motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively, different motors could assoc. with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-assocd. structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addn., dynein puncta often rapidly reverse their direction. Dynein stably assocs. with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo assocn. but rather by switching activity of motors assocd. with the cargo. These observations suggest that both plus- and minus-end-directed motors assoc. with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.
- 454Sieczkarski, S. B.; Whittaker, G. R. Dissecting Virus Entry via Endocytosis. J. Gen. Virol. 2002, 83, 1535– 1545, DOI: 10.1099/0022-1317-83-7-1535454Dissecting virus entry via endocytosisSieczkarski, Sara B.; Whittaker, Gary R.Journal of General Virology (2002), 83 (7), 1535-1545CODEN: JGVIAY; ISSN:0022-1317. (Society for General Microbiology)A review. Numerous virus families utilize endocytosis to infect host cells, mediating virus internalization as well as trafficking to the site of replication. Recent research has demonstrated that viruses employ the full endocytic capabilities of the cell. The endocytic pathways utilized include clathrin-mediated endocytosis, caveolae, macropinocytosis and novel non-clathrin, non-caveolae pathways. The tools to study endocytosis and, consequently, virus entry are becoming more effective and specific as the amt. of information on endocytic component structure and function increases. The use of inhibitory drugs, although still quite common, often leads to non-specific disruptions in the cell. Mol. inhibitors in the form of dominant-neg. proteins have surpassed the use of chem. inhibitors in terms of specificity to individual pathways. Dominant-neg. mols. are derived from both structural proteins of endocytosis, such as dynamin and caveolin, and regulatory proteins, primarily small GTPases and kinases. This review focuses on the exptl. approaches taken to examine virus entry and provides both classic examples and recent research on a variety of virus families.
- 455Boulant, S.; Stanifer, M.; Lozach, P. Y. Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis. Viruses 2015, 7, 2794– 2815, DOI: 10.3390/v7062747455Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosisBoulant, Steeve; Stanifer, Megan; Lozach, Pierre-YvesViruses (2015), 7 (6), 2794-2815CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)During viral infection the first challenge that viruses have to overcome is gaining access to the intracellular compartment. The infection process starts when the virus contacts the surface of the host cell. A complex series of events ensues, including diffusion at the host cell membrane surface, binding to receptors, signaling, internalization, and delivery of the genetic information. The focus of this review is on the very initial steps of virus entry, from receptor binding to particle uptake into the host cell. We will discuss how viruses find their receptor, move to sub-membranous regions permissive for entry, and how they hijack the receptor-mediated signaling pathway to promote their internalization.
- 456Mudhakir, D.; Harashima, H. Learning from the Viral Journey: How to Enter Cells and How to Overcome Intracellular Barriers to Reach the Nucleus. AAPS J. 2009, 11, 65– 77, DOI: 10.1208/s12248-009-9080-9456Learning from the viral journey: how to enter cells and how to overcome intracellular barriers to reach the nucleusMudhakir Diky; Harashima HideyoshiThe AAPS journal (2009), 11 (1), 65-77 ISSN:.Viruses deliver their genome into host cells where they subsequently replicate and multiply. A variety of relevant strategies have evolved by which viruses gain intracellular access and utilize cellular machinery for the synthesis of their genome. Therefore, the viral journey provides insight into the cell's trafficking machinery and how it can be best exploited to improve nonviral gene delivery systems. This review summarizes viral internalization pathways and intracellular trafficking of viruses, with an emphasis on the endosomal escape processes of nonenveloped viruses. Intracellular events from viral entry through nuclear delivery of the viral complementary DNA are also discussed.
- 457Damm, E. M.; Pelkmans, L. Systems Biology of Virus Entry in Mammalian Cells. Cell. Microbiol. 2006, 8, 1219– 1227, DOI: 10.1111/j.1462-5822.2006.00745.x457Systems biology of virus entry in mammalian cellsDamm, Eva-Maria; Pelkmans, LucasCellular Microbiology (2006), 8 (8), 1219-1227CODEN: CEMIF5; ISSN:1462-5814. (Blackwell Publishing Ltd.)A review. In this article, the authors define systems biol. of virus entry in mammalian cells as the discipline that combines several approaches to comprehensively understand the collective phys. behavior of virus entry routes, and to understand the coordinated operation of the functional modules and mol. machineries that lead to this phys. behavior. Clearly, these are extremely ambitious aims, but recent developments in different life science disciplines slowly allow us to set them as realistic, although very distant, goals. Besides classical approaches to obtain high-resoln. information of the mols., particles and machines involved, the authors require approaches that can monitor collective behavior of many mols., particles and machines simultaneously, to reveal design principles of the systems as a whole. Here the authors will discuss approaches that fall in the latter category, namely time-lapse imaging and single-particle tracking (SPT) combined with computational anal. and modeling, and genome-wide RNA interference approaches to reveal the host components required for virus entry. These techniques should in the future allow us to assign host genes to the systems' functions and characteristics, and allow emergence-driven, in silico assembly of networks that include interactions with increasing hierarchy (mols.-multiprotein complexes-vesicles and organelles), and kinetics and subcellular spatiality, to allow realistic simulations of virus entry in real time.
- 458Chang, K.; Baginski, J.; Hassan, S. F.; Volin, M.; Shukla, D.; Tiwari, V. Filopodia and Vruses: An Analysis of Membrane Processes in Entry Mechanisms. Front. Microbiol. 2016, 7, 300, DOI: 10.3389/fmicb.2016.00300458Filopodia and Viruses: An Analysis of Membrane Processes in Entry MechanismsChang Kenneth; Baginski John; Volin Michael; Tiwari Vaibhav; Hassan Samer F; Shukla DeepakFrontiers in microbiology (2016), 7 (), 300 ISSN:1664-302X.Filopodia are thin, actin rich bundles protruding from cell plasma membranes, serving physiological purposes, such as probing the environment and facilitating cell-to-cell adhesion. Recent studies have highlighted that actively polymerized filopodial-protrusions are exploited during virus entry, trafficking, spread, and the development of clinical pathology of viral diseases. These observations have caused a surge in investigation of the key determinants of filopodial induction and their influence on cell topography including receptor expression for viral entry. It is now very clear that filopodia can provide unique opportunities for many viruses to invade host cells vertically during primary infection, or horizontally during virus spread from cell-to-cell. These emerging concepts can explain the unprecedented ability of viruses to invade both nearby and long-distant host cells, a feature that may directly contribute to viral tropism. In this review, we summarize the significance of filopodia in viral diseases and discuss future therapeutic possibilities to precisely target filopodial-flyovers to prevent or control infectious diseases.
- 459Shinya, K.; Ebina, M.; Yamada, S.; Ono, M.; Kasai, N.; Kawaoka, Y. Avian Flu: Influenza Virus Receptors in the Human Airway. Nature 2006, 440, 435– 436, DOI: 10.1038/440435a459Influenza virus receptors in the human airway: avian and human flu viruses seem to target different regions of a patient's respiratory tract.Shinya, Kyoko; Ebina, Masahito; Yamada, Shinya; Ono, Masao; Kasai, Noriyuki; Kawaoka, YoshihiroNature (London, United Kingdom) (2006), 440 (7083), 435-436CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)H5N1 influenza viruses derived from humans and birds target different regions of a patient's respiratory tract. Although the viruses preferentially recognizing SAα2,3Gal can be transmitted from birds to humans, they can replicate only efficiently in the lower parts of respiratory tract. The may explain the phenomenon that H5N1 viruses rarely infect and spread between humans although they can replicate efficiently in the lungs.
- 460Ibricevic, A.; Pekosz, A.; Walter, M. J.; Newby, C.; Battaile, J. T.; Brown, E. G.; Holtzman, M. J.; Brody, S. L. Influenza Virus Receptor Specificity and Cell Tropism in Mouse and Human Airway Epithelial Cells. J. Virol. 2006, 80, 7469– 7480, DOI: 10.1128/JVI.02677-05460Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cellsIbricevic, Aida; Pekosz, Andrew; Walter, Michael J.; Newby, Celeste; Battaile, John T.; Brown, Earl G.; Holtzman, Michael J.; Brody, Steven L.Journal of Virology (2006), 80 (15), 7469-7480CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an α2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an α2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the α2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The α2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, α2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the α2,3-linked SA receptor.
- 461Lakadamyali, M.; Rust, M. J.; Zhuang, X. Endocytosis of Influenza Viruses. Microbes Infect. 2004, 6, 929– 936, DOI: 10.1016/j.micinf.2004.05.002461Endocytosis of influenza virusesLakadamyali, Melike; Rust, Michael J.; Zhuang, XiaoweiMicrobes and Infection (2004), 6 (10), 929-936CODEN: MCINFS; ISSN:1286-4579. (Elsevier B.V.)A review. Receptor-mediated endocytosis is known to play an important role in the entry of many viruses into host cells. However, the exact internalization mechanism has, until recently, remained poorly understood for many medically important viruses, including influenza. Developments in real-time imaging of single viruses as well as the use of dominant-neg. mutants to selectively block specific endocytic pathways have improved our understanding of the influenza infection process.
- 462Mercer, J.; Schelhaas, M.; Helenius, A. Virus Entry by Endocytosis. Annu. Rev. Biochem. 2010, 79, 803– 833, DOI: 10.1146/annurev-biochem-060208-104626462Virus entry by endocytosisMercer, Jason; Schelhaas, Mario; Helenius, AriAnnual Review of Biochemistry (2010), 79 (), 803-833CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Although viruses are simple in structure and compn., their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biol. of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
- 463Nisole, S.; Saib, A. Early Steps of Retrovirus Replicative Cycle. Retrovirology 2004, 1, 9, DOI: 10.1186/1742-4690-1-9463Early steps of retrovirus replicative cycleNisole Sebastien; Saib AliRetrovirology (2004), 1 (), 9 ISSN:.During the last two decades, the profusion of HIV research due to the urge to identify new therapeutic targets has led to a wealth of information on the retroviral replication cycle. However, while the late stages of the retrovirus life cycle, consisting of virus replication and egress, have been partly unraveled, the early steps remain largely enigmatic. These early steps consist of a long and perilous journey from the cell surface to the nucleus where the proviral DNA integrates into the host genome. Retroviral particles must bind specifically to their target cells, cross the plasma membrane, reverse-transcribe their RNA genome, while uncoating the cores, find their way to the nuclear membrane and penetrate into the nucleus to finally dock and integrate into the cellular genome. Along this journey, retroviruses hijack the cellular machinery, while at the same time counteracting cellular defenses. Elucidating these mechanisms and identifying which cellular factors are exploited by the retroviruses and which hinder their life cycle, will certainly lead to the discovery of new ways to inhibit viral replication and to improve retroviral vectors for gene transfer. Finally, as proven by many examples in the past, progresses in retrovirology will undoubtedly also provide some priceless insights into cell biology.
- 464Spear, P. G.; Eisenberg, R. J.; Cohen, G. H. Three Classes of Cell Surface Receptors for Alphaherpesvirus Entry. Virology 2000, 275, 1– 8, DOI: 10.1006/viro.2000.0529464Three classes of cell surface receptors for alphaherpesvirus entrySpear, Patricia G.; Eisenberg, Roselyn J.; Cohen, Gary H.Virology (2000), 275 (1), 1-8CODEN: VIRLAX; ISSN:0042-6822. (Academic Press)A review with 66 refs. (c) 2000 Academic Press.
- 465Bailey, C. J.; Crystal, R. G.; Leopold, P. L. Association of Adenovirus with the Microtubule Organizing Center. J. Virol. 2003, 77, 13275– 13287, DOI: 10.1128/JVI.77.24.13275-13287.2003465Association of adenovirus with the microtubule organizing centerBailey, Christopher J.; Crystal, Ronald G.; Leopold, Philip L.Journal of Virology (2003), 77 (24), 13275-13287CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an assocn. with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and assocs. with the microtubule organizing center (MTOC). To test this hypothesis, we established an exptl. system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymn. of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-assocd. Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymg. agent nocodazole, but not to the microfilament-depolymg. agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissocn. of Ad from microtubules likely requires nuclear factors.
- 466Kielian, M.; Rey, F. A. Virus Membrane-Fusion Proteins: More Than One Way to Make a Hairpin. Nat. Rev. Microbiol. 2006, 4, 67– 76, DOI: 10.1038/nrmicro1326466Virus membrane-fusion proteins: more than one way to make a hairpinKielian, Margaret; Rey, Felix A.Nature Reviews Microbiology (2006), 4 (1), 67-76CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Structure-function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. In both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. This review will focus in particular on the properties of the more recently described class II proteins.
- 467Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D. C. Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation. Biophys. J. 2013, 104, 2373– 2382, DOI: 10.1016/j.bpj.2013.04.005467Tracking Image Correlation: Combining Single-Particle Tracking and Image CorrelationDupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D. C.Biophysical Journal (2013), 104 (11), 2373-2382CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The interactions and coordination of biomols. are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quant. global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomols., usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization anal. along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle's position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addn., the cross-correlation anal. shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.
- 468Alenquer, M.; Vale-Costa, S.; Etibor, T. A.; Ferreira, F.; Sousa, A. L.; Amorim, M. J. Influenza A Virus Ribonucleoproteins Form Liquid Organelles at Endoplasmic Reticulum Exit Sites. Nat. Commun. 2019, 10, 1629, DOI: 10.1038/s41467-019-09549-4468Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sitesAlenquer Marta; Vale-Costa Silvia; Etibor Temitope Akhigbe; Ferreira Filipe; Sousa Ana Laura; Amorim Maria Joao; Sousa Ana LauraNature communications (2019), 10 (1), 1629 ISSN:.Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.
- 469Jo, S.; Kawaguchi, A.; Takizawa, N.; Morikawa, Y.; Momose, F.; Nagata, K. Involvement of Vesicular Trafficking System in Membrane Targeting of the Progeny Influenza Virus Genome. Microbes Infect. 2010, 12, 1079– 1084, DOI: 10.1016/j.micinf.2010.06.011469Involvement of vesicular trafficking system in membrane targeting of the progeny influenza virus genomeJo, Shuichi; Kawaguchi, Atsushi; Takizawa, Naoki; Morikawa, Yuko; Momose, Fumitaka; Nagata, KyosukeMicrobes and Infection (2010), 12 (12-13), 1079-1084CODEN: MCINFS; ISSN:1286-4579. (Elsevier Masson SAS)The genome of influenza type A virus consists of single-stranded RNAs of neg. polarity. Progeny viral RNA (vRNA) replicated in the nucleus is nuclear-exported, and finally transported to the budding site beneath the plasma membrane. However, the precise process of the membrane targeting of vRNA is unclear, although viral proteins and cytoskeleton are thought to play roles. Here, we have visualized the translocation process of progeny vRNA using fluorescence in situ hybridization method. Our results provide an evidence of the involvement of vesicular trafficking in membrane targeting of progeny vRNA independent of that of viral membrane proteins.
- 470Avilov, S. V.; Moisy, D.; Naffakh, N.; Cusack, S. Influenza A Virus Progeny vRNP Trafficking in Live Infected Cells Studied with the Virus-Encoded Fluorescently Tagged PB2 Protein. Vaccine 2012, 30, 7411– 7417, DOI: 10.1016/j.vaccine.2012.09.077470Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 proteinAvilov, Sergiy V.; Moisy, Dorothee; Naffakh, Nadia; Cusack, StephenVaccine (2012), 30 (51), 7411-7417CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, the authors have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells. Here, using this virus, the authors studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection. In agreement with recent reports, upon nuclear export, progeny vRNPs accumulate in the particles contg. Rab11, a multifunctional protein involved in vesicle trafficking which resides at recycling endosomes. Fluorescence resonance energy transfer microscopy indicated a distance <10 nm between PB2 and Rab11, suggesting that a direct interaction occurs. Single particle tracking anal. showed that most of the motions of vRNP-pos. particles in infected cells are slow, while rapid directional motions intermittently occur. Anal. focused on these intermittent motions indicated that depolymn. of either microtubules or actin filaments moderately reduced their occurrence, while disruption of both cytoskeleton components in combination suppressed the rapid motions entirely. Thus, the split-GFP based virus enabled the authors to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of vRNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.
- 471Amorim, M. J.; Bruce, E. A.; Read, E. K. C.; Foeglein, A.; Mahen, R.; Stuart, A. D.; Digard, P. A Rab11-and Microtubule-Dependent Mechanism for Cytoplasmic Transport of Influenza A Virus Viral RNA. J. Virol. 2011, 85, 4143– 4156, DOI: 10.1128/JVI.02606-10471A Rab11- and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNAAmorim, Maria Joao; Bruce, Emily A.; Read, Eliot K. C.; Foeglein, Agnes; Mahen, Robert; Stuart, Amanda D.; Digard, PaulJournal of Virology (2011), 85 (9), 4143-4156CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Copptn. expts. showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.
- 472Hendrix, J.; Baumgartel, V.; Schrimpf, W.; Ivanchenko, S.; Digman, M. A.; Gratton, E.; Krausslich, H. G.; Muller, B.; Lamb, D. C. Live-Cell Observation of Cytosolic HIV-1 Assembly onset Reveals RNA-Interacting Gag Oligomers. J. Cell Biol. 2015, 210, 629– 646, DOI: 10.1083/jcb.201504006472Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomersHendrix, Jelle; Baumgaertel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kraeusslich, Hans-Georg; Mueller, Barbara; Lamb, Don C.Journal of Cell Biology (2015), 210 (4), 629-646CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Redn. of mobility is assocd. with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was obsd. on the seconds timescale that oligomerized in a concn.-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.
- 473Jose, J.; Tang, J.; Taylor, A. B.; Baker, T. S.; Kuhn, R. J. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells. Viruses 2015, 7, 6182– 6199, DOI: 10.3390/v7122926473Fluorescent protein-tagged sindbis virus E2 glycoprotein allows single particle analysis of virus budding from live cellsJose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.Viruses (2015), 7 (12), 6182-6199CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examd. the virions by transmission electron cryo-microscopy and detd. the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, resp. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry anal., contributed to a 10-fold redn. in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking expts. We used the FP-tagged virus for high-resoln. live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close assocn. with filopodial extensions.
- 474Baumgartel, V.; Muller, B.; Lamb, D. C. Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly. Viruses 2012, 4, 777– 799, DOI: 10.3390/v4050777474Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assemblyBaumgartel Viola; Muller Barbara; Lamb Don CViruses (2012), 4 (5), 777-99 ISSN:.Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.
- 475Perlman, M.; Resh, M. D. Identification of an Intracellular Trafficking and Assembly Pathway for HIV-1 Gag. Traffic 2006, 7, 731– 745, DOI: 10.1111/j.1398-9219.2006.00428.x475Identification of an intracellular trafficking and assembly pathway for HIV-1 GagPerlman, Mira; Resh, Marilyn D.Traffic (Oxford, United Kingdom) (2006), 7 (6), 731-745CODEN: TRAFFA; ISSN:1398-9219. (Blackwell Publishing Ltd.)Retroviral Gag proteins are membrane-bound poly-proteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle prodn. is detd. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amts. of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag assocn. with MVB-like compartments regulates the site of HIV-1 budding and particle formation.
- 476Gunzenhauser, J.; Olivier, N.; Pengo, T.; Manley, S. Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions. Nano Lett. 2012, 12, 4705– 4710, DOI: 10.1021/nl3021076476Quantitative super-resolution imaging reveals protein stoichiometry and nanoscale morphology of assembling HIV-Gag virionsGunzenhauser, Julia; Olivier, Nicolas; Pengo, Thomas; Manley, SulianaNano Letters (2012), 12 (9), 4705-4710CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The HIV structural protein Gag assembles to form spherical particles of radius ∼70 nm. During the assembly process, the no. of Gag proteins increases over several orders of magnitude from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as std. fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, the authors demonstrate an approach using super-resoln. fluorescence imaging that permits quant. morphol. and mol. counting anal. over a wide range of protein cluster sizes. The authors applied this technique to the anal. of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions.
- 477Fogarty, K. H.; Chen, Y.; Grigsby, I. F.; Macdonald, P. J.; Smith, E. M.; Johnson, J. L.; Rawson, J. M.; Mansky, L. M.; Mueller, J. D. Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color z-Scan Fluorescence Fluctuation Spectroscopy. Biophys. J. 2011, 100, 1587– 1595, DOI: 10.1016/j.bpj.2011.02.008477Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation SpectroscopyFogarty, Keir H.; Chen, Yan; Grigsby, Iwen F.; MacDonald, Patrick J.; Smith, Elizabeth M.; Johnson, Jolene L.; Rawson, Jonathan M.; Mansky, Louis M.; Mueller, Joachim D.Biophysical Journal (2011), 100 (6), 1587-1595CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness anal. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation vol., cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addn. of a distinctly colored ref. protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concn.-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.
- 478Chen, Y.; Wu, B.; Musier-Forsyth, K.; Mansky, L. M.; Mueller, J. D. Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry. Biophys. J. 2009, 96, 1961– 1969, DOI: 10.1016/j.bpj.2008.10.067478Fluorescence fluctuation spectroscopy on viral-like particles reveals variable Gag stoichiometryChen, Yan; Wu, Bin; Musier-Forsyth, Karin; Mansky, Louis M.; Mueller, Joachim D.Biophysical Journal (2009), 96 (5), 1961-1969CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Fluorescence fluctuation spectroscopy dets. the brightness, size, and concn. of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation vol. Brightness provides a measure of the no. of fluorescently labeled proteins within a complex and has been used previously to det. the stoichiometry of small oligomers in cells. We extend brightness anal. to large macromol. protein complexes contg. thousands of proteins and det. their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to det. the stoichiometry of HIV-1 Gag within the particles. Control expts. establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The expts. further show that the brightness scales linearly with the amt. of labeled Gag within the particle. Brightness anal. shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not const., but varies with the amt. of transfected DNA plasmid. We obsd. HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP prodn. This study shows that brightness anal. has the potential to become an important tool for investigating large mol. complexes by providing quant. information about their size and compn.
- 479Sardo, L.; Hatch, S. C.; Chen, J.; Nikolaitchik, O.; Burdick, R. C.; Chen, D.; Westlake, C. J.; Lockett, S.; Pathak, V. K.; Hu, W. S. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly. J. Virol. 2015, 89, 10832– 10840, DOI: 10.1128/JVI.01146-15479Dynamics of HIV-1 RNA near the plasma membrane during virus assemblySardo, Luca; Hatch, Steven C.; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C.; Chen, De; Westlake, Christopher J.; Lockett, Stephen; Pathak, Vinay K.; Hu, Wei-ShauJournal of Virology (2015), 89 (21), 10832-10840CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)To increase our understanding of the events that lead to HIV-1 genome packaging, we examd. the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we obsd. that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions.
- 480Rahman, S. A.; Koch, P.; Weichsel, J.; Godinez, W. J.; Schwarz, U.; Rohr, K.; Lamb, D. C.; Krausslich, H. G.; Muller, B. Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy. J. Virol. 2014, 88, 7904– 7914, DOI: 10.1128/JVI.00431-14480Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopyRahman Sheikh Abdul; Koch Peter; Krausslich Hans-Georg; Muller Barbara; Weichsel Julian; Schwarz Ulrich; Godinez William J; Rohr Karl; Lamb Don CJournal of virology (2014), 88 (14), 7904-14 ISSN:.Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells. Importance: HIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.
- 481Baumgartel, V.; Ivanchenko, S.; Dupont, A.; Sergeev, M.; Wiseman, P. W.; Krausslich, H. G.; Brauchle, C.; Muller, B.; Lamb, D. C. Live-Cell Visualization of Dynamics of HIV Budding Site Interactions with an ESCRT Component. Nat. Cell Biol. 2011, 13, 469– 474, DOI: 10.1038/ncb2215481Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT componentBaumgartel Viola; Ivanchenko Sergey; Dupont Aurelie; Sergeev Mikhail; Wiseman Paul W; Krausslich Hans-Georg; Brauchle Christoph; Muller Barbara; Lamb Don CNature cell biology (2011), 13 (4), 469-74 ISSN:.HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.
- 482Dimitrov, D. S.; Willey, R. L.; Sato, H.; Chang, L. J.; Blumenthal, R.; Martin, M. A. Quantitation of Human Immunodeficiency Virus Type 1 Infection Kinetics. J. Virol. 1993, 67, 2182– 2190, DOI: 10.1128/JVI.67.4.2182-2190.1993482Quantitation of human immunodeficiency virus type 1 infection kineticsDimitrov, Dimiter S.; Willey, Ronald L.; Sato, Hironori; Chang, Lung Ji; Blumenthal, Robert; Martin, Malcolm A.Journal of Virology (1993), 67 (4), 2182-90CODEN: JOVIAM; ISSN:0022-538X.Tissue culture infections of CD4-pos. human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in 3 stages: (1) a period following the initiation of an infection during which no detectable virus is produced; (2) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (3) a final period when virus prodn. declines. Equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection were derived. The analyses indicated that the crit. parameter affecting the kinetics of HIV-1 infection is the infection rate const. k = lnn/ti, where n is the no. of infectious virions produced by one cell (about 102) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was the finding that the infectivity of HIV-1 during cell-to-cell transmission is 102 to 103 times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. It was also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small no. of infectious particles produced per cycle.
- 483Johnson, D. C.; Huber, M. T. Directed Egress of Animal Viruses Promotes Cell-to-Cell Spread. J. Virol. 2002, 76, 1– 8, DOI: 10.1128/JVI.76.1.1-8.2002483Directed egress of animal viruses promotes cell-to-cell spreadJohnson, David C.; Huber, Mary T.Journal of Virology (2002), 76 (1), 1-8CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A review focuses on direct cell-to-cell spread of animal viruses in solid tissues. Three interesting examples of how animal viruses have organized their egress strategies to promote cell-to-cell spread are described. These examples include alpha herpesviruses, HIV, and poxviruses. The alpha herpesviruses provide fascinating examples of viruses that replicate in polarized cells, epithelial cells, and neurons and imitate intracellular sorting pathways to direct nascent virions to cell junctions, promoting infection of adjacent epithelial cells and directed spread within the nervous system. HIV normally replicates in lymphocytes and macrophages, cells that are not usually considered polarized. Poxviruses can induce the formation of actin tails that launch virus particles from the cell surface on the tips of microvilli toward neighboring cells.
- 484Sattentau, Q. Avoiding the Void: Cell-to-Cell Spread of Human Viruses. Nat. Rev. Microbiol. 2008, 6, 815– 826, DOI: 10.1038/nrmicro1972484Avoiding the void: cell-to-cell spread of human virusesSattentau, QuentinNature Reviews Microbiology (2008), 6 (11), 815-826CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. The initial stages of animal virus infection are generally described as the binding of free virions to permissive target cells followed by entry and replication. Although this route of infection is undoubtedly important, many viruses that are pathogenic for humans, including HIV-1, herpes simplex virus and measles, can also move between cells without diffusing through the extracellular environment. Cell-to-cell spread not only facilitates rapid viral dissemination, but may also promote immune evasion and influence disease. The author discusses the various mechanisms by which viruses move directly between cells and the implications of this for viral dissemination and pathogenesis.
- 485Sherer, N. M.; Lehmann, M. J.; Jimenez-Soto, L. F.; Horensavitz, C.; Pypaert, M.; Mothes, W. Retroviruses Can Establish Filopodial Bridges for Efficient Cell-to-Cell Transmission. Nat. Cell Biol. 2007, 9, 310– 315, DOI: 10.1038/ncb1544485Retroviruses can establish filopodial bridges for efficient cell-to-cell transmissionSherer, Nathan M.; Lehmann, Maik J.; Jimenez-Soto, Luisa F.; Horensavitz, Christina; Pypaert, Marc; Mothes, WaltherNature Cell Biology (2007), 9 (3), 310-315CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)The spread of retroviruses between cells is estd. to be 2-3 orders of magnitude more efficient when cells can phys. interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virol. or infectious synapses. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong assocn. of the viral envelope glycoprotein (Env) in an infected cell with the receptor mols. in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.
- 486Mothes, W.; Sherer, N. M.; Jin, J.; Zhong, P. Virus Cell-to-Cell Transmission. J. Virol. 2010, 84, 8360– 8368, DOI: 10.1128/JVI.00443-10486Virus cell-to-cell transmissionMothes, Walther; Sherer, Nathan M.; Jin, Jing; Zhong, PengJournal of Virology (2010), 84 (17), 8360-8368CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)A review. Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion mols., and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections.
- 487Eugenin, E. A.; Gaskill, P. J.; Berman, J. W. Tunneling Nanotubes (TNT) Are Induced by HIV-Infection of Macrophages: A Potential Mechanism for Intercellular HIV Trafficking. Cell. Immunol. 2009, 254, 142– 148, DOI: 10.1016/j.cellimm.2008.08.005487Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: A potential mechanism for intercellular HIV traffickingEugenin, E. A.; Gaskill, P. J.; Berman, J. W.Cellular Immunology (2009), 254 (2), 142-148CODEN: CLIMB8; ISSN:0008-8749. (Elsevier B.V.)Cell to cell communication is essential for the organization/coordination of multicellular systems and cellular development. Cellular communication is mediated by sol. factors, including growth factors, neurotransmitters, cytokines/chemokines, gap junctions, and the recently described tunneling nanotubes (TNT). TNT are long cytoplasmatic bridges that enable long range directed communication between cells. The proposed function for TNT is the cell-to-cell transfer of large cellular structures such as vesicles and organelles. We demonstrate that HIV-infection of human macrophages results in an increased no. of TNT, and show HIV particles within these structures. We propose that HIV "highjacks" TNT communication to spread HIV through an intercellular route between communicated cells, contributing to the pathogenesis of AIDS.
- 488Gousset, K.; Schiff, E.; Langevin, C.; Marijanovic, Z.; Caputo, A.; Browman, D. T.; Chenouard, N.; de Chaumont, F.; Martino, A.; Enninga, J. Prions Hijack Tunnelling Nanotubes for Intercellular Spread. Nat. Cell Biol. 2009, 11, 328– 336, DOI: 10.1038/ncb1841488Prions hijack tunnelling nanotubes for intercellular spreadGousset, Karine; Schiff, Edwin; Langevin, Christelle; Marijanovic, Zrinka; Caputo, Anna; Browman, Duncan T.; Chenouard, Nicolas; de Chaumont, Fabrice; Martino, Angelo; Enninga, Jost; Olivo-Marin, Jean-Christophe; Maennel, Daniela; Zurzolo, ChiaraNature Cell Biology (2009), 11 (3), 328-336CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)In variant Creutzfeldt-Jakob disease, prions (PrPSc) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunneling nanotubes (TNTs) have been identified between cells, both in vitro and in vivo, and may represent a conserved means of cell-to-cell communication. Here we show that TNTs allow transfer of exogenous and endogenous PrPSc between infected and naive neuronal CAD cells. Significantly, transfer of endogenous PrPSc aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrPSc spreading in neuronal cells. In addn., we detected the transfer of labeled PrPSc from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS. We therefore propose that TNTs are involved in the spreading of PrPSc within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.
- 489Sowinski, S.; Jolly, C.; Berninghausen, O.; Purbhoo, M. A.; Chauveau, A.; Köhler, K.; Oddos, S.; Eissmann, P.; Brodsky, F. M.; Hopkins, C. Membrane Nanotubes Physically Connect T Cells over Long Distances Presenting a Novel Route for HIV-1 Transmission. Nat. Cell Biol. 2008, 10, 211– 219, DOI: 10.1038/ncb1682489Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmissionSowinski, Stefanie; Jolly, Clare; Berninghausen, Otto; Purbhoo, Marco A.; Chauveau, Anne; Koehler, Karsten; Oddos, Stephane; Eissmann, Philipp; Brodsky, Frances M.; Hopkins, Colin; Oenfelt, Bjoern; Sattentau, Quentin; Davis, Daniel M.Nature Cell Biology (2008), 10 (2), 211-219CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Transmission of HIV-1 via intercellular connections has been estd. as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virol. synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.
- 490Kumar, A.; Kim, J. H.; Ranjan, P.; Metcalfe, M. G.; Cao, W.; Mishina, M.; Gangappa, S.; Guo, Z.; Boyden, E. S.; Zaki, S. Influenza Virus Exploits Tunneling Nanotubes for Cell-to-Cell Spread. Sci. Rep. 2017, 7, 40360, DOI: 10.1038/srep40360490Influenza virus exploits tunneling nanotubes for cell-to-cell spreadKumar, Amrita; Kim, Jin Hyang; Ranjan, Priya; Metcalfe, Maureen G.; Cao, Weiping; Mishina, Margarita; Gangappa, Shivaprakash; Guo, Zhu; Boyden, Edward S.; Zaki, Sherif; York, Ian; Garcia-Sastre, Adolfo; Shaw, Michael; Sambhara, SuryaprakashScientific Reports (2017), 7 (), 40360CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naive cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naive cell, resulting in productive viral replication in the naive cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.
- 491Nzounza, P.; Chazal, M.; Guedj, C.; Schmitt, A.; Masse, J. M.; Randriamampita, C.; Pique, C.; Ramirez, B. C. The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells. PLoS One 2012, 7, e30130 DOI: 10.1371/journal.pone.0030130491The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cellsNzounza, Patrycja; Chazal, Maxime; Guedj, Chloe; Schmitt, Alain; Masse, Jean-Marc; Randriamampita, Clotilde; Pique, Claudine; Ramirez, Bertha CeciliaPLoS One (2012), 7 (1), e30130CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Background: Cell-to-cell virus transmission of human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virol. synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunol. synapse (IS). We have previously identified the human homolog of the Drosophila Disks Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a neg. regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodol./Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quant. cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. Conclusion: Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.
- 492Igakura, T.; Stinchcombe, J. C.; Goon, P. K.; Taylor, G. P.; Weber, J. N.; Griffiths, G. M.; Tanaka, Y.; Osame, M.; Bangham, C. R. Spread of HTLV-I between Lymphocytes by Virus-Induced Polarization of the Cytoskeleton. Science 2003, 299, 1713– 1716, DOI: 10.1126/science.1080115492Spread of HTLV-I Between Lymphocytes by Virus-Induced Polarization of the CytoskeletonIgakura, Tadahiko; Stinchcombe, Jane C.; Goon, Peter K. C.; Taylor, Graham P.; Weber, Jonathan N.; Griffiths, Gillian M.; Tanaka, Yuetsu; Osame, Mitsuhiro; Bangham, Charles R. M.Science (Washington, DC, United States) (2003), 299 (5613), 1713-1716CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell contact is required for efficient transmission of human T cell leukemia virus-type 1 (HTLV-I) between cells and between individuals, because naturally infected lymphocytes produce virtually no cell-free infectious HTLV-I particles. However, the mechanism of cell-to-cell spread of HTLV-I is not understood. We show here that cell contact rapidly induces polarization of the cytoskeleton of the infected cell to the cell-cell junction. HTLV-I core (Gag protein) complexes and the HTLV-I genome accumulate at the cell-cell junction and are then transferred to the uninfected cell. Other lymphotropic viruses, such as HIV-1, may similarly subvert normal T cell physiol. to allow efficient propagation between cells.
- 493Barnard, A. L.; Igakura, T.; Tanaka, Y.; Taylor, G. P.; Bangham, C. R. Engagement of Specific T-Cell Surface Molecules Regulates Cytoskeletal Polarization in HTLV-1–Infected Lymphocytes. Blood 2005, 106, 988– 995, DOI: 10.1182/blood-2004-07-2850493Engagement of specific T-cell surface molecules regulates cytoskeletal polarization in HTLV-1-infected lymphocytesBarnard, Amanda L.; Igakura, Tadahiko; Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.Blood (2005), 106 (3), 988-995CODEN: BLOOAW; ISSN:0006-4971. (American Society of Hematology)Cell-cell contact is required for efficient transmission of human T-lymphotropic virus type 1 (HTLV-1). An HTLV-1-infected cell polarizes its microtubule-organizing center (MTOC) toward the cell-cell junction; HTLV-1 core (Gag) complexes and the HTLV-1 genome accumulate at the point of contact and are then transferred to the uninfected cell. However, the mechanisms involved in this cytoskeletal polarization and transport of HTLV-1 complexes are unknown. Here, we tested the hypothesis that engagement of a specific T-cell surface ligand is synergistic with HTLV-1 infection in causing polarization of the MTOC to the cell contact region. We show that antibodies to intercellular adhesion mol.-1 (ICAM-1; CD54) caused MTOC polarization at a higher frequency in HTLV-1-infected cells. ICAM-1 is upregulated on HTLV-1-infected cells, and, in turn, ICAM-1 on the cell surface upregulates HTLV-1 gene expression. We propose that a pos. feedback loop involving ICAM-1 and HTLV-1 Tax protein facilitates the formation of the virol. synapse and contributes to the T-cell tropism of HTLV-1. In contrast, MTOC polarization induced in T cells by antibodies to CD3 or CD28 was significantly inhibited by HTLV-1 infection.
- 494Johnson, D. C.; Webb, M.; Wisner, T. W.; Brunetti, C. Herpes Simplex Virus gE/gI Sorts Nascent Virions to Epithelial Cell Junctions, Promoting Virus Spread. J. Virol. 2001, 75, 821– 833, DOI: 10.1128/JVI.75.2.821-833.2001494Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spreadJohnson, David C.; Webb, Mike; Wisner, Todd W.; Brunetti, CraigJournal of Virology (2001), 75 (2), 821-833CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. GE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, μ1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles. Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.
- 495Rustom, A.; Saffrich, R.; Markovic, I.; Walther, P.; Gerdes, H. H. Nanotubular Highways for Intercellular Organelle Transport. Science 2004, 303, 1007– 1010, DOI: 10.1126/science.1093133495Nanotubular Highways for Intercellular Organelle TransportRustom, Amin; Saffrich, Rainer; Markovic, Ivanka; Walther, Paul; Gerdes, Hans-HermannScience (Washington, DC, United States) (2004), 303 (5660), 1007-1010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chem. synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small mols. Accordingly, we propose a novel biol. principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.
- 496Davis, D. M.; Sowinski, S. Membrane Nanotubes: Dynamic Long-Distance Connections between Animal Cells. Nat. Rev. Mol. Cell Biol. 2008, 9, 431– 436, DOI: 10.1038/nrm2399496Membrane nanotubes: dynamic long-distance connections between animal cellsDavis, Daniel M.; Sowinski, StefanieNature Reviews Molecular Cell Biology (2008), 9 (6), 431-436CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)Membrane nanotubes are thin extensions of the plasma membrane that connect cells transiently and might facilitate intercellular communication. Recent studies have revealed considerable heterogeneity in their structure, formation, mode of cargo transport and functional properties, depending on the cell types involved. Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication (for example, by trafficking vesicles or transmitting calcium-mediated signals), but they can also contribute to pathologies (for example, by directing the spread of viruses). Recent data have revealed considerable heterogeneity in their structures, processes of formation and functional properties, in part dependent on the cell types involved. Despite recent progress in this young research field, further research is sorely needed.
- 497Efros, A. L.; Nesbitt, D. J. Origin and Control of Blinking in Quantum Dots. Nat. Nanotechnol. 2016, 11, 661– 671, DOI: 10.1038/nnano.2016.140497Origin and control of blinking in quantum dotsEfros, Alexander L.; Nesbitt, David J.Nature Nanotechnology (2016), 11 (8), 661-671CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)A review. Semiconductor nanocrystals offer an enormous diversity of potential device applications, based on their size-tunable luminescence, high optical stability and bottom-up chem. approaches to self-assembly. The promise of such applications can be seriously limited by luminescence intermittency in nanocrystal emission, i.e., blinking, arising from the escape of either 1 or both of the photoexcited carriers to the nanocrystal surface. In the 1st scenario, the remaining nanocrystal charge quenches luminescence via nonradiative Auger recombination, whereas for the other, the exciton probably is intercepted before thermalization and does not contribute to the luminescence. This summarizes the current understanding of the mechanisms responsible for nanocrystal blinking kinetics as well as core-shell engineering efforts to control such phenomena. Softening of the core-shell confinement potential strongly suppresses nonradiative Auger processes in charged nanocrystals, with successful nonblinking implementations demonstrated in CdSe-CdS core-thick-shell nanocrystals and their modifications.
- 498Marchuk, K.; Guo, Y.; Sun, W.; Vela, J.; Fang, N. High-Precision Tracking with Non-Blinking Quantum Dots Resolves Nanoscale Vertical Displacement. J. Am. Chem. Soc. 2012, 134, 6108– 6111, DOI: 10.1021/ja301332t498High-Precision Tracking with Non-blinking Quantum Dots Resolves Nanoscale Vertical DisplacementMarchuk, Kyle; Guo, Yijun; Sun, Wei; Vela, Javier; Fang, NingJournal of the American Chemical Society (2012), 134 (14), 6108-6111CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Novel nonblinking quantum dots (NBQDs) were utilized in three-dimensional super-localization, high-precision tracking applications under an automated scanning-angle total internal reflection fluorescence microscope (SA-TIRFM). NBQDs were randomly attached to stationary microtubules along the radial axis under gliding assay conditions. By automatically scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves were obtained. Fit with theor. decay functions, the abs. vertical positions were detd. with sub-10-nm localization precision. The emission intensity profile of the NBQDs attached to kinesin-propelled microtubules was used to resolve the self-rotation of gliding microtubules within a small vertical distance of ∼50 nm. The authors demonstrate the applicability of NBQDs in high-precision fluorescence imaging expts.
- 499Keller, A. M.; Ghosh, Y.; DeVore, M. S.; Phipps, M. E.; Stewart, M. H.; Wilson, B. S.; Lidke, D. S.; Hollingsworth, J. A.; Werner, J. H. 3-Dimensional Tracking of Non-blinking ’Giant’ Quantum Dots in Live Cells. Adv. Funct. Mater. 2014, 24, 4796– 4803, DOI: 10.1002/adfm.2014003494993-Dimensional Tracking of Non-blinking 'Giant' Quantum Dots in Live CellsKeller, Aaron M.; Ghosh, Yagnaseni; DeVore, Matthew S.; Phipps, Mary E.; Stewart, Michael H.; Wilson, Bridget S.; Lidke, Diane S.; Hollingsworth, Jennifer A.; Werner, James H.Advanced Functional Materials (2014), 24 (30), 4796-4803CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or 'blinking,' which causes ambiguity in particle trajectory anal. and limits tracking duration. Here, non-blinking 'giant' quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to com. available QDs. A time-gated photon-pair correlation anal. is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coeff. and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, esp. in cases where changes in cellular dynamics are occurring on the time scale of several minutes.
- 500Patterson, G.; Davidson, M.; Manley, S.; Lippincott-Schwartz, J. Superresolution Imaging Using Single-Molecule Localization. Annu. Rev. Phys. Chem. 2010, 61, 345– 367, DOI: 10.1146/annurev.physchem.012809.103444500Superresolution imaging using single-molecule localizationPatterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, JenniferAnnual Review of Physical Chemistry (2010), 61 (), 345-368CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews Inc.)A review. Superresoln. imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resoln. of light microscopy by over an order of magnitude (∼10-20-nm resoln.), allowing biol. processes to be described at the mol. scale. Here, we discuss a form of superresoln. microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent mols. In this single-mol.-based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresoln. imaging: from three-dimensional, multicolor mol. localization to tracking of nanometric structures and mols. in living cells. Single-mol.-based superresoln. imaging thus offers exciting possibilities for obtaining mol.-scale information on biol. events occurring at variable timescales.
- 501Lee, A.; Tsekouras, K.; Calderon, C.; Bustamante, C.; Presse, S. Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis. Chem. Rev. 2017, 117, 7276– 7330, DOI: 10.1021/acs.chemrev.6b00729501Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data AnalysisLee, Antony; Tsekouras, Konstantinos; Calderon, Christopher; Bustamante, Carlos; Presse, SteveChemical Reviews (Washington, DC, United States) (2017), 117 (11), 7276-7330CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Super-resoln. microscopy provides direct insight into fundamental biol. processes occurring at length scales smaller than light's diffraction limit. The anal. of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data anal. methods starting from an overview of basic statistical techniques underlying the anal. of super-resoln. and, more broadly, imaging data. We subsequently break down the anal. of super-resoln. data into four problems: the localization problem, the counting problem, the linking problem, and what we've termed the interpretation problem.
- 502Huang, B.; Bates, M.; Zhuang, X. Super-Resolution Fluorescence Microscopy. Annu. Rev. Biochem. 2009, 78, 993– 1016, DOI: 10.1146/annurev.biochem.77.061906.092014502Super-resolution fluorescence microscopyHuang, Bo; Bates, Mark; Zhuang, XiaoweiAnnual Review of Biochemistry (2009), 78 (), 993-1016CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)A review. Achieving a spatial resoln. that is not limited by the diffraction of light, recent developments of super-resoln. fluorescence microscopy techniques allow the observation of many biol. structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resoln. fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biol. structures and processes.
- 503Pan, W.; Dong, Z.; Li, F.; Meng, W.; Feng, L.; Niu, X.; Li, C.; Luo, Q.; Li, Z.; Sun, C. Visualizing Influenza Virus Infection in Living Mice. Nat. Commun. 2013, 4, 2369, DOI: 10.1038/ncomms3369503Visualizing influenza virus infection in living micePan Weiqi; Dong Zhenyuan; Li Feng; Meng Weixu; Feng Liqiang; Niu Xuefeng; Li Chufang; Luo Qinfang; Li Zhengfeng; Sun Caijun; Chen LingNature communications (2013), 4 (), 2369 ISSN:.Preventing and treating influenza virus infection remain a challenge because of incomplete understanding of the host-pathogen interactions, limited therapeutics and lack of a universal vaccine. So far, methods for monitoring the course of infection with influenza virus in real time in living animals are lacking. Here we report the visualization of influenza viral infection in living mice using an engineered replication-competent influenza A virus carrying luciferase reporter gene. After intranasal inoculation, bioluminescence can be detected in the chest and nasopharyngeal passage of living mice. The intensity of bioluminescence in the chest correlates with the dosage of infection and the viral load in the lung. Bioluminescence in the chest of infected mice diminishes on antiviral treatment. This work provides a novel approach that enables real-time study of influenza virus infection and effects of antiviral therapeutics in living animals.
- 504Pan, H.; Zhang, P.; Gao, D.; Zhang, Y.; Li, P.; Liu, L.; Wang, C.; Wang, H.; Ma, Y.; Cai, L. Noninvasive Visualization of Respiratory Viral Infection Using Bioorthogonal Conjugated Near-Infrared-Emitting Quantum Dots. ACS Nano 2014, 8, 5468– 5477, DOI: 10.1021/nn501028b504Noninvasive Visualization of Respiratory Viral Infection Using Bioorthogonal Conjugated Near-Infrared-Emitting Quantum DotsPan, Hong; Zhang, Pengfei; Gao, Duyang; Zhang, Yijuan; Li, Ping; Liu, Lanlan; Wang, Ce; Wang, Hanzhong; Ma, Yifan; Cai, LintaoACS Nano (2014), 8 (6), 5468-5477CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Highly pathogenic avian influenza A viruses are emerging pandemic threats in human beings. Monitoring the in vivo dynamics of avian influenza viruses is extremely important for understanding viral pathogenesis and developing antiviral drugs. Although a no. of technologies have been applied for tracking viral infection in vivo, most of them are laborious with unsatisfactory detection sensitivity. Herein the authors labeled avian influenza H5N1 pseudotype virus (H5N1p) with near-IR (NIR)-emitting QDs by bioorthogonal chem. The conjugation of QDs onto H5N1p was highly efficient with superior stability both in vitro and in vivo. Furthermore, QD-labeled H5N1p (QD-H5N1p) demonstrated bright and sustained fluorescent signals in mouse lung tissues, allowing the authors to visualize respiratory viral infection in a noninvasive and real-time manner. The fluorescence signals of QD-H5N1p in lung were correlated with the severity of virus infection and significantly attenuated by antiviral agents, such as oseltamivir carboxylate and mouse antiserum against H5N1p. The biodistribution of QD-H5N1p in lungs and other organs could be easily quantified by measuring fluorescent signals and cadmium concn. of virus-conjugated QDs in tissues. Hence, virus labeling with NIR QDs provides a simple, reliable, and quant. strategy for tracking respiratory viral infection and for antiviral drug screening.
- 505Zong, W.; Wu, R.; Li, M.; Hu, Y.; Li, Y.; Li, J.; Rong, H.; Wu, H.; Xu, Y.; Lu, Y. Fast High-Resolution Miniature Two-Photon Microscopy for Brain Imaging in Freely Behaving Mice. Nat. Methods 2017, 14, 713– 719, DOI: 10.1038/nmeth.4305505Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving miceZong, Weijian; Wu, Runlong; Li, Mingli; Hu, Yanhui; Li, Yijun; Li, Jinghang; Rong, Hao; Wu, Haitao; Xu, Yangyang; Lu, Yang; Jia, Hongbo; Fan, Ming; Zhou, Zhuan; Zhang, Yunfeng; Wang, Aimin; Chen, Liangyi; Cheng, HepingNature Methods (2017), 14 (7), 713-719CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-detd. behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resoln., miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resoln. (0.64 microm laterally and 3.35 microm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.
- 506Wang, K.; Sun, W.; Richie, C. T.; Harvey, B. K.; Betzig, E.; Ji, N. Direct Wavefront Sensing for High-Resolution in Vivo Imaging in Scattering Tissue. Nat. Commun. 2015, 6, 7276, DOI: 10.1038/ncomms8276506Direct wavefront sensing for high-resolution in vivo imaging in scattering tissueWang, Kai; Sun, Wenzhi; Richie, Christopher T.; Harvey, Brandon K.; Betzig, Eric; Ji, NaNature Communications (2015), 6 (), 7276CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-IR guide stars. The method enables in vivo two-photon morphol. and functional imaging down to 700 μm inside the mouse brain.
- 507Hong, G.; Antaris, A. L.; Dai, H. Near-Infrared Fluorophores for Biomedical Imaging. Nat. Biomed. Eng. 2017, 1, 0010 DOI: 10.1038/s41551-016-0010507Near-infrared fluorophores for biomedical imagingHong, Guosong; Antaris, Alexander L.; Dai, HongjieNature Biomedical Engineering (2017), 1 (1), 0010CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)A review. In this Review, we cover recent progress made on NIR fluorescence imaging in both the 700-900 nm NIR-I and the 1,000-1,700 nm NIR-II windows by highlighting an increasingly developing palette of biocompatible NIR fluorophores that span the entire NIR window and include inorg. nanoparticles, org. macromols. and small mols. with tunable emission wavelengths. Together with advances in imaging instrumentation allowing for the efficient detection of long-wavelength NIR photons, recently developed NIR fluorophores have fuelled biomedical imaging from contrast-enhanced imaging of anatomical structures and mol. imaging of specific biomarkers to functional imaging of physiol. activities, both for preclin. animal studies and clin. diagnostics and interventions.
- 508Ding, F.; Zhan, Y. B.; Lu, X. J.; Sun, Y. Recent Advances in Near-Infrared II Fluorophores for Multifunctional Biomedical Imaging. Chem. Sci. 2018, 9, 4370– 4380, DOI: 10.1039/C8SC01153B508Recent advances in near-infrared II fluorophores for multifunctional biomedical imagingDing, Feng; Zhan, Yibei; Lu, Xiaoju; Sun, YaoChemical Science (2018), 9 (19), 4370-4380CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. In recent years, owing to unsatisfactory clin. imaging clarity and depths in the living body for early diagnosis and prognosis, novel imaging modalities with high bioimaging performance have been actively explored. The remarkable headway made in the second near-IR region (NIR-II, 1000-1700 nm) has promoted the development of biomedical imaging significantly. NIR-II fluorescence imaging possesses a no. of merits which prevail over the traditional and NIR-I (400-900 nm) imaging modalities in fundamental research, such as reduced photon scattering, as well as auto-fluorescence and improved penetration depth. Functional probes for instant and precise feedback of in vivo information are at the core of this modality for superb imaging. Herein, we review the recently developed fluorophores including carbon nanotubes, org. small mols., quantum dots, conjugated polymers and rare-earth-doped materials to present superior and multifunctionality of biomedical imaging in the NIR-II regions (1000-1700 nm).
- 509Smith, A. M.; Mancini, M. C.; Nie, S. Bioimaging: Second Window for in Vivo Imaging. Nat. Nanotechnol. 2009, 4, 710, DOI: 10.1038/nnano.2009.326509Second window for in vivo imagingSmith, Andrew M.; Mancini, Michael C.; Nie, ShumingNature Nanotechnology (2009), 4 (11), 710-711CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Enhanced fluorescence from carbon nanotubes and advances in near-IR cameras have opened up a new wavelength window for small animal imaging.
- 510Zhang, J. J.; Lin, Y.; Zhou, H.; He, H.; Ma, J. J.; Luo, M. Y.; Zhang, Z. L.; Pang, D. W. Cell Membrane-Camouflaged NIR II Fluorescent Ag2Te Quantum Dots-Based Nanobioprobes for Enhanced in Vivo Homotypic Tumor Imaging. Adv. Healthcare Mater. 2019, 8, e1900341 DOI: 10.1002/adhm.201900341There is no corresponding record for this reference.
- 511Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared Region. Angew. Chem., Int. Ed. 2012, 51, 9818– 9821, DOI: 10.1002/anie.201206059511In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared RegionHong, Guosong; Robinson, Joshua T.; Zhang, Yejun; Diao, Shuo; Antaris, Alexander L.; Wang, Qiangbin; Dai, HongjieAngewandte Chemie, International Edition (2012), 51 (39), 9818-9821, S9818/1-S9818/11CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors have developed biocompatible, heavy-metal free 6PEG-Ag2S QDs as an imaging contrast agent that are brightly fluorescent in the NIR-II window. Imaging with these NIR-II QDs afforded deep inner organ registration, dynamic tumor contrast, and fast tumor detection. The in vivo pharmacokinetics of the QDs was studied, suggesting an unprecedented degree of accumulation of 6PEG-Ag2S QDs in the tumor(> 10% ID/g) through the EPR effect. The short-term excretion profile of the 6PEG-Ag2S QDs suggested biliary clearance as the main clearance pathway. Further studies of genotoxicity and reproductive toxicity will be used to evaluate the potential of this new type of NIR-II fluorophores for pre-clin. use.
- 512Jiang, P.; Zhu, C. N.; Zhang, Z. L.; Tian, Z. Q.; Pang, D. W. Water-Soluble Ag2S Quantum Dots for Near-Infrared Fluorescence Imaging in Vivo. Biomaterials 2012, 33, 5130– 5135, DOI: 10.1016/j.biomaterials.2012.03.059512Water-soluble Ag2S quantum dots for near-infrared fluorescence imaging in vivoJiang, Peng; Zhu, Chun-Nan; Zhang, Zhi-Ling; Tian, Zhi-Quan; Pang, Dai-WenBiomaterials (2012), 33 (20), 5130-5135CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)A one-step method for synthesizing water-sol. Ag2S quantum dots terminated with carboxylic acid group has been reported. The crystal structure and surface of the prepd. Ag2S quantum dots were characterized. The prepd. Ag2S quantum dots exhibited bright photoluminescence and excellent photostabilities. The photoluminescence emissions could be tuned from visible region to near-IR (NIR) region (from 510 nm to 1221 nm). Ultra-small sized Ag2S nanoclusters were synthesized with high initial monomer concn. in the current system. The in vivo imaging expts. of nude mice showed that the NIR photoluminescence of the prepd. Ag2S quantum dots could penetrate the body of mice. Compared to the conventional NIR quantum dots, the Ag2S quantum dots don't contain toxic elements to body (such as Cd and Pb), thus, the prepd. Ag2S quantum dots could serve as excellent NIR optical imaging probes and would open the opportunity to study nanodiagnostics and imaging in vivo.