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Dynamic Excimer (DYNEX) Imaging of Lipid Droplets
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  • M. Carmen Gonzalez-Garcia
    M. Carmen Gonzalez-Garcia
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
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  • Carmen Salto-Giron
    Carmen Salto-Giron
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    More by Carmen Salto-Giron
  • Pilar Herrero-Foncubierta
    Pilar Herrero-Foncubierta
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    Departamento de Quimica Organica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva sn, 18071 Granada, Spain
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  • Tomás Peña-Ruiz
    Tomás Peña-Ruiz
    Departamento de Química Física y Analítica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas, 23071 Jaén, Spain
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  • Maria Dolores Giron-Gonzalez
    Maria Dolores Giron-Gonzalez
    Departamento de Bioquimica y Biologia Molecular II, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
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    Orcidhttps://orcid.org/0000-0001-9638-988X
  • Rafael Salto-Gonzalez
    Rafael Salto-Gonzalez
    Departamento de Bioquimica y Biologia Molecular II, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
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    Orcidhttps://orcid.org/0000-0002-7044-3611
  • Angel Perez-Lara
    Angel Perez-Lara
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany
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  • Amparo Navarro
    Amparo Navarro
    Departamento de Química Física y Analítica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas, 23071 Jaén, Spain
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    Orcidhttps://orcid.org/0000-0001-9620-6668
  • Emilio Garcia-Fernandez*
    Emilio Garcia-Fernandez
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    *Email: [email protected]
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  • Angel Orte*
    Angel Orte
    Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-1905-4183
Open PDFSupporting Information (2)

ACS Sensors

Cite this: ACS Sens. 2021, 6, 10, 3632–3639
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https://doi.org/10.1021/acssensors.1c01206
Published September 9, 2021

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Unraveling cellular physiological processes via luminescent probes that target specific cellular microenvironments is quite challenging due to the uneven distribution of probes. Herein, we designed a new dynamic excimer (DYNEX) imaging method that involves the sensitive detection of nanosecond-scale dynamic molecular contacts of a fluorescent acridone derivative and reveals the cell microenvironment polarity. Using our method, we specifically tracked cell lipid droplets in fibroblast colon carcinoma cells. These organelles play a central role in metabolic pathways, acting as energy reservoirs in regulatory processes. DYNEX imaging provides the inner polarity of cell lipid droplets, which can be related to lipid contents and metabolic dysfunctions. This new methodology will inspire development of novel multidimensional fluorescent sensors that are able to provide target-specific and orthogonal information at the nanosecond scale.

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Fluorescence microscopy is an extensively used platform to characterize membrane and membraneless organelles due to its high sensitivity, accuracy, and noninvasiveness. Specific chemically driven targeting of these cell organelles is based on their cellular microenvironment, which is defined by different parameters, such as macromolecules, salt types and concentration, temperature, pH, viscosity, and polarity. (1) Thus, the development of new sensors to monitor the dynamics of these parameters is key to understanding many cellular physiological processes and diseases in biology. In this work, we employed a chemically simple, fluorescent probe, N-(3-(benzyloxy)propyl)-2-methoxy-acridin-9-one (1, Figure 1), to study the polarity of lipid droplets. These organelles act as lipid storage units and are involved in prominent trafficking pathways, dynamically interacting with practically all subcellular structures. Importantly, dysregulation of lipid droplets has been associated with several diseases, such as lipodystrophies, fatty liver disease, or atherosclerosis. (2,3) Therefore, lipid droplets are interesting target organelles in the study of altered metabolic states, such as highly proliferative tumor cells. (2) Additionally, microenvironment polarity is an intrinsically complex parameter involving the medium acting as a continuous phase and specific molecular interactions of dispersion forces, dipole-dipole interactions, and hydrogen bonds, affecting enzymatic activity, membrane transport, and cellular proliferation. (4) Thus, microenvironment polarity inside lipid droplets reveals different cellular metabolic states, either physiological or pathological. (5−9)

Figure 1

Figure 1. TD-CAM-B3LYP-D3/6-31+G** calculation results of the molecular geometry and HOMO and LUMO (isocontour plots 0.02 au) of an antiparallel excimer of acridone 1 in water solution.

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In a previous work, we designed a robust and quantitative methodology to obtain intracellular polarity in terms of the ET30 parameter by multiparametric microscopy and simultaneously employing two orthogonal imaging approaches, namely, time-gated ratiometric and fluorescence lifetime imaging. (10) The ET30 parameter, defined as the difference in energy between the excited state and the ground state of the pyridinium-N-phenolate of betaine, has an advantage over other polarity parameters combining general solvent effects and hydrogen-bonding interactions. (11) Our method employs acridone derivatives as fluorescent reporters because of their excellent photophysical properties, which include long lifetimes (7–22 ns), high quantum yields (>0.6), and great pH stability at physiological levels. Importantly, the long fluorescence lifetime of these probes overcomes the problems of interferences caused by cellular autofluorescence by applying suitable time gates to filter out short-lived, interfering photons after excitation. (12) Moreover, the subcellular localization of acridones can be finely tailored by incorporating directing molecules into the structural groups that do not alter the photophysical properties or performance of the dyes. (13) Another interesting and distinctive feature of these acridone derivatives is their excited-state dynamics, including the excited-state formation of an excimer in solution. (14)
Taking advantage of this property, herein, we propose a new approach to organelle-specific tracking using a new layer of information based on the kinetics of the acridone excited state. This segmentation approach is subsequently combined with a previously validated method to probe polarity microenvironments, (10) resulting in a novel imaging approach. The novelty of this DYNamic EXcimer (DYNEX) imaging method relies on the sensitive and specific detection of the dynamic excited-state formation of excimers of acridone 1 inside live cells. We used this feature as a tool to clearly identify, track, and specifically segment lipid droplets, the organelles in which we detected such dynamics. This adds a new and complementary layer of information to conventional fluorescence intensity and lifetime imaging.

Results and Discussion

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Excited-State Dynamics

To develop the DYNEX method, we first thoroughly investigated the excimer formation dynamics of compound 1 in different solvents using a combination of spectroscopic and computational tools. Time-dependent density functional theory (TD-DFT) calculations, using the TD-CAM-B3LYP/6-31+G** level of theory (see the Supporting Information, SI, for details), were performed to understand excimer formation at a molecular level, taking into account dispersion interactions in the dimer by including an empirical dispersion correction (GD3). (15) Since 1 has a bulky N-substituent in the acridone core, we also studied unsubstituted 4-methoxyacridone and other simple derivatives (Chart S1 in the Supporting Information, SI). Modeling of such simpler molecular systems helped to optimize the different configurations of the dimers in excited states as well as to investigate whether the hydrophobicity of the benzyl radical in 1 is involved in the excimer stability. Four different excimer arrangements were found in both water and cyclohexane (Figure S1 and Table S1 in the SI), with the structure with antiparallel packing being the most stable. Importantly, the lowest unoccupied molecular orbital (LUMO) wavefunction presented a clear overlap delocalized between the two acridone monomers, while in the highest occupied molecular orbital (HOMO), the molecular orbitals were isolated on the individual molecules (Figure 1). This result is a clear indication of the formation of excited-state π–π interactions and, hence, of excimer formation. Some interatomic distances between carbon atoms are shown in Figure 1, which are within the interval of previous studies for aromatic compounds, 3.1–3.4 Å. (16)
We also investigated excimer formation using absorption, fluorescence (Figure S2), and time-resolved emission spectroscopy (TRES). TRES is an extremely valuable tool that allows following the nanosecond-scale temporal evolution of the emission spectrum, identifying excited-state dynamics, such as solvent relaxation or excited-state reactions. (14,17,18) Although the formation of excimers of 4-methoxyacridone derivatives in aqueous solution has been previously established, (14) it was not confirmed for acridone 1 due to its low solubility in water. This is an important feature since the formation of excimers will be favored in nonaqueous environments. Therefore, we first studied the excited-state dynamics of acridone 1 in acetonitrile (ET30 = 45.6 kcal mol–1) and methanol (ET30 = 55.4 kcal mol–1). In acetonitrile, acridone 1 presented two decay times: a long lifetime of 10.26 ns and a short lifetime of 0.98 ns. The TRES spectra (Figure 2A) exhibited an isoemissive point at 467 nm in the initial 1.5 ns, which indicated the excited-state transformation of the initially excited species into a different product. The species-associated emission spectra (SAEMS), as obtained from TRES measurements, represent the emission profiles linked to each one of the detected decay times. In a mixture of emissive dyes with different fluorescence lifetimes, the SAEMS would represent the spectrum of each form. However, in the presence of an excited-state reaction, the SAEMS provide information on the spectral distribution of the reagent, or directly excited species, and the final product arising during the excited-state reaction. (17) The analysis of SAEMS clearly showed negative pre-exponential factors associated with the short decay time (rise time) from 460 to 500 nm (Figure 2B), indicating that the excited-state product has preferential emission in this spectral range. Similar results, confirming the formation of excimers, were found for acridone 1 dissolved in other solvents (Figures S3 and S4). The photophysical properties of acridone 1 responded mainly to microenviroment polarity, also including acidity or basicity of the solvent. (19) In contrast, the emission features of the dye are almost invariable with other factors, such as pH (in the near-neutral range), (14) viscosity (since the fluorophore does not have a rotorlike structure, see Figure S5), or the presence of typical biologically relevant species, such as metal ions, peroxide, sulfides, and sulfites (Figure S6).

Figure 2

Figure 2. (A) TRES spectra of 1 dissolved in acetonitrile (λex = 375 nm) at 0, 0.5, 0.8, 1, 1.2, 1.5, 3, 5, 8, 10, 15, 20, and 25 ns after the excitation pulse. (B) SAEMS of 1 in acetonitrile, associated with the longest lifetime (blue) and the shortest lifetime (red).

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DYNEX Imaging of Cellular Lipid Droplets

Next, we aimed to actively seek direct evidence of dynamic excimer formation of acridone 1 inside the fibroblast colon cell line CCD-18Co.
The formation of excited-state emissive species, such as an excimer, is unequivocally characterized by the appearance of short rise times in the emission kinetics, i.e., a negative pre-exponential factor associated with the short decay time in the fluorescence decay trace. (14) This type of fluorophore dynamics has not yet been detected inside live cells. To do so, only time-resolved fluorimetry, working in single photon timing (SPT) mode, is capable of reliably detecting the rise time; hence, fluorescence lifetime imaging microscopy (FLIM) is the tool of choice. For the unprecedented detection of photophysical nanosecond kinetics of acridone 1 in live cells, we analyzed FLIM images, adjusting the fluorescence decays to a biexponential model. Importantly, we found specific subcellular areas in which the pre-exponential (amplitude) associated with the short decay time, termed as A2, was consistently negative (Figure 3A,B). This is an unequivocal feature of nanosecond excited-state dynamics, (14,20) as explained earlier.

Figure 3

Figure 3. (A) FLIM image of dye 1 in CCD-18Co cells and segmentation procedure using DYNEX. Pixels with negative A2 amplitude are represented in blue. (B) Distribution of amplitudes of the short decay time (A2) of the image in panel (A). (C) Representative colocalization imaging of 1 (green channel) with LipidTOX (red channel). Scale bar of 10 μm. (D) Correlation plot (left) and intensity profiles of lines 1, 2, and 3 (right) from the green and red channels of panel (C).

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The DYNEX imaging approach consists of an image segmentation procedure, which selects only pixels with negative amplitude of the short decay time. When the DYNEX criterion was applied to the collected FLIM images of 1 in live cells, it highlighted specifically spherical organelles, whereas the dye that had spread through the cytosol and other organelles was mostly monomeric and was thus filtered out by DYNEX. Figure 3A shows the segmentation of lipid droplets using DYNEX, whereas Figure 3B shows the distribution of amplitude values of the short decay time (A2) from the whole image in Figure 3A. Additional videos and animations illustrating the DYNEX segmentation method can be found as Supporting Videos S1–S4. The organelles selected by DYNEX imaging were morphologically spherical, and we identified them as lipid droplets through colocalization experiments with different organelle trackers (Figures 3C,D, S7–S10, and Table S2 in the SI). Therefore, using DYNEX, we were capable of specifically identifying lipid droplets within the cytoplasm.
It is important to note that for this approach to work properly, the long decay time of the fluorophore must be as large as possible. Hence, acridone derivatives are especially interesting candidates. (14) Nevertheless, among the different tested N-substituted-4-methoxyacridone derivatives as intracellular probes, (19) strikingly, we unequivocally detected intracellular dynamic excimer formation in acridone 1 bearing an N-(3-(benzyloxy)propyl) radical. We did not find evidence of dynamic excimers in live-cell imaging using N-(3-(hydroxy)propyl)- or N-(3-(methoxy)propyl)-4-methoxyacridones. Importantly, the fact that the detection of an acridone excimer in cellular lipid droplets is specific to dye 1 confirms that it is not an instrumental artifact due to detector jitter. We hypothesized that the higher hydrophobicity of the benzyl radical promoted accumulation of the dye in lipid droplets, thus facilitating the formation of the excimer. These results can be reconciled by comparing the lipophilicity of these three dyes. The consensus, simulated log P value (partition coefficient octanol/water) is 4.24 for acridone 1, whereas it is 2.67 and 3.01 for N-(3-(hydroxy)propyl)- and N-(3-(methoxy)propyl)-4-methoxyacridone, respectively, as obtained using the SwissADME tool. (21) Therefore, the larger log P value of acridone 1 supports further accumulation in lipid droplets compared to the other studied dyes. The higher local concentration of the dye in these lipid droplets allows nanosecond, excited-state encounters between different molecules of the dye and, hence, the formation of actual dynamic excimers. Excimer formation, as a second-order reaction, is a concentration-dependent process. However, we did not find variations in the application of the DYNEX segmentation criterion with typical dye concentrations used for cell imaging in the sub-μM range. The negative amplitude values for A2 detected were high (Figure 3B), thus supporting that the methodology could be safely applied at much lower concentrations of dye 1.
Next, we employed DYNEX imaging, combined with a validated multiparametric microscopy approach for determining microenvironment polarity, (10) to compare the polarity inside lipid droplets in the human colon cell line CCD-18Co and the mouse colon carcinoma cell line CT26.WT. The polarity in lipid droplets is an indirect estimation of the lipid content and composition and can be related to altered metabolic states. (22) For these measurements, once lipid droplet pixels were segmented using DYNEX, we extracted polarity ET30 values (Figure 4) via multiparametric microscopy by combining the time-gated intensity ratio and fluorescence lifetime information (10) (see the SI for experimental details and Figures S11–S13). Heretofore, there have been some efforts to characterize intracellular polarity employing fluorescence microscopy techniques using emission spectra, (23) fluorescence intensity, (24) and ratiometric measures. (25−27) Nevertheless, biological samples present important difficulties for quantitative imaging that are overcome by our methodology. (10)Table 1 and Figure S14 show the ET30 values recovered from lipid droplets of CCD-18Co and CT26.WT cells. The average ET30 values were 46.1 ± 0.1 and 45.6 ± 0.2 kcal mol–1 for CCD-18Co and CT26.WT cells, respectively. These values are consistent with droplets formed with neutral lipids surrounded by polar phospholipids. A close inspection of the droplet images (Figures 4B,C and S15) clearly exhibited lower polarity in the inner core of the droplets, in agreement with the aforementioned arrangement. (26) Interestingly, compared with the CCD-18Co line, the tumoral CT26.WT cell line exhibited lipid droplets with slightly lower polarity, consistent with previous works. (8,24,26−29) This observation supports the idea of a higher lipid content in the tumoral line due to altered, proliferative metabolism.

Figure 4

Figure 4. (A) Schematic of the DYNEX concept: selective formation of dynamic excimers of dye 1 in lipid droplets. (B, C) FLIM image (left panels), DYNEX segmentation of lipid droplets (central panels), and ET30 imaging (right panels) of 1 in CCD-18Co cells (B) and tumor CT26.WT cells (C). Scale bars represent 10 μm for full images and 2 μm for zoomed images.

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Table 1. ET30 Values in Lipid Droplets
cell linetreatment ET30a (kcal mol–1)
CCD-18Co  46.1 ± 0.1
sucrose50 mM46.8 ± 0.1c
 100 mM47.1 ± 0.2c
chloroquine25–100 μM43.70 ± 0.09d
CT26.WT  45.6 ± 0.2e
sucrose50 mM46.9 ± 0.1c
 100 mM46.7 ± 0.2c
chloroquine25–100 μM42.3 ± 0.1d
a

Errors are indicated as the standard error of the mean, s.e.m.

b

Statistical comparison of populations was performed using the Mann–Whitney, Holm–Bonferroni, and Holm–Sidak statistical tests.

c

Significantly different from the untreated control (p < 0.001).

d

Significantly different from the untreated control (p < 10–25).

e

Significantly different from untreated CCD-18Co cells (p < 0.15).

Although many different alternatives for fluorescently tagging lipid droplets are available, (30) few probes are capable of quantitatively reporting lipid content and polarity. During the production phase of our work, several polarity probes specific for lipid droplets have been reported. (31−33) In these recent works, the polarity of lipid droplets is quantified through conventional ratiometric imaging based on the spectral shift of solvatochromic probes. Klymchenko and colleagues further defined an arbitrary lipid order parameter to scale relative polarity changes, (31) but absolute quantitative polarity values were not reported. In other cases, polarity changes in lipid droplets were only visualized but not quantified using fluorogenic reactions of solvatochromic probes. (32) Interestingly, a coumarin–quinolinium dyad has been recently reported to respond to polarity changes using ratiometric and FLIM imaging. (33) The response of this dye toward polarity was calibrated in terms of the dielectric constant, ε; however, when used in live cells, the detected intensity ratio values were out of the calibration values, making it impossible to quantify polarity. These recent reports illustrate the far superiority and advantages of our approach for in cellulo quantification of absolute polarity. First, DYNEX imaging allows specific filtering of pixels in lipid droplets without the need for an additional organelle tracker. A second important advantage of our method is that the simultaneous use of multidimensional information, lifetime, and time-gated intensity ratio provides robust and absolute determination of the polarity, in terms of the ET30 parameter, free from potential interferences from cellular autofluorescence and other sources of light, such as scatter. For comparison purposes, Table S3 in the SI gathers other fluorescent polarity probes for lipid droplets, the basis of the imaging approach employed, and whether the sensor is capable of reporting absolute polarity values.
We further exploited the potential of DYNEX to measure how the polarity of lipid droplets was altered upon treatment with sucrose and chloroquine. Sucrose is known to increase membrane stability, interacting through hydroxyl groups and hence altering the polarity of intracellular compartments. (34,35) On the other hand, chloroquine causes enhanced lipid production, potentially decreasing the polarity of lipid droplets. (22,34) After confirming that sucrose did not have any adverse effect on the photophysical properties of dye 1 (Figure S16), our results show that treatment with sucrose resulted in a significant increase in the polarity of lipid droplets (p < 0.001) for treatments with 50 and 100 mM sucrose in both CCD-18Co and tumoral CT26.WT cells (Table 1, Figures 5, S17, and S18). Being a disaccharide, the internalization of sucrose strongly depends on the cellular type, usually running via endocytosis to lysosomes. For instance, the polarity of lysosomes has been shown to notably change upon sucrose treatment in MCF-7 breast cancer cells, (34) but the effect was less noticeable in 293T cells and even lower in HepG2 cells, (35) using 80 mM sucrose in either case. The two cell lines that we used in our work are colon cells, with sufficient disaccharide receptors for endocytosis, and the increase in polarity of lipid droplets that we have detected is then in line with previous results, considering the metabolic role of such organelles.

Figure 5

Figure 5. ET30 values in lipid droplets of CCD-18Co cells (A) and tumor CT26.WT cells (B) upon treatment with sucrose and chloroquine. Each individual value corresponds to lipid droplet regions from different cells from >10 different images.

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In contrast, treatment with chloroquine resulted in significantly more hydrophobic lipid droplets in both CCD-18Co and CT26.WT cell lines (Table 1, Figures 5 and S19). This result indicated a higher production of lipids stored in the corresponding organelles. (22)

Conclusions

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In this work, we have demonstrated that segmentation by novel DYNEX imaging is a powerful approach for quantifying microenvironment polarity in cellular lipid droplets. The detection of an actual dynamic excimer inside live cells, as photophysically characterized by negative amplitudes on a decay time, i.e., a rise time, has not been previously reported. The fact that we detected a rise time is an unequivocal demonstration of such excited-state dynamics, as is well known in the photophysics field. (14,36) For this advance, the extraordinary behavior of acridone dyes was a turning point in terms of both a well-defined excimer reaction, as evidenced in our computational and experimental studies, and a long decay time, leaving enough time, on the nanosecond timescale, to detect the formation of excited-state interactions using FLIM microscopy in SPT mode. In this design, the solvatochromism of the acridone core is able to resolve the microenvironment polarity, while the N-substituent allows convenient placement of the probe in the correct organelle and facilitates excimer formation.
In the past, changes in fluorescence associated with the formation of excimers, such as quenching of monomer emission or red-shifted excimer emission, have been exploited for some time in imaging for sensing purposes, (37−40) for instance, of metallic ions, such as Al3+, (38) Zn2+, (40) and Hg2+, (39) and biomolecules, such as heparin, (37) cysteine, (40) and phosphate. (40) Most of these works employed common dyes with well-known excimer emission, such as pyrene (38−40) or perylene. (37) However, all of these works were based on the usual red-shifted emission of the excimers and used ratiometric intensity approaches (37,38) or off/on behavior (39,40) to illustrate the detection of a different emissive species. Since these works used steady-state intensity, one cannot provide kinetic information on the dynamics of the dye nor unequivocally confirm whether the red-shifted emissive species was dynamically formed in the excited state (an actual excimer) or was preformed in the ground state (static aggregates). The detection of red-shifted, enhanced emission upon dimerization or aggregation of certain dyes is well known and has given rise to widespread studies on aggregation-induced emission (AIE). (41) To the best of our knowledge, direct measurement of environment-dependent excited-state excimer kinetics inside live cells has not been previously employed in fluorescence imaging. Therefore, our observations are important not only from the point of view of the photophysics of a dye detected intracellularly but also because they allowed us to develop a novel segmentation procedure via DYNEX imaging, an approach that exploits the use of time-resolved excited-state kinetic information to gain a new layer for image filtering in fluorescence microscopy, complementary to other common fluorescence parameters.
In this regard, similar methodologies of imaging analysis and filtering based on excited-state kinetics of the fluorescent probe can be foreseen. In the past, we successfully developed fluorescent sensors for phosphate imaging, based on excited-state proton transfer reactions of xanthene derivatives. (42,43) Therefore, we envisage that the appropriate combination of rich dye’s photophysics and time-resolved fluorescence imaging tools, such as FLIM microscopy, will pave the way for the design of new multidimensional fluorescent sensors able to provide target-specific and orthogonal information, including spectral, intensity, lifetime, and excited-state kinetic changes, among others.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.1c01206.

  • Detailed materials and methods; supporting results from quantum chemistry calculations (Chart S1, Figure S1, and Table S1); supporting spectroscopic results (Figures S2–S6); subcellular localization study (Figures S7–S10 and Table S2); additional DYNEX images and results (Figures S11–S15); comparison of different lipid droplet polarity probes (Table S3); and sucrose and chloroquine treatments (Figures S16–S19) (PDF)

  • Supporting Videos S1–S4 illustrating the DYNEX segmentation method (ZIP)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Emilio Garcia-Fernandez - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Email: [email protected]
    • Angel Orte - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Orcidhttps://orcid.org/0000-0003-1905-4183;  Email: [email protected]
  • Authors
    • M. Carmen Gonzalez-Garcia - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    • Carmen Salto-Giron - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain
    • Pilar Herrero-Foncubierta - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Departamento de Quimica Organica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva sn, 18071 Granada, Spain
    • Tomás Peña-Ruiz - Departamento de Química Física y Analítica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas, 23071 Jaén, Spain
    • Maria Dolores Giron-Gonzalez - Departamento de Bioquimica y Biologia Molecular II, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Orcidhttps://orcid.org/0000-0001-9638-988X
    • Rafael Salto-Gonzalez - Departamento de Bioquimica y Biologia Molecular II, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Orcidhttps://orcid.org/0000-0002-7044-3611
    • Angel Perez-Lara - Departamento de Fisicoquimica, Unidad de Excelencia en Quimica Aplicada a Biomedicina y Medioambiente (UEQ), Facultad de Farmacia, Universidad de Granada, Campus de Cartuja sn, 18071 Granada, Spain;  Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany
    • Amparo Navarro - Departamento de Química Física y Analítica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas, 23071 Jaén, Spain;  Orcidhttps://orcid.org/0000-0001-9620-6668
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was funded by grants CTQ2017-85658-R (MICIU/AEI/ERDF), FQM-337 (Junta de Andalucía), and PIUJA 2019-20 (Universidad de Jaén). We acknowledge the Universidad de Granada (Spain) microscopy central facilities (CIC-UGR) and computing time from CSIRC-UGR. M.C.G.-G. thanks MICIU/AEI for a predoctoral fellowship. Funding for open access charge: Universidad de Granada/CBUA. We are indebted to Prof. Reinhard Jahn for his generous support.

Abbreviations

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DYNEX

dynamic excimer imaging

TD-DFT

time-dependent density functional theory

FLIM

fluorescence lifetime imaging microscopy

TRES

time-resolved emission spectroscopy

SAEMS

species-associated emission spectra

References

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This article references 43 other publications.

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    Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye
    Crovetto, Luis; Orte, Angel; Paredes, Jose M.; Resa, Sandra; Valverde, Javier; Castello, Fabio; Miguel, Delia; Cuerva, Juan M.; Talavera, Eva M.; Alvarez-Pez, Jose M.
    Journal of Physical Chemistry A (2015), 119 (44), 10854-10862CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
    Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophys. behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity const. of the dye, pKN-A, was detd. by absorbance and steady-state fluorescence at very low buffer concns. Next, we detd. whether the addn. of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aq. solns. at near-neutral pH. For this anal., both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES expts. demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate consts. were detd. by global anal. of the fluorescence decay traces recorded as a function of pH, and buffer concn. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate const. for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity const. pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we detd. whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.
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  19. 19
    Gonzalez-Garcia, M. C.; Peña-Ruiz, T.; Herrero-Foncubierta, P.; Miguel, D.; Giron, M. D.; Salto, R.; Cuerva, J. M.; Navarro, A.; Garcia-Fernandez, E.; Orte, A. Orthogonal cell polarity imaging by multiparametric fluorescence microscopy. Sens. Actuators, B 2020, 309, 127770  DOI: 10.1016/j.snb.2020.127770
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    Orthogonal cell polarity imaging by multiparametric fluorescence microscopy
    Gonzalez-Garcia, M. Carmen; Pena-Ruiz, Tomas; Herrero-Foncubierta, Pilar; Miguel, Delia; Giron, Maria D.; Salto, Rafael; Cuerva, Juan M.; Navarro, Amparo; Garcia-Fernandez, Emilio; Orte, Angel
    Sensors and Actuators, B: Chemical (2020), 309 (), 127770CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    The cellular microenvironment is a complex medium due to high concns. of proteins and an intertwined framework of cellular organelles. In particular, cellular micro-polarity controls several biol. processes, since it modulates hydrophobic/hydrophilic interactions and, hence, recognition, signalling and binding events. In this work, we have developed an unprecedented methodol. to construct accurate environment polarity images using multiparametric fluorescence microscopy, via a multi-linear calibration of orthogonal parameters: the fluorescence lifetime and the spectral shift of a series of solvatochromic dyes. For this approach, we have synthesized and fully characterized N-substituted 2-methoxy-9-acridone dyes as suitable bioimaging polarity probes. However, to fully comprehend the complex links between microenvironment polarity and the dyes' properties, we have endeavoured a multidisciplinary approach, in which we have studied the photophysics of our fluorophores using spectroscopic tools and state-of-the-art computational chem. This profound knowledge permitted to use these dyes as intracellular polarity probes, quant. and robustly probing the microenvironment of different cellular compartments. Our new methodol. may pave the way to further developments in accurate sensing of cellular microenvironment parameters.
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  20. 20
    Orte, A.; Bermejo, R.; Talavera, E. M.; Crovetto, L.; Alvarez-Pez, J. M. 2′,7′-Difluorofluorescein Excited-State Proton Reactions: Correlation between Time-Resolved Emission and Steady-State Fluorescence Intensity. J. Phys. Chem. A 2005, 109, 2840– 2846,  DOI: 10.1021/jp044681m
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    20
    2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity
    Orte, Angel; Bermejo, Ruperto; Talavera, Eva M.; Crovetto, Luis; Alvarez-Pez, Jose M.
    Journal of Physical Chemistry A (2005), 109 (12), 2840-2846CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
    The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in soln. Since biomols. in general have some chem. groups that can act as proton acceptors/donors and are usually dissolved in buffer solns. which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (2005), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concns., analyzed by means of global compartmental anal. In this article we use the rate consts. and the cor. pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and assocd. pre-exponentials at different acetate buffer concns. and const. pH and compare these theor. calcd. values with new exptl. data. We also calc. the steady-state fluorescence intensity vs pH and vs acetate buffer concn. (at const. pH) and compare these calcd. emission values with the exptl. data previously published. The agreement between the exptl. and simulated data is excellent.
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    Daina, A.; Michielin, O.; Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717  DOI: 10.1038/srep42717
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    SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules
    Daina Antoine; Michielin Olivier; Zoete Vincent; Michielin Olivier; Michielin Olivier
    Scientific reports (2017), 7 (), 42717 ISSN:.
    To be effective as a drug, a potent molecule must reach its target in the body in sufficient concentration, and stay there in a bioactive form long enough for the expected biologic events to occur. Drug development involves assessment of absorption, distribution, metabolism and excretion (ADME) increasingly earlier in the discovery process, at a stage when considered compounds are numerous but access to the physical samples is limited. In that context, computer models constitute valid alternatives to experiments. Here, we present the new SwissADME web tool that gives free access to a pool of fast yet robust predictive models for physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry friendliness, among which in-house proficient methods such as the BOILED-Egg, iLOGP and Bioavailability Radar. Easy efficient input and interpretation are ensured thanks to a user-friendly interface through the login-free website http://www.swissadme.ch. Specialists, but also nonexpert in cheminformatics or computational chemistry can predict rapidly key parameters for a collection of molecules to support their drug discovery endeavours.
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  22. 22
    Bik, E.; Mateuszuk, L.; Orleanska, J.; Baranska, M.; Chlopicki, S.; Majzner, K. Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium. Int. J. Mol. Sci. 2021, 22, 2401  DOI: 10.3390/ijms22052401
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    Chloroquine-induced accumulation of autophagosomes and lipids in the endothelium
    Bik, Ewelina; Mateuszuk, Lukasz; Orleanska, Jagoda; Baranska, Malgorzata; Chlopicki, Stefan; Majzner, Katarzyna
    International Journal of Molecular Sciences (2021), 22 (5), 2401CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome-lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid compn. of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodol. based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chem. changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a ref. method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concns. of 1-30μM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, resp. The CQ-induced endothelial response was assocd. with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochem. changes offers a better understanding of the autophagy mechanism in the endothelial cells.
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    Pal, K.; Kumar, P.; Koner, A. L. Deciphering interior polarity of lysosome in live cancer and normal cells using spectral scanning microscopy. J. Photochem. Photobiol., B 2020, 206, 111848  DOI: 10.1016/j.jphotobiol.2020.111848
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    Deciphering interior polarity of lysosome in live cancer and normal cells using spectral scanning microscopy
    Pal, Kaushik; Kumar, Prashant; Koner, Apurba Lal
    Journal of Photochemistry and Photobiology, B: Biology (2020), 206 (), 111848CODEN: JPPBEG; ISSN:1011-1344. (Elsevier B.V.)
    A lysosome specific, pH tolerant, and polarity-sensitive fluorescent probe (LyPol) is designed and synthesized for the detn. of lysosomal polarity in live cells. LyPol possesses an intramol. charge transfer (ICT) properties with high quantum yield in water and in other polar solvents such as methanol, ethanol, DMSO, acetonitrile, etc. The fluorescence maxima and lifetime increase linearly with a non-specific manner with an increase in the polarity of its surrounding environment. A morpholine group connected with an alkyl linker acts as a lysosome directing moiety, which is attached to the fluorescent core of LyPol. The selective localization of LyPol inside the lysosome was confirmed with live-cell confocal imaging. Further, the spectral scanning confocal technique was utilized to det. the emission spectrum of LyPol inside lysosome, and the polarity turns out to be quite lower as compared to water. Moreover, the combined spectroscopic and live-cell microscopy confirms that the interior of the lysosome is significantly non-polar in cancer cells compared to normal cells. We believe that this report on the measuring polarity inside the biol. system with a solvatofluorochromic probe will be of immense interest to researchers working in the multidisciplinary area of biophysics, microscopy, chem. biol., and organelle biol.
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    Gai, F.; Zuo, Y.; Lin, W. Detecting lipid droplets polarity: Silicone-based unique fluorescent probe for cancer diagnosis in living cells. Talanta 2021, 225, 122059  DOI: 10.1016/j.talanta.2020.122059
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    Detecting lipid droplets polarity: Silicone-based unique fluorescent probe for cancer diagnosis in living cells
    Gai, Fengqing; Zuo, Yujing; Lin, Weiying
    Talanta (2021), 225 (), 122059CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    Fluorescent probes for monitoring polarity of lipid droplets (LDs) are essential tools in pathol. research, esp. cancer related. Herein, we have designed a biocompatible and novel fluorescent probe (TDCQ) with intramol. charge transfer mechanism, which consists of a naphthalimide moiety accepting electron and a triphenylamine fragment providing electron. In view of polarity-sensitivity and AIE characteristic, TDCQ specially aggregates on the LDs in cells by remarkable green dots fluorescent. Due to the variation of LDs nos. in normal cells and cancer cells, the probe emits stronger green fluorescence in cancer cells but weaker in normal cells. Moreover, TDCQ has outstanding photostability and low toxicity, permitting green fluorescence to persist for a valid time in cells. This article demonstrates that the capacity of TDCQ for facilitating the in-depth study of LDs and applying to the identification of cancer cells.
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    Danylchuk, D. I.; Jouard, P.-H.; Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. J. Am. Chem. Soc. 2021, 143, 912– 924,  DOI: 10.1021/jacs.0c10972
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    25
    Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
    Danylchuk, Dmytro I.; Jouard, Pierre-Henri; Klymchenko, Andrey S.
    Journal of the American Chemical Society (2021), 143 (2), 912-924CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi app., plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission max., as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy expts. with ref. organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quant. description of the lipid order in each organelle of interest. Cholesterol extn./enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mech. stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to phys. and chem. stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.
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    Collot, M.; Bou, S.; Fam, T. K.; Richert, L.; Mély, Y.; Danglot, L.; Klymchenko, A. S. Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push–Pull Fluorophore. Anal. Chem. 2019, 91, 1928– 1935,  DOI: 10.1021/acs.analchem.8b04218
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    Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore
    Collot, Mayeul; Bou, Sophie; Fam, Tkhe Kyong; Richert, Ludovic; Mely, Yves; Danglot, Lydia; Klymchenko, Andrey S.
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (3), 1928-1935CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiol. processes as well as diseases. Herein the authors synthesized a push-pull mol. named DAF (Di-Me Aniline Furaldehyde) that possesses a strong pos. solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.
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    Dai, Y.; Zhan, Z.; Li, Q.; Liu, R.; Lv, Y. Simultaneous monitoring of polarity changes of lipid droplets and lysosomes with two-photon fluorescent probes. Anal. Chim. Acta 2020, 1136, 34– 41,  DOI: 10.1016/j.aca.2020.08.033
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    Simultaneous monitoring of polarity changes of lipid droplets and lysosomes with two-photon fluorescent probes
    Dai, Yongcheng; Zhan, Zixuan; Li, Qiuyan; Liu, Rui; Lv, Yi
    Analytica Chimica Acta (2020), 1136 (), 34-41CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    Intracellular polarity is an essential feature of cell physiol. state and abnormal polarity changes of various organelles are related to many diseases. Thus, monitoring of polarity changes of multiple subcellular in living cells contributes to understanding different physiol. and pathol. processes more accurately. However, most of the previous reports on polarity probes mainly monitored the polarity of a single organelle. Therefore, we designed and synthesized two unique polarity-sensitive fluorescent probes LDs-TPFP and Lyso-TPFP, which can be selectively located in lipid droplets (LDs) and lysosomes resp., to obtain more subcellular information in living cells. Thanks to the strong intramol.-charge-transfer (ICT) characteristics of probes, the fluorescence intensity and emission wavelength would change with the polarity of the surroundings of cells. Moreover, LDs-TPFP and Lyso-TPFP exhibits large Stokes shift and excellent biocompatibility. Through fluorescence imaging, the probes can effectively distinguish normal cells from cancer cells. In addn., the results of two-photon confocal fluorescence imaging indicated that LDs and lysosomes have discrepant polarity change behaviors under different physiol. conditions.
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    Yin, J.; Peng, M.; Lin, W. Two-photon fluorescence imaging of lipid drops polarity toward cancer diagnosis in living cells and tissue. Sens. Actuators, B 2019, 288, 251– 258,  DOI: 10.1016/j.snb.2019.02.122
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    Two-photon fluorescence imaging of lipid drops polarity toward cancer diagnosis in living cells and tissue
    Yin, Junling; Peng, Min; Lin, Weiying
    Sensors and Actuators, B: Chemical (2019), 288 (), 251-258CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Monitoring the polarity of intracellular lipid droplets (LDs) is of great importance toward cancer diagnosis. Herein, we have rationally designed a novel LDs-targeting and polarity-sensitive fluorescent probe CBMC, which contains carbazole as an electron-donating group and malononitrile as an electron-accepting group forming intramol. charge transfer mechanism. Due to its excellent solvatochromism, the probe emits stronger fluorescence in normal cells but weaker in cancer cells. By means of CBMC, normal tissues with stronger fluorescence have been obviously pointed out from cancerous tissues. For cancer therapy, the probe CBMC could be an ideal candidate to evaluate tumor, because the tumor area would decrease while the normal tissue area would increase with treatment time. Compared with existing probes with turn-off signal to normal cells, such a new probe with turn on type in normal cells could provide more reliable information with advantages of lower background interference, higher signal to noise ratio, and higher sensitivity. This probe CBMC will be a powerful tool to be potentially applied in tumor diagnosis in the case of treatment.
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    Chowdhury, R.; Jana, B.; Saha, A.; Ghosh, S.; Bhattacharyya, K. Confocal microscopy of cytoplasmic lipid droplets in a live cancer cell: number, polarity, diffusion and solvation dynamics. MedChemComm 2014, 5, 536– 539,  DOI: 10.1039/C3MD00269A
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    Confocal microscopy of cytoplasmic lipid droplets in a live cancer cell: number, polarity, diffusion and solvation dynamics
    Chowdhury, Rajdeep; Jana, Batakrishna; Saha, Abhijit; Ghosh, Surajit; Bhattacharyya, Kankan
    MedChemComm (2014), 5 (4), 536-539CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)
    Time resolved confocal microscopy indicates that the cytoplasmic lipid droplets (CLDs) in live cells (normal and cancer lung cells) are less polar, and exhibit slower diffusion (motility) and solvation dynamics than the cytoplasm. The no. of CLDs in a human lung cancer cell (A549) is ∼10 times higher than in a non-cancer lung fibroblast cell (WI38). This may result in accumulation of non-polar cell signaling agents in the CLDs of the cancer cell.
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    Fam, T. K.; Klymchenko, A. S.; Collot, M. Recent Advances in Fluorescent Probes for Lipid Droplets. Materials 2018, 11, 1768  DOI: 10.3390/ma11091768
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    Recent advances in fluorescent probes for lipid droplets
    Fam, Tkhe Kyong; Klymchenko, Andrey S.; Collot, Mayeul
    Materials (2018), 11 (9), 1768/1-1768/19CODEN: MATEG9; ISSN:1996-1944. (MDPI AG)
    Lipid droplets (LDs) are organelles that serve as the storage of intracellular neutral lipids. LDs regulate many physiol. processes. They recently attracted attention after extensive studies showed their involvement in metabolic disorders and diseases such as obesity, diabetes, and cancer. Therefore, it is of the highest importance to have reliable imaging tools. In this review, we focus on recent advances in the development of selective fluorescent probes for LDs. Their photophys. properties are described, and their advantages and drawbacks in fluorescence imaging are discussed. At last, we review the reported applications using these probes including two-photon excitation, in vivo and tissue imaging, as well as LDs tracking.
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    Danylchuk, D. I.; Jouard, P.-H.; Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. J. Am. Chem. Soc. 2021, 143, 912– 924,  DOI: 10.1021/jacs.0c10972
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    31
    Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
    Danylchuk, Dmytro I.; Jouard, Pierre-Henri; Klymchenko, Andrey S.
    Journal of the American Chemical Society (2021), 143 (2), 912-924CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi app., plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission max., as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy expts. with ref. organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quant. description of the lipid order in each organelle of interest. Cholesterol extn./enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mech. stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to phys. and chem. stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.
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    Fan, L.; Wang, X.; Zan, Q.; Fan, L.; Li, F.; Yang, Y.; Zhang, C.; Shuang, S.; Dong, C. Lipid Droplet-Specific Fluorescent Probe for In Vivo Visualization of Polarity in Fatty Liver, Inflammation, and Cancer Models. Anal. Chem. 2021, 93, 8019– 8026,  DOI: 10.1021/acs.analchem.1c01125
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    Lipid Droplet-Specific Fluorescent Probe for In Vivo Visualization of Polarity in Fatty Liver, Inflammation, and Cancer Models
    Fan, Li; Wang, Xiaodong; Zan, Qi; Fan, Lifang; Li, Feng; Yang, Yongming; Zhang, Caihong; Shuang, Shaomin; Dong, Chuan
    Analytical Chemistry (Washington, DC, United States) (2021), 93 (22), 8019-8026CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Elucidating the intrinsic relationship between diseases and lipid droplet (LD) polarity remains a great challenge owing to the lack of the research on multiple disease models. Until now, the visualization of abnormal LD polarity in models of inflammation and clin. cancer patient samples has not been achieved. To meet the urgent challenge, we facilely synthesized a robust LD-specific and polarity-sensitive fluorescent probe (LD-TTP), which consists of a triphenylamine segment as an electron-donor group (D) and a pyridinium as an electron-acceptor moiety (A), forming a typical D-π-A mol. configuration. Owing to the unique intramol. charge transfer effect, LD-TTP exhibits high sensitivity to polarity change in the linear range from Δf = 0.258 to 0.312, with over 278-fold fluorescence enhancement. Moreover, we revealed that LD-TTP possessed satisfactory ability for sensitively monitoring LD-polarity changes in living cells. Using LD-TTP, we first demonstrated the detection of LD-polarity changes in fatty liver tissues and inflammatory living mice via confocal laser scanning fluorescence imaging. Surprisingly, the visualization of LD polarity has been achieved not only at the cellular levels and living organs but also in surgical specimens from cancer patients, thus holding great potential in the clin. diagnosis of human cancer. All these features render LD-TTP an effective tool for medical diagnosis of LD polarity-related diseases.
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  33. 33
    Wang, K.-N.; Liu, L.-Y.; Mao, D.; Xu, S.; Tan, C.-P.; Cao, Q.; Mao, Z.-W.; Liu, B. A Polarity-Sensitive Ratiometric Fluorescence Probe for Monitoring Changes in Lipid Droplets and Nucleus during Ferroptosis. Angew. Chem., Int. Ed. 2021, 60, 15095– 15100,  DOI: 10.1002/anie.202104163
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    A Polarity-Sensitive Ratiometric Fluorescence Probe for Monitoring Changes in Lipid Droplets and Nucleus during Ferroptosis
    Wang, Kang-Nan; Liu, Liu-Yi; Mao, Duo; Xu, Shidang; Tan, Cai-Ping; Cao, Qian; Mao, Zong-Wan; Liu, Bin
    Angewandte Chemie, International Edition (2021), 60 (27), 15095-15100CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Ferroptosis regulates cell death through reactive oxygen species (ROS)-assocd. lipid peroxide accumulation, which is expected to affect the structure and polarity of lipid droplets (LDs), but with no clear evidence. Herein, we report the first example of an LD/nucleus dual-targeted ratiometric fluorescent probe, CQPP, for monitoring polarity changes in the cellular microenvironment. Due to the donor-acceptor structure of CQPP, it offers ratiometric fluorescence emission and fluorescence lifetime signals that reflect polarity variations. Using nucleus imaging as a ref., CQPP was applied to report the increase in LD polarity and the homogenization of polarity between LDs and cytoplasm in the ferroptosis model. This LD/nucleus dual-targeted fluorescent probe shows the great potential of using fluorescence imaging to study ferroptosis and ferroptosis-related diseases.
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    Zhu, H.; Fan, J.; Mu, H.; Zhu, T.; Zhang, Z.; Du, J.; Peng, X. d-PET-controlled “off-on” Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes. Sci. Rep. 2016, 6, 35627  DOI: 10.1038/srep35627
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    d-PET-controlled "off-on" Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes
    Zhu, Hao; Fan, Jiangli; Mu, Huiying; Zhu, Tao; Zhang, Zhen; Du, Jianjun; Peng, Xiaojun
    Scientific Reports (2016), 6 (), 35627CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Polarity-sensitive fluorescent probes are powerful chem. tools for studying biomol. structures and activities both in vitro and in vivo. However, the lack of "off-on" polarity-sensing probes has limited the accurate monitoring of biol. processes that involve an increase in local hydrophilicity. Here, we design and synthesize a series of "off-on" polarity-sensitive fluorescent probes BP series consisting of the difluoroboron dippyomethene (BODIPY) fluorophore connected to a quaternary ammonium moiety via different carbon linkers. All these probes showed low fluorescence quantum yields in nonpolar soln. but became highly fluorescent in polar media. BP-2, which contains a two-carbon linker and a tri-Me quaternary ammonium, displayed a fluorescence intensity and quantum yield that were both linearly correlated with solvent polarity. In addn., BP-2 exhibited high sensitivity and selectivity for polarity over other environmental factors and a variety of biol. relevant species. BP-2 can be synthesized readily via an unusual Mannich reaction followed by methylation. Using electrochem. combined with theor. calcns., we demonstrated that the "off-on" sensing behavior of BP-2 is primarily due to the polarity-dependent donor-excited photoinduced electron transfer (d-PET) effect. Live-cell imaging established that BP-2 enables the detection of local hydrophilicity within lysosomes under conditions of lysosomal dysfunction.
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    Hu, L.; Shi, D.; Li, X.; Zhu, J.; Mao, F.; Li, X.; Xia, C.; Jiang, B.; Guo, Y.; Li, J. Curcumin-based polarity fluorescent probes: Design strategy and biological applications. Dyes Pigm. 2020, 177, 108320  DOI: 10.1016/j.dyepig.2020.108320
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    Curcumin-based polarity fluorescent probes: Design strategy and biological applications
    Hu, Linghao; Shi, Donglei; Li, Xinming; Zhu, Jin; Mao, Fei; Li, Xiaokang; Xia, Conglong; Jiang, Bei; Guo, Yuan; Li, Jian
    Dyes and Pigments (2020), 177 (), 108320CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)
    Polarity is a crucial characteristic of indispensable organelles, which has been indicated to be closely related to many pathol. and biol. processes. Hence, a novel design strategy of curcumin-based polarity fluorescent probes was presented. Curcumin attracted much attention due to its environment-sensitivity and optical properties. The authors reformed the structure of curcumin by attaching a galactose moiety that was used to increase the water soly. and regulate the probe working mode. Furthermore, the authors also introduced a diethylamino group, which played a substantial role in adjusting the polarity specificity and sensitivity of the probes. The authors finally screened two polarity-specific fluorescent probes 0301 and 0302. Both probes exhibited remarkable changes in fluorescence from red to green upon increasing polarity of environment. Using 0301 in confocal imaging, the authors obtained a clear and exact view of lysosomal polarity in different cells, suggesting that the lysosomal polarity in normal cells is higher than that in cancer cells. Also, the authors use 0301 to monitor variations in lysosomal polarity in live cells induced by dimethylsulfoxide and sucrose, indicating that the authors' probe could specifically monitor the changes of polarity in live cells to elucidate polarity-related biol. processes.
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    Orte, A.; Crovetto, L.; Talavera, E. M.; Boens, N.; Alvarez-Pez, J. M. Absorption and Emission Study of 2′,7′-Difluorofluorescein and Its Excited-State Buffer-Mediated Proton Exchange Reactions. J. Phys. Chem. A 2005, 109, 734– 747,  DOI: 10.1021/jp046786v
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    Absorption and emission study of 2',7'-difluorofluorescein and its excited-state buffer-mediated proton exchange reactions
    Orte, Angel; Crovetto, Luis; Talavera, Eva M.; Boens, Noeel; Alvarez-Pez, Jose M.
    Journal of Physical Chemistry A (2005), 109 (5), 734-747CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
    We present steady-state emission evidence that acetate buffer species promote an excited-state proton transfer between neutral, monoanionic, and dianionic forms of 2',7'-difluorofluorescein. The time course of the excited species in this reaction was characterized through time-resolved fluorescence measurements, and the kinetics of the reaction were solved by using global compartmental anal. A previous identifiability study on the compartmental system set the conditions to design the fluorescence decay surface. This is the first exptl. system, studied within this kinetic model, solved under identifiability conditions through global compartmental anal. The recovered rate const. values for deactivation were 2.94 × 108 s-1 for the monoanion and 2.47 × 108 s-1 for the dianion, whereas the rate const. values of the buffer-mediated excited-state reaction were 9.70 × 108 and 1.79 × 108 M-1 s-1 for the deprotonation and protonation, resp. With these values, a pKa* = 4.02 was obtained. In this work, we addnl. provide an absorption study, including acid-base equil., detn. of ground-state pKa values (1.02, 3.61, and 4.69), and recovery of molar absorption coeffs. of every prototropic species, including absorption and NMR evidence for the existence of three tautomers in neutral species. Steady-state emission spectra of 2',7'-difluorofluorescein in aq. soln. are also described, where the strong photoacid behavior of the cation is noteworthy.
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    Li, J.; Xu, J.; Guo, W.; Zhong, W.; Li, Q.; Tan, L.; Shang, L. Ratiometric fluorescence sensors for heparin and heparinase based on enhanced excimer emission of perylene probe induced by cationic silver nanoparticles. Sens. Actuators, B 2020, 305, 127422  DOI: 10.1016/j.snb.2019.127422
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    Ratiometric fluorescence sensors for heparin and heparinase based on enhanced excimer emission of perylene probe induced by cationic silver nanoparticles
    Li, Juanmin; Xu, Jie; Guo, Wenfeng; Zhong, Wencheng; Li, Qiang; Tan, Lili; Shang, Li
    Sensors and Actuators, B: Chemical (2020), 305 (), 127422CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Herein, cationic MUTAB-Ag NPs of different sizes were synthesized and their interactions with a neg. charged perylene probe, N,N'-bis(6-caproic acid)-3,4:9,10-perylenediimide (PDI-COOH), were investigated. MUTAB-Ag NPs could result in significant monomer quenching and excimer formation by inducing probe aggregation due to the strong electrostatic attractive interactions between them. Moreover, significant excimer emission could be obsd. at extremely low probe concn. when induced by larger MUTAB-Ag NPs. A possible mechanism of metal-enhanced fluorescence was proposed, which is evidenced by the strong dependence between surface plasmon resonance absorption spectra of MUTAB-Ag NPs and the excimer emission spectra of PDI-COOH, together with the sharp decrease in fluorescence lifetime of excimer compared with that induced by poly(allylamine). Consequently, ratiometric fluorescence sensors for heparin and heparinase were developed based on the Ag NP-induced excimer emission of PDI-COOH. The adsorption of heparin on the surface of MUTAB-Ag NPs led to the release of monomer, while the degrdn. of heparin by heparinase facilitated the formation of PDI-COOH excimer. The monomer-excimer transition signal was harnessed for heparin and heparinase sensing, which exhibited good sensitivity and excellent selectivity. Finally, application of the present system for detection of heparin and heparinase in human serum sample was successfully demonstrated.
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    Das, S.; Sahana, A.; Banerjee, A.; Lohar, S.; Safin, D. A.; Babashkina, M. G.; Bolte, M.; Garcia, Y.; Hauli, I.; Mukhopadhyay, S. K.; Das, D. Ratiometric fluorescence sensing and intracellular imaging of Al3+ ions driven by an intramolecular excimer formation of a pyrimidine–pyrene scaffold. Dalton Trans. 2013, 42, 4757– 4763,  DOI: 10.1039/C3DT32908A
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    Ratiometric fluorescence sensing and intracellular imaging of Al3+ ions driven by an intramolecular excimer formation of a pyrimidine-pyrene scaffold
    Das, Sudipta; Sahana, Animesh; Banerjee, Arnab; Lohar, Sisir; Safin, Damir A.; Babashkina, Maria G.; Bolte, Michael; Garcia, Yann; Hauli, Ipsit; Mukhopadhyay, Subhra Kanti; Das, Debasis
    Dalton Transactions (2013), 42 (14), 4757-4763CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
    An Al3+ selective ratiometric fluorescent probe (L) has been synthesized by condensation of 4,5-diaminopyrimidine with 1-pyrenecarboxaldehyde. The structure of L has been confirmed by single crystal X-ray anal. In the presence of Al3+, L exhibits a considerable excimer emission at 445 nm along with the decrease of its monomer emission at 368 nm in DMSO-H2O (4 : 1, vol./vol.). The lowest detection limit for Al3+ is 0.24 μM. Furthermore, L can permeate through the cell membrane and detects intracellular Al3+ ions under a fluorescence microscope.
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    Razi, S. S.; Ali, R.; Srivastava, P.; Misra, A. Smart excimer fluorescence probe for visual detection, cell imaging and extraction of Hg2+. RSC Adv. 2015, 5, 79538– 79547,  DOI: 10.1039/C5RA13021B
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    Smart excimer fluorescence probe for visual detection, cell imaging and extraction of Hg2+
    Razi, Syed S.; Ali, Rashid; Srivastava, Priyanka; Misra, Arvind
    RSC Advances (2015), 5 (97), 79538-79547CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    Smart pyrene-based simple fluorescent probes 2 and 4 were designed, synthesized and characterized by different spectroscopic methods. The photophys. properties of the probes and their affinity towards different metal ions in phosphate buffer were investigated. Upon selective interaction with Hg2+, the mol. probes showed enhanced static excimer emission at 506 nm along with a naked-eye detectable chromo- and fluoro-genic response. Probe 2 sensitively showed a high limit of detection (3.4 pM) for Hg2+ in the soln. The pyrene silicate deriv., 4, was utilized to detect and ext. Hg2+ in soln. as well as in the solid state. The data obtained from NMR and ESI-MS spectroscopy supported the postulate that the mode of interaction of the probe with Hg2+ involves the N and O atoms of the -C=N and -OH functional groups to complex Hg2+ in 2 : 1 stoichiometry. Moreover, probe 2 exhibited excellent selectivity for Hg2+ in protein medium (BSA/HSA) and was used to detect Hg2+ in live HeLa cells, on test paper strips and in real contaminated water samples.
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    Upadhyay, Y.; Anand, T.; Babu, L. T.; Paira, P.; Crisponi, G.; Sk, A. K.; Kumar, R.; Sahoo, S. K. Three-in-one type fluorescent sensor based on a pyrene pyridoxal cascade for the selective detection of Zn(II), hydrogen phosphate and cysteine. Dalton Trans. 2018, 47, 742– 749,  DOI: 10.1039/C7DT04234E
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    Three-in-one type fluorescent sensor based on a pyrene pyridoxal cascade for the selective detection of Zn(II), hydrogen phosphate and cysteine
    Upadhyay, Yachana; Anand, Thangaraj; Babu, Lavanya Thilak; Paira, Priyankar; Crisponi, Guido; S K, Ashok Kumar; Kumar, Rajender; Sahoo, Suban K.
    Dalton Transactions (2018), 47 (3), 742-749CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
    A novel fluorescent receptor L was synthesized by Schiff base condensation of 1-pyrenemethylamine with the vitamin B6 cofactor pyridoxal. The receptor L is highly selective and sensitive towards Zn2+ ions among other tested metal ions. Upon interaction with Zn2+, the receptor L showed a distinct fluorescence enhancement at 485 nm due to the excimer formation leading to the fluorescent color change from blue to bluish-green. Subsequently, when the in situ generated ZnL2 complex interacted with various anions and amino acids, the addn. of H2PO4- and cysteine reinstated the fluorescence of the receptor L due to the demetalation of Zn2+ from the ZnL2 complex. Accordingly, the receptor L was developed for the highly selective, specific and sensitive detection of three important bioactive analytes, i.e., Zn2+, H2PO4- and cysteine with a detection limit down to 2.3 × 10-6 M, 2.18 × 10-7 M and 1.59 × 10-7 M, resp. Addnl., the receptor L was applied to the detection of intracellular Zn2+ ions in live HeLa cells.
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    Gao, M.; Tang, B. Z. Fluorescent Sensors Based on Aggregation-Induced Emission: Recent Advances and Perspectives. ACS Sens. 2017, 2, 1382– 1399,  DOI: 10.1021/acssensors.7b00551
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    Fluorescent Sensors Based on Aggregation-Induced Emission: Recent Advances and Perspectives
    Gao, Meng; Tang, Ben Zhong
    ACS Sensors (2017), 2 (10), 1382-1399CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    A review. Fluorescent sensors with advantages of excellent sensitivity, rapid response, and easy operation are emerging as powerful tools in environmental monitoring, biol. research, and disease diagnosis. However, conventional fluorophores featured with π-planar structures usually suffer from serious self-quenching in the aggregated state, poor photostability, and small Stokes' shift. In contrast to conventional aggregation-caused quenching (ACQ) fluorophores, the newly emerged aggregation-induced emission fluorogens (AIEgens) are featured with high emission efficiency in the aggregated state, which provide unique opportunities for various sensing applications with advantages of high signal-to-noise ratio, strong photostability, and large Stokes' shift. In this review, the authors will first briefly give an introduction of the AIE concept and the turn-on sensing principles. Then, the authors will discuss the recent examples of AIE sensors according to types of analytes. Finally, the authors will give a perspective on the future developments of AIE sensors. The authors hope this review will inspire more endeavors to devote to this emerging world.
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    Zhang, Q.; Xiao, K.; Paredes, J. M.; Mamonova, T.; Sneddon, W. B.; Liu, H.; Wang, D.; Li, S.; McGarvey, J. C.; Uehling, D.; Al-awar, R.; Joseph, B.; Jean-Alphonse, F.; Orte, A.; Friedman, P. A. Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport. J. Biol. Chem. 2019, 294, 4546– 4571,  DOI: 10.1074/jbc.RA119.007421
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    Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport
    Zhang, Qiangmin; iao, Kunhong; Paredes, Jose M.; Mamonova, Tatyana; Sneddon, W. Bruce; Liu, Hongda; Wang, Dawei; Li, Sheng; McGarvey, Jennifer C.; Uehling, David; Al-awar, Rima; Joseph, Babu; Jean-Alphonse, Frederic; Orte, Angel; Friedman, Peter A.
    Journal of Biological Chemistry (2019), 294 (12), 4546-4571CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Na+-Hα exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium- phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS anal., we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290. Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we obsd. that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange M S revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochem. mechanisms underlying PTH control of phosphate transport.
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    Paredes, J. M.; Giron, M. D.; Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Salto, R.; Alvarez-Pez, J. M. Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM). J. Phys. Chem. B 2013, 117, 8143– 8149,  DOI: 10.1021/jp405041c
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    Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM)
    Paredes, Jose M.; Giron, Maria D.; Ruedas-Rama, Maria J.; Orte, Angel; Crovetto, Luis; Talavera, Eva M.; Salto, Rafael; Alvarez-Pez, Jose M.
    Journal of Physical Chemistry B (2013), 117 (27), 8143-8149CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Phosphate ions play important roles in signal transduction and energy storage in biol. systems. However, robust chem. sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein deriv. dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivs. at approx. physiol. pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concn. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodol. has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alk. phosphatase activity in the extracellular medium as a marker of the differentiation process.
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  • Abstract

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    Figure 1

    Figure 1. TD-CAM-B3LYP-D3/6-31+G** calculation results of the molecular geometry and HOMO and LUMO (isocontour plots 0.02 au) of an antiparallel excimer of acridone 1 in water solution.

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    Figure 2

    Figure 2. (A) TRES spectra of 1 dissolved in acetonitrile (λex = 375 nm) at 0, 0.5, 0.8, 1, 1.2, 1.5, 3, 5, 8, 10, 15, 20, and 25 ns after the excitation pulse. (B) SAEMS of 1 in acetonitrile, associated with the longest lifetime (blue) and the shortest lifetime (red).

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    Figure 3

    Figure 3. (A) FLIM image of dye 1 in CCD-18Co cells and segmentation procedure using DYNEX. Pixels with negative A2 amplitude are represented in blue. (B) Distribution of amplitudes of the short decay time (A2) of the image in panel (A). (C) Representative colocalization imaging of 1 (green channel) with LipidTOX (red channel). Scale bar of 10 μm. (D) Correlation plot (left) and intensity profiles of lines 1, 2, and 3 (right) from the green and red channels of panel (C).

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    Figure 4

    Figure 4. (A) Schematic of the DYNEX concept: selective formation of dynamic excimers of dye 1 in lipid droplets. (B, C) FLIM image (left panels), DYNEX segmentation of lipid droplets (central panels), and ET30 imaging (right panels) of 1 in CCD-18Co cells (B) and tumor CT26.WT cells (C). Scale bars represent 10 μm for full images and 2 μm for zoomed images.

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    Figure 5

    Figure 5. ET30 values in lipid droplets of CCD-18Co cells (A) and tumor CT26.WT cells (B) upon treatment with sucrose and chloroquine. Each individual value corresponds to lipid droplet regions from different cells from >10 different images.

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  • References

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      Chemical Society Reviews (2014), 43 (13), 4563-4601CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Environment-related parameters, including viscosity, polarity, temp., hypoxia, and pH, play pivotal roles in controlling the phys. or chem. behaviors of local mols. In particular, in a biol. environment, such factors predominantly det. the biol. properties of the local environment or reflect corresponding status alterations. Abnormal changes in these factors would cause cellular malfunction or become a hallmark of the occurrence of severe diseases. Therefore, in recent years, they have increasingly attracted research interest from the fields of chem. and biol. chem. With the emergence of fluorescence sensing and imaging technol., several fluorescent chemosensors have been designed to respond to such parameters and to further map their distributions and variations in vitro/in vivo. In this work, we have reviewed a no. of various environment-responsive chemosensors related to fluorescent recognition of viscosity, polarity, temp., hypoxia, and pH that have been reported thus far.
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      A review. Lipid droplets are storage organelles at the center of lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, which is enclosed by a phospholipid monolayer that is decorated by a specific set of proteins. Originating from the endoplasmic reticulum, lipid droplets can assoc. with most other cellular organelles through membrane contact sites. It is becoming apparent that these contacts between lipid droplets and other organelles are highly dynamic and coupled to the cycles of lipid droplet expansion and shrinkage. Importantly, lipid droplet biogenesis and degrdn., as well as their interactions with other organelles, are tightly coupled to cellular metab. and are crit. to buffer the levels of toxic lipid species. Thus, lipid droplets facilitate the coordination and communication between different organelles and act as vital hubs of cellular metab.
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      Welte, Michael A.
      Current Biology (2015), 25 (11), R470-R481CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      A review. Lipid droplets are the intracellular sites for neutral lipid storage. They are crit. for lipid metab. and energy homeostasis, and their dysfunction has been linked to many diseases. Accumulating evidence suggests that the roles lipid droplets play in biol. are significantly broader than previously anticipated. Lipid droplets are the source of mols. important in the nucleus: they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors. Lipid droplets have also emerged as important nodes for fatty acid trafficking, both inside the cell and between cells. In immunity, new roles for droplets, not directly linked to lipid metab., have been uncovered, with evidence that they act as assembly platforms for specific viruses and as reservoirs for proteins that fight intracellular pathogens. Until recently, knowledge about droplets in the nervous system has been minimal, but now there are multiple links between lipid droplets and neurodegeneration: many candidate genes for hereditary spastic paraplegia also have central roles in lipid-droplet formation and maintenance, and mitochondrial dysfunction in neurons can lead to transient accumulation of lipid droplets in neighboring glial cells, an event that may, in turn, contribute to neuronal damage. As the cell biol. and biochem. of lipid droplets become increasingly well understood, the next few years should yield many new mechanistic insights into these novel functions of lipid droplets.
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      Combining hydrophilic and hydrophobic environment sensitive dyes to detect a wide range of cellular polarity
      Park, Sang Jun; Juvekar, Vinayak; Jo, Jae Hyung; Kim, Hwan Myung
      Chemical Science (2020), 11 (2), 596-601CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Intracellular polarity is an important parameter of pathol. and biol. phenomena of cells; abnormal polarities are assocd. with diabetes, neurol. diseases, and cancer. However, previously reported polarity probes have issues with quant. detecting intracellular polarities, can measure only a limited range of polarities, and can only detect specific intracellular regions. Here, we developed a novel two-dye system, RPS-1, that contains a new "turn-on" polarity probe (Dye1) based on a spiropyran intramol. ring closing-opening system activated in polar protic solvents, and a benzothiadiazole contg. dye (Dye3), which emits only in non-polar solvents with a large stoke shift. Individually, Dye1 and Dye3 selectively localized to lysosome and lipid droplets, resp.; however, combining these dyes, which have completely different characteristics, via a piperazine linker resulted in the staining of various intracellular organelles. Therefore, as Dye1 and Dye3 have the same absorption but different emissions, combining them resulted in a ratiometric polarity probe that could quant. measure a wider polarity range inside the cell using a single excitation source. In addn., ratiometric imaging using our RPS-1 probe to quant. detect the distribution of polarity in different cell lines indicated that lysosomes were the most polar organelles in the cell.
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      Ashoka, A. H.; Ashokkumar, P.; Kovtun, Y. P.; Klymchenko, A. S. Solvatochromic Near-Infrared Probe for Polarity Mapping of Biomembranes and Lipid Droplets in Cells under Stress. J. Phys. Chem. Lett. 2019, 10, 2414– 2421,  DOI: 10.1021/acs.jpclett.9b00668
      5
      Solvatochromic Near-Infrared Probe for Polarity Mapping of Biomembranes and Lipid Droplets in Cells under Stress
      Ashoka, Anila Hoskere; Ashokkumar, Pichandi; Kovtun, Yuriy P.; Klymchenko, Andrey S.
      Journal of Physical Chemistry Letters (2019), 10 (10), 2414-2421CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Can polarity-sensitive fluorescent dyes monitor the response of live cells to fundamental stress conditions, such as deprivation from nutrition and oxidative stress. To address this question, we developed a push-pull dioxaborine probe (DXB-NIR) for biomembranes and lipid droplets featuring strong solvatochromism in the far-red to near-IR region, high fluorescence brightness, photostability, and two-photon absorption cross section, reaching 13800 GM at 930 nm. In model membranes, DXB-NIR exhibits unprecedented 80 nm shift between liq. ordered and disordered membrane phases, allowing robust imaging of sepd. membrane microdomains. Two-color imaging of live cells with DXB-NIR enables polarity mapping in plasma membranes, endoplasmic reticulum, and lipid droplets, which reveals that starvation and oxidative stress produce an increase in the local polarity, and this change is different for each of the studied cell compartments. Thus, by pushing the limits of existing solvatochromic dyes, we introduce a concept of polarity mapping for monitoring the response of cells to stress.
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      Yin, J.; Peng, M.; Ma, Y.; Guo, R.; Lin, W. Rational design of a lipid-droplet-polarity based fluorescent probe for potential cancer diagnosis. Chem. Commun. 2018, 54, 12093– 12096,  DOI: 10.1039/C8CC07398H
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      Rational design of a lipid-droplet-polarity based fluorescent probe for potential cancer diagnosis
      Yin, Junling; Peng, Min; Ma, Yanyan; Guo, Rui; Lin, Weiying
      Chemical Communications (Cambridge, United Kingdom) (2018), 54 (85), 12093-12096CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A robust fluorescent probe CTPA has been rationally designed for cancer diagnosis by monitoring lipid drop (LD) polarity and no. variation. With the outstanding properties of CTPA, we have shown that the diagnosis of cancer can be achieved not only at the cellular levels but also in organs and living mice for the first time.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVahs77M&md5=2d702f1363a9443308c6b76665a8633f
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      Collot, M.; Fam, T. K.; Ashokkumar, P.; Faklaris, O.; Galli, T.; Danglot, L.; Klymchenko, A. S. Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues. J. Am. Chem. Soc. 2018, 140, 5401– 5411,  DOI: 10.1021/jacs.7b12817
      7
      Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues
      Collot, Mayeul; Fam, Tkhe Kyong; Ashokkumar, Pichandi; Faklaris, Orestis; Galli, Thierry; Danglot, Lydia; Klymchenko, Andrey S.
      Journal of the American Chemical Society (2018), 140 (16), 5401-5411CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiol. processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein the authors present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate deriv. These so-called StatoMerocyanines (SMCy)(I, n = 1-3) fluoresce from yellow to the near-IR (NIR) in oil with an impressive fluorescence enhancement compared to aq. media. Addnl., SMCy display remarkably high molar absorptivities (up to 390,000 M-1 cm-1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, esp. SMCy5.5 (up to 13,300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication.
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      Ghosh, C.; Nandi, S.; Bhattacharyya, K. Probing micro-environment of lipid droplets in a live breast cell: MCF7 and MCF10A. Chem. Phys. Lett. 2017, 670, 27– 31,  DOI: 10.1016/j.cplett.2016.12.068
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      Probing micro-environment of lipid droplets in a live breast cell: MCF7 and MCF10A
      Ghosh, Catherine; Nandi, Somen; Bhattacharyya, Kankan
      Chemical Physics Letters (2017), 670 (), 27-31CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
      Local environment of the lipid droplets inside the breast cancer cells, MCF7 and in non-malignant breast cells, MCF10A is monitored using time-resolved confocal microscopy. For this study, a coumarin-based dye C153 has been used. The local polarity and the solvation dynamics indicate that a cytoplasmic lipid droplet is less polar and displays slower solvation dynamics compared to the cytosol. Significant differences in terms of no. of lipid droplets, polarity and solvation dynamics are obsd. between the cancer cell (MCF7) and its non-malignant cell (MCF10A).
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    9. 9
      Lu, B.; Yin, J.; Liu, C.; Lin, W. Lipid droplet polarity decreases during the pathology of muscle injury as revealed by a polarity sensitive sensor. Spectrochim. Acta, Part A 2021, 262, 120149  DOI: 10.1016/j.saa.2021.120149
      9
      Lipid droplet polarity decreases during the pathology of muscle injury as revealed by a polarity sensitive sensor
      Lu, Bingli; Yin, Junling; Liu, Cong; Lin, Weiying
      Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (2021), 262 (), 120149CODEN: SAMCAS; ISSN:1386-1425. (Elsevier B.V.)
      Revealing the relationship between lipid droplets (LDs)polarity and disease is indispensable in clinicopathol. diagnosis. So far, muscle injury is often ignored as it is not life-threatening as cardiovascular and cerebrovascular diseases, making the exploration of the internal relationship between muscle injury and LDs polarity a gray area. Herein, a fluorescent probe (CCB) with powerful polar-sensitive as well as precise LDs targeting was designed for visualizing the LDs polarity in the pathol. of muscle injury. By means of the probe CCB, the identification of cancer cells and the monitoring of LDs polarity changes in dysfunctional cells were successfully realized. Furthermore, the penetration ability of CCB in tissues of mice was tested to verify the applicability of the probe in organisms. Importantly, by CCB, the relationship between muscle damage and LDs polarity was explored, revealing that muscle damage caused a significant decrease in LDs polarity accompanied by a significant increase in fluorescence. Most importantly, it is the first time to reveal the relationship between muscle damage and LDs polarity. Therefore, the probe CCB will be a powerful monitoring platform for diagnosing related diseases caused by abnormal LDs polarity.
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    10. 10
      Gonzalez-Garcia, M. C.; Herrero-Foncubierta, P.; Garcia-Fernandez, E.; Orte, A. Building Accurate Intracellular Polarity Maps through Multiparametric Microscopy. Methods Protoc. 2020, 3, 78  DOI: 10.3390/mps3040078
      10
      Building accurate intracellular polarity maps through multiparametric microscopy
      Gonzalez-Garcia, M. Carmen; Herrero-Foncubierta, Pilar; Garcia-Fernandez, Emilio; Orte, Angel
      Methods and Protocols (2020), 3 (4), 78CODEN: MPERC6; ISSN:2409-9279. (MDPI AG)
      The precise knowledge of intracellular polarity, a physiol. parameter that involves complex and intertwined intracellular mechanisms, may be relevant in the study of important diseases like cancer or Alzheimer's. In this tech. note, we illustrate our recently developed, accurate method for obtaining intracellular polarity maps employing potent fluorescence microscopy techniques. Our method is based on the selection of appropriate luminescent probes, in which several emission properties vary with microenvironment polarity, specifically spectral shifts and luminescence lifetime. A multilinear calibration is performed, correlating polarity vs. spectral shift vs. luminescence lifetime, to generate a powerful and error-free 3D space for reliable interpolation of microscopy data. Multidimensional luminescence microscopy is then used to obtain simultaneously spectral shift and luminescence lifetime images, which are then interpolated in the 3D calibration space, resulting in accurate, quant. polarity maps.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlvFCls7w%253D&md5=572b6fd2e25e185afa71671bb71de2ca
    11. 11
      Cerón-Carrasco, J. P.; Jacquemin, D.; Laurence, C.; Planchat, A.; Reichardt, C.; Sraïdi, K. Solvent polarity scales: determination of new ET(30) values for 84 organic solvents. J. Phys. Org. Chem. 2014, 27, 512– 518,  DOI: 10.1002/poc.3293
      11
      Solvent polarity scales: determination of new ET(30) values for 84 organic solvents
      Ceron-Carrasco, Jose P.; Jacquemin, Denis; Laurence, Christian; Planchat, Aurelien; Reichardt, Christian; Sraidi, Khadija
      Journal of Physical Organic Chemistry (2014), 27 (6), 512-518CODEN: JPOCEE; ISSN:0894-3230. (John Wiley & Sons Ltd.)
      The solvent polarity parameter ET(30) is newly measured from the solvatochromism of the betaine dye 30 for 84 solvents and re-measured for 186 addnl. ones. The results are organized in a database. It is shown that the validity of linear solvation energy relationships used for the detn. of secondary ET(30) values is limited to non-hydrogen-bond donor solvents. Relationships with the chain length n are given for the detn. of tertiary ET(30) values of the homologous H(CH2)nY solvent series. The parameter ET(30) is orthogonal to the function of the refractive index (n2 - 1) / (2n2 + 1). For non hydrogen-bond donor solvents, this allows to enter ET(30) as an almost pure electrostatic parameter in a new linear solvation energy relationship. Copyright © 2014 John Wiley & Sons, Ltd.
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      Garcia-Fernandez, E.; Pernagallo, S.; González-Vera, J. A.; Ruedas-Rama, M. J.; Díaz-Mochón, J. J.; Orte, A. Time-Gated Luminescence Acquisition for Biochemical Sensing: miRNA Detection. In Fluorescence in Industry; Pedras, B., Ed.; Springer International Publishing: Cham, 2019; Vol. 18, pp 213– 267.
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      Herrero-Foncubierta, P.; González-García, M. D. C.; Resa, S.; Paredes, J. M.; Ripoll, C.; Girón, M. D.; Salto, R.; Cuerva, J. M.; Orte, A.; Miguel, D. Simple and non-charged long-lived fluorescent intracellular organelle trackers. Dyes Pigm. 2020, 183, 108649  DOI: 10.1016/j.dyepig.2020.108649
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      Simple and non-charged long-lived fluorescent intracellular organelle trackers
      Herrero-Foncubierta, Pilar; Gonzalez-Garcia, Maria del Carmen; Resa, Sandra; Paredes, Jose Manuel; Ripoll, Consuelo; Giron, Maria D.; Salto, Rafael; Cuerva, Juan M.; Orte, Angel; Miguel, Delia
      Dyes and Pigments (2020), 183 (), 108649CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)
      The preferential accumulation of long-lived acridone derivs. in mitochondria were evaluated, based on a new concept of non-protonable and non-charged group carriers. In this sense, the thiophene ring has been proved to act, for the first time, as mitochondria targeting group. Moreover, the unique long lifetime of the dyes has allowed the detn. of the polarity of the organelles by fluorescence lifetime imaging microscopy (FLIM), without causing remarkable cytotoxicity. The neutral nature of the vectoring group favors the synthetic process and prevents the modification of different organelle parameters as membrane potential, making these probes excellent candidates to organelle tracking.
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      Gonzalez-Garcia, M. C.; Herrero-Foncubierta, P.; Castro, S.; Resa, S.; Alvarez-Pez, J. M.; Miguel, D.; Cuerva, J. M.; Garcia-Fernandez, E.; Orte, A. Coupled Excited-State Dynamics in N-Substituted 2-Methoxy-9-Acridones. Front. Chem. 2019, 7, 129  DOI: 10.3389/fchem.2019.00129
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      Coupled excited-state dynamics in n-substituted 2-methoxy-9-acridones
      Gonzalez-Garcia, M. Carmen; Herrero-Foncubierta, Pilar; Castro, Silvia; Resa, Sandra; Alvarez-Pez, Jose M.; Miguel, Delia; Cuerva, Juan M.; Garcia-Fernandez, Emilio; Orte, Angel
      Frontiers in Chemistry (Lausanne, Switzerland) (2019), 7 (), 129pp.CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
      The synthesizes and fully characterization of different 2-methoxy-9-acridone dyes was reported. Their transient fluorescence emission spectra exhibited a complex dynamic behavior that can be linked to several excited-state reactions. A thorough study of the excited-state dynamics of these dyes was performed by means of time-resolved fluorimetry supported by computational calcns. All this allowed to establish a multistate kinetic scheme, involving an ESPT reaction coupled to an excimer formation process. Rich dynamics was unraveled behind this complex behavior, which provided a better understanding of the excited states of these dyes.
      >> More from SciFinder ®
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      Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 2010, 132, 154104  DOI: 10.1063/1.3382344
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      A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu
      Grimme, Stefan; Antony, Jens; Ehrlich, Stephan; Krieg, Helge
      Journal of Chemical Physics (2010), 132 (15), 154104/1-154104/19CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The method of dispersion correction as an add-on to std. Kohn-Sham d. functional theory (DFT-D) has been refined regarding higher accuracy, broader range of applicability, and less empiricism. The main new ingredients are atom-pairwise specific dispersion coeffs. and cutoff radii that are both computed from first principles. The coeffs. for new eighth-order dispersion terms are computed using established recursion relations. System (geometry) dependent information is used for the first time in a DFT-D type approach by employing the new concept of fractional coordination nos. (CN). They are used to interpolate between dispersion coeffs. of atoms in different chem. environments. The method only requires adjustment of two global parameters for each d. functional, is asymptotically exact for a gas of weakly interacting neutral atoms, and easily allows the computation of at. forces. Three-body nonadditivity terms are considered. The method has been assessed on std. benchmark sets for inter- and intramol. noncovalent interactions with a particular emphasis on a consistent description of light and heavy element systems. The mean abs. deviations for the S22 benchmark set of noncovalent interactions for 11 std. d. functionals decrease by 15%-40% compared to the previous (already accurate) DFT-D version. Spectacular improvements are found for a tripeptide-folding model and all tested metallic systems. The rectification of the long-range behavior and the use of more accurate C6 coeffs. also lead to a much better description of large (infinite) systems as shown for graphene sheets and the adsorption of benzene on an Ag(111) surface. For graphene it is found that the inclusion of three-body terms substantially (by about 10%) weakens the interlayer binding. We propose the revised DFT-D method as a general tool for the computation of the dispersion energy in mols. and solids of any kind with DFT and related (low-cost) electronic structure methods for large systems. (c) 2010 American Institute of Physics.
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      Kołaski, M.; Arunkumar, C. R.; Kim, K. S. Aromatic Excimers: Ab Initio and TD-DFT Study. J. Chem. Theory Comput. 2013, 9, 847– 856,  DOI: 10.1021/ct300350m
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      Aromatic Excimers: Ab Initio and TD-DFT Study
      Kolaski, Maciej; Arunkumar, C. R.; Kim, Kwang S.
      Journal of Chemical Theory and Computation (2013), 9 (1), 847-856CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Excited dimers (excimers) formed by arom. mols. are important in biol. systems as well as in chem. sensing. The structure of many biol. systems is governed by excimer formation. Since theor. studies of such systems provide important information about mutual arrangement of arom. mols. in structural biol., we carried out extensive calcns. on the benzene excimer using EOM-CCSD, RI-CC2, CASPT2, and TD-DFT approaches. For the benzene excimer, we evaluate the reliability of the TD-DFT method based on the B3LYP, PBE, PBE0, and ωPBEh functionals. We extended the calcns. to naphthalene, anthracene, and pyrene excimers. We find that nearly parallel stacked forms are the min. energy structure. On the basis of the benzene to pyrene excimers, we might roughly est. the equil. layer-to-layer distance for bilayer-long arenes in the first singlet excited state, which is predicted to be bound.
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      Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006.
      There is no corresponding record for this reference.
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      Crovetto, L.; Orte, A.; Paredes, J. M.; Resa, S.; Valverde, J.; Castello, F.; Miguel, D.; Cuerva, J. M.; Talavera, E. M.; Alvarez-Pez, J. M. Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye. J. Phys. Chem. A 2015, 119, 10854– 10862,  DOI: 10.1021/acs.jpca.5b07898
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      Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye
      Crovetto, Luis; Orte, Angel; Paredes, Jose M.; Resa, Sandra; Valverde, Javier; Castello, Fabio; Miguel, Delia; Cuerva, Juan M.; Talavera, Eva M.; Alvarez-Pez, Jose M.
      Journal of Physical Chemistry A (2015), 119 (44), 10854-10862CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
      Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophys. behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity const. of the dye, pKN-A, was detd. by absorbance and steady-state fluorescence at very low buffer concns. Next, we detd. whether the addn. of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aq. solns. at near-neutral pH. For this anal., both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES expts. demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate consts. were detd. by global anal. of the fluorescence decay traces recorded as a function of pH, and buffer concn. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate const. for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity const. pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we detd. whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1Cqtr%252FE&md5=59c6e66d5e184537f325cdbdecd1fbaf
    19. 19
      Gonzalez-Garcia, M. C.; Peña-Ruiz, T.; Herrero-Foncubierta, P.; Miguel, D.; Giron, M. D.; Salto, R.; Cuerva, J. M.; Navarro, A.; Garcia-Fernandez, E.; Orte, A. Orthogonal cell polarity imaging by multiparametric fluorescence microscopy. Sens. Actuators, B 2020, 309, 127770  DOI: 10.1016/j.snb.2020.127770
      19
      Orthogonal cell polarity imaging by multiparametric fluorescence microscopy
      Gonzalez-Garcia, M. Carmen; Pena-Ruiz, Tomas; Herrero-Foncubierta, Pilar; Miguel, Delia; Giron, Maria D.; Salto, Rafael; Cuerva, Juan M.; Navarro, Amparo; Garcia-Fernandez, Emilio; Orte, Angel
      Sensors and Actuators, B: Chemical (2020), 309 (), 127770CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      The cellular microenvironment is a complex medium due to high concns. of proteins and an intertwined framework of cellular organelles. In particular, cellular micro-polarity controls several biol. processes, since it modulates hydrophobic/hydrophilic interactions and, hence, recognition, signalling and binding events. In this work, we have developed an unprecedented methodol. to construct accurate environment polarity images using multiparametric fluorescence microscopy, via a multi-linear calibration of orthogonal parameters: the fluorescence lifetime and the spectral shift of a series of solvatochromic dyes. For this approach, we have synthesized and fully characterized N-substituted 2-methoxy-9-acridone dyes as suitable bioimaging polarity probes. However, to fully comprehend the complex links between microenvironment polarity and the dyes' properties, we have endeavoured a multidisciplinary approach, in which we have studied the photophysics of our fluorophores using spectroscopic tools and state-of-the-art computational chem. This profound knowledge permitted to use these dyes as intracellular polarity probes, quant. and robustly probing the microenvironment of different cellular compartments. Our new methodol. may pave the way to further developments in accurate sensing of cellular microenvironment parameters.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1yju7k%253D&md5=eb487b23fbac97e5731297806d03e3d7
    20. 20
      Orte, A.; Bermejo, R.; Talavera, E. M.; Crovetto, L.; Alvarez-Pez, J. M. 2′,7′-Difluorofluorescein Excited-State Proton Reactions: Correlation between Time-Resolved Emission and Steady-State Fluorescence Intensity. J. Phys. Chem. A 2005, 109, 2840– 2846,  DOI: 10.1021/jp044681m
      20
      2',7'-Difluorofluorescein excited-state proton reactions: correlation between time-resolved emission and steady-state fluorescence intensity
      Orte, Angel; Bermejo, Ruperto; Talavera, Eva M.; Crovetto, Luis; Alvarez-Pez, Jose M.
      Journal of Physical Chemistry A (2005), 109 (12), 2840-2846CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
      The presence of excited-state buffer-mediated proton exchange reactions influences the steady-state fluorescence signals from dyes in soln. Since biomols. in general have some chem. groups that can act as proton acceptors/donors and are usually dissolved in buffer solns. which can also behave as appropriate proton acceptors/donors, the excited-state proton exchange reactions may result in distorted steady-state fluorescence signals. In a previous paper (2005), we evaluated kinetic and other pertinent parameters for the excited-state proton reactions of the prototropic forms of 2',7'-difluorofluorescein (Oregon Green 488, OG488), recording a fluorescence decay surface at different pH values and acetate buffer concns., analyzed by means of global compartmental anal. In this article we use the rate consts. and the cor. pre-exponential factors from the previously recorded fluorescence decay traces to simulate the decay times and assocd. pre-exponentials at different acetate buffer concns. and const. pH and compare these theor. calcd. values with new exptl. data. We also calc. the steady-state fluorescence intensity vs pH and vs acetate buffer concn. (at const. pH) and compare these calcd. emission values with the exptl. data previously published. The agreement between the exptl. and simulated data is excellent.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXitVWrur0%253D&md5=efe78b21f3a2eb83af6d9d3f78972b28
    21. 21
      Daina, A.; Michielin, O.; Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717  DOI: 10.1038/srep42717
      21
      SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules
      Daina Antoine; Michielin Olivier; Zoete Vincent; Michielin Olivier; Michielin Olivier
      Scientific reports (2017), 7 (), 42717 ISSN:.
      To be effective as a drug, a potent molecule must reach its target in the body in sufficient concentration, and stay there in a bioactive form long enough for the expected biologic events to occur. Drug development involves assessment of absorption, distribution, metabolism and excretion (ADME) increasingly earlier in the discovery process, at a stage when considered compounds are numerous but access to the physical samples is limited. In that context, computer models constitute valid alternatives to experiments. Here, we present the new SwissADME web tool that gives free access to a pool of fast yet robust predictive models for physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry friendliness, among which in-house proficient methods such as the BOILED-Egg, iLOGP and Bioavailability Radar. Easy efficient input and interpretation are ensured thanks to a user-friendly interface through the login-free website http://www.swissadme.ch. Specialists, but also nonexpert in cheminformatics or computational chemistry can predict rapidly key parameters for a collection of molecules to support their drug discovery endeavours.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czisFSrtg%253D%253D&md5=9715b8cb8a34b17c4c73ff69a5a8cc50
    22. 22
      Bik, E.; Mateuszuk, L.; Orleanska, J.; Baranska, M.; Chlopicki, S.; Majzner, K. Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium. Int. J. Mol. Sci. 2021, 22, 2401  DOI: 10.3390/ijms22052401
      22
      Chloroquine-induced accumulation of autophagosomes and lipids in the endothelium
      Bik, Ewelina; Mateuszuk, Lukasz; Orleanska, Jagoda; Baranska, Malgorzata; Chlopicki, Stefan; Majzner, Katarzyna
      International Journal of Molecular Sciences (2021), 22 (5), 2401CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome-lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid compn. of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodol. based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chem. changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a ref. method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concns. of 1-30μM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, resp. The CQ-induced endothelial response was assocd. with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochem. changes offers a better understanding of the autophagy mechanism in the endothelial cells.
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    23. 23
      Pal, K.; Kumar, P.; Koner, A. L. Deciphering interior polarity of lysosome in live cancer and normal cells using spectral scanning microscopy. J. Photochem. Photobiol., B 2020, 206, 111848  DOI: 10.1016/j.jphotobiol.2020.111848
      23
      Deciphering interior polarity of lysosome in live cancer and normal cells using spectral scanning microscopy
      Pal, Kaushik; Kumar, Prashant; Koner, Apurba Lal
      Journal of Photochemistry and Photobiology, B: Biology (2020), 206 (), 111848CODEN: JPPBEG; ISSN:1011-1344. (Elsevier B.V.)
      A lysosome specific, pH tolerant, and polarity-sensitive fluorescent probe (LyPol) is designed and synthesized for the detn. of lysosomal polarity in live cells. LyPol possesses an intramol. charge transfer (ICT) properties with high quantum yield in water and in other polar solvents such as methanol, ethanol, DMSO, acetonitrile, etc. The fluorescence maxima and lifetime increase linearly with a non-specific manner with an increase in the polarity of its surrounding environment. A morpholine group connected with an alkyl linker acts as a lysosome directing moiety, which is attached to the fluorescent core of LyPol. The selective localization of LyPol inside the lysosome was confirmed with live-cell confocal imaging. Further, the spectral scanning confocal technique was utilized to det. the emission spectrum of LyPol inside lysosome, and the polarity turns out to be quite lower as compared to water. Moreover, the combined spectroscopic and live-cell microscopy confirms that the interior of the lysosome is significantly non-polar in cancer cells compared to normal cells. We believe that this report on the measuring polarity inside the biol. system with a solvatofluorochromic probe will be of immense interest to researchers working in the multidisciplinary area of biophysics, microscopy, chem. biol., and organelle biol.
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    24. 24
      Gai, F.; Zuo, Y.; Lin, W. Detecting lipid droplets polarity: Silicone-based unique fluorescent probe for cancer diagnosis in living cells. Talanta 2021, 225, 122059  DOI: 10.1016/j.talanta.2020.122059
      24
      Detecting lipid droplets polarity: Silicone-based unique fluorescent probe for cancer diagnosis in living cells
      Gai, Fengqing; Zuo, Yujing; Lin, Weiying
      Talanta (2021), 225 (), 122059CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Fluorescent probes for monitoring polarity of lipid droplets (LDs) are essential tools in pathol. research, esp. cancer related. Herein, we have designed a biocompatible and novel fluorescent probe (TDCQ) with intramol. charge transfer mechanism, which consists of a naphthalimide moiety accepting electron and a triphenylamine fragment providing electron. In view of polarity-sensitivity and AIE characteristic, TDCQ specially aggregates on the LDs in cells by remarkable green dots fluorescent. Due to the variation of LDs nos. in normal cells and cancer cells, the probe emits stronger green fluorescence in cancer cells but weaker in normal cells. Moreover, TDCQ has outstanding photostability and low toxicity, permitting green fluorescence to persist for a valid time in cells. This article demonstrates that the capacity of TDCQ for facilitating the in-depth study of LDs and applying to the identification of cancer cells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXptlCltw%253D%253D&md5=2355b3aa2cacc7fa69dd94816ddcb21a
    25. 25
      Danylchuk, D. I.; Jouard, P.-H.; Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. J. Am. Chem. Soc. 2021, 143, 912– 924,  DOI: 10.1021/jacs.0c10972
      25
      Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
      Danylchuk, Dmytro I.; Jouard, Pierre-Henri; Klymchenko, Andrey S.
      Journal of the American Chemical Society (2021), 143 (2), 912-924CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi app., plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission max., as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy expts. with ref. organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quant. description of the lipid order in each organelle of interest. Cholesterol extn./enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mech. stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to phys. and chem. stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmsFCitw%253D%253D&md5=467fda3ceb4e1f4b20f495e619a06fe8
    26. 26
      Collot, M.; Bou, S.; Fam, T. K.; Richert, L.; Mély, Y.; Danglot, L.; Klymchenko, A. S. Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push–Pull Fluorophore. Anal. Chem. 2019, 91, 1928– 1935,  DOI: 10.1021/acs.analchem.8b04218
      26
      Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore
      Collot, Mayeul; Bou, Sophie; Fam, Tkhe Kyong; Richert, Ludovic; Mely, Yves; Danglot, Lydia; Klymchenko, Andrey S.
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (3), 1928-1935CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiol. processes as well as diseases. Herein the authors synthesized a push-pull mol. named DAF (Di-Me Aniline Furaldehyde) that possesses a strong pos. solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.
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    27. 27
      Dai, Y.; Zhan, Z.; Li, Q.; Liu, R.; Lv, Y. Simultaneous monitoring of polarity changes of lipid droplets and lysosomes with two-photon fluorescent probes. Anal. Chim. Acta 2020, 1136, 34– 41,  DOI: 10.1016/j.aca.2020.08.033
      27
      Simultaneous monitoring of polarity changes of lipid droplets and lysosomes with two-photon fluorescent probes
      Dai, Yongcheng; Zhan, Zixuan; Li, Qiuyan; Liu, Rui; Lv, Yi
      Analytica Chimica Acta (2020), 1136 (), 34-41CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      Intracellular polarity is an essential feature of cell physiol. state and abnormal polarity changes of various organelles are related to many diseases. Thus, monitoring of polarity changes of multiple subcellular in living cells contributes to understanding different physiol. and pathol. processes more accurately. However, most of the previous reports on polarity probes mainly monitored the polarity of a single organelle. Therefore, we designed and synthesized two unique polarity-sensitive fluorescent probes LDs-TPFP and Lyso-TPFP, which can be selectively located in lipid droplets (LDs) and lysosomes resp., to obtain more subcellular information in living cells. Thanks to the strong intramol.-charge-transfer (ICT) characteristics of probes, the fluorescence intensity and emission wavelength would change with the polarity of the surroundings of cells. Moreover, LDs-TPFP and Lyso-TPFP exhibits large Stokes shift and excellent biocompatibility. Through fluorescence imaging, the probes can effectively distinguish normal cells from cancer cells. In addn., the results of two-photon confocal fluorescence imaging indicated that LDs and lysosomes have discrepant polarity change behaviors under different physiol. conditions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFWmt7jK&md5=2f612c803792ec81378d84185e20d1cb
    28. 28
      Yin, J.; Peng, M.; Lin, W. Two-photon fluorescence imaging of lipid drops polarity toward cancer diagnosis in living cells and tissue. Sens. Actuators, B 2019, 288, 251– 258,  DOI: 10.1016/j.snb.2019.02.122
      28
      Two-photon fluorescence imaging of lipid drops polarity toward cancer diagnosis in living cells and tissue
      Yin, Junling; Peng, Min; Lin, Weiying
      Sensors and Actuators, B: Chemical (2019), 288 (), 251-258CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Monitoring the polarity of intracellular lipid droplets (LDs) is of great importance toward cancer diagnosis. Herein, we have rationally designed a novel LDs-targeting and polarity-sensitive fluorescent probe CBMC, which contains carbazole as an electron-donating group and malononitrile as an electron-accepting group forming intramol. charge transfer mechanism. Due to its excellent solvatochromism, the probe emits stronger fluorescence in normal cells but weaker in cancer cells. By means of CBMC, normal tissues with stronger fluorescence have been obviously pointed out from cancerous tissues. For cancer therapy, the probe CBMC could be an ideal candidate to evaluate tumor, because the tumor area would decrease while the normal tissue area would increase with treatment time. Compared with existing probes with turn-off signal to normal cells, such a new probe with turn on type in normal cells could provide more reliable information with advantages of lower background interference, higher signal to noise ratio, and higher sensitivity. This probe CBMC will be a powerful tool to be potentially applied in tumor diagnosis in the case of treatment.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktlKmtbs%253D&md5=04f8f5fd0d8b86eeb07f5882c782fe7e
    29. 29
      Chowdhury, R.; Jana, B.; Saha, A.; Ghosh, S.; Bhattacharyya, K. Confocal microscopy of cytoplasmic lipid droplets in a live cancer cell: number, polarity, diffusion and solvation dynamics. MedChemComm 2014, 5, 536– 539,  DOI: 10.1039/C3MD00269A
      29
      Confocal microscopy of cytoplasmic lipid droplets in a live cancer cell: number, polarity, diffusion and solvation dynamics
      Chowdhury, Rajdeep; Jana, Batakrishna; Saha, Abhijit; Ghosh, Surajit; Bhattacharyya, Kankan
      MedChemComm (2014), 5 (4), 536-539CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)
      Time resolved confocal microscopy indicates that the cytoplasmic lipid droplets (CLDs) in live cells (normal and cancer lung cells) are less polar, and exhibit slower diffusion (motility) and solvation dynamics than the cytoplasm. The no. of CLDs in a human lung cancer cell (A549) is ∼10 times higher than in a non-cancer lung fibroblast cell (WI38). This may result in accumulation of non-polar cell signaling agents in the CLDs of the cancer cell.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXkvF2it7s%253D&md5=7ffd3ad1efcebbe84ac6963ba7fa34d8
    30. 30
      Fam, T. K.; Klymchenko, A. S.; Collot, M. Recent Advances in Fluorescent Probes for Lipid Droplets. Materials 2018, 11, 1768  DOI: 10.3390/ma11091768
      30
      Recent advances in fluorescent probes for lipid droplets
      Fam, Tkhe Kyong; Klymchenko, Andrey S.; Collot, Mayeul
      Materials (2018), 11 (9), 1768/1-1768/19CODEN: MATEG9; ISSN:1996-1944. (MDPI AG)
      Lipid droplets (LDs) are organelles that serve as the storage of intracellular neutral lipids. LDs regulate many physiol. processes. They recently attracted attention after extensive studies showed their involvement in metabolic disorders and diseases such as obesity, diabetes, and cancer. Therefore, it is of the highest importance to have reliable imaging tools. In this review, we focus on recent advances in the development of selective fluorescent probes for LDs. Their photophys. properties are described, and their advantages and drawbacks in fluorescence imaging are discussed. At last, we review the reported applications using these probes including two-photon excitation, in vivo and tissue imaging, as well as LDs tracking.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKksLjM&md5=be7d5917a54fb266f8daafb5fd0429bb
    31. 31
      Danylchuk, D. I.; Jouard, P.-H.; Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. J. Am. Chem. Soc. 2021, 143, 912– 924,  DOI: 10.1021/jacs.0c10972
      31
      Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
      Danylchuk, Dmytro I.; Jouard, Pierre-Henri; Klymchenko, Andrey S.
      Journal of the American Chemical Society (2021), 143 (2), 912-924CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi app., plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission max., as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy expts. with ref. organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quant. description of the lipid order in each organelle of interest. Cholesterol extn./enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mech. stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to phys. and chem. stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmsFCitw%253D%253D&md5=467fda3ceb4e1f4b20f495e619a06fe8
    32. 32
      Fan, L.; Wang, X.; Zan, Q.; Fan, L.; Li, F.; Yang, Y.; Zhang, C.; Shuang, S.; Dong, C. Lipid Droplet-Specific Fluorescent Probe for In Vivo Visualization of Polarity in Fatty Liver, Inflammation, and Cancer Models. Anal. Chem. 2021, 93, 8019– 8026,  DOI: 10.1021/acs.analchem.1c01125
      32
      Lipid Droplet-Specific Fluorescent Probe for In Vivo Visualization of Polarity in Fatty Liver, Inflammation, and Cancer Models
      Fan, Li; Wang, Xiaodong; Zan, Qi; Fan, Lifang; Li, Feng; Yang, Yongming; Zhang, Caihong; Shuang, Shaomin; Dong, Chuan
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (22), 8019-8026CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Elucidating the intrinsic relationship between diseases and lipid droplet (LD) polarity remains a great challenge owing to the lack of the research on multiple disease models. Until now, the visualization of abnormal LD polarity in models of inflammation and clin. cancer patient samples has not been achieved. To meet the urgent challenge, we facilely synthesized a robust LD-specific and polarity-sensitive fluorescent probe (LD-TTP), which consists of a triphenylamine segment as an electron-donor group (D) and a pyridinium as an electron-acceptor moiety (A), forming a typical D-π-A mol. configuration. Owing to the unique intramol. charge transfer effect, LD-TTP exhibits high sensitivity to polarity change in the linear range from Δf = 0.258 to 0.312, with over 278-fold fluorescence enhancement. Moreover, we revealed that LD-TTP possessed satisfactory ability for sensitively monitoring LD-polarity changes in living cells. Using LD-TTP, we first demonstrated the detection of LD-polarity changes in fatty liver tissues and inflammatory living mice via confocal laser scanning fluorescence imaging. Surprisingly, the visualization of LD polarity has been achieved not only at the cellular levels and living organs but also in surgical specimens from cancer patients, thus holding great potential in the clin. diagnosis of human cancer. All these features render LD-TTP an effective tool for medical diagnosis of LD polarity-related diseases.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtFOht73P&md5=e9906eaa71ca252aae25048a73a21514
    33. 33
      Wang, K.-N.; Liu, L.-Y.; Mao, D.; Xu, S.; Tan, C.-P.; Cao, Q.; Mao, Z.-W.; Liu, B. A Polarity-Sensitive Ratiometric Fluorescence Probe for Monitoring Changes in Lipid Droplets and Nucleus during Ferroptosis. Angew. Chem., Int. Ed. 2021, 60, 15095– 15100,  DOI: 10.1002/anie.202104163
      33
      A Polarity-Sensitive Ratiometric Fluorescence Probe for Monitoring Changes in Lipid Droplets and Nucleus during Ferroptosis
      Wang, Kang-Nan; Liu, Liu-Yi; Mao, Duo; Xu, Shidang; Tan, Cai-Ping; Cao, Qian; Mao, Zong-Wan; Liu, Bin
      Angewandte Chemie, International Edition (2021), 60 (27), 15095-15100CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Ferroptosis regulates cell death through reactive oxygen species (ROS)-assocd. lipid peroxide accumulation, which is expected to affect the structure and polarity of lipid droplets (LDs), but with no clear evidence. Herein, we report the first example of an LD/nucleus dual-targeted ratiometric fluorescent probe, CQPP, for monitoring polarity changes in the cellular microenvironment. Due to the donor-acceptor structure of CQPP, it offers ratiometric fluorescence emission and fluorescence lifetime signals that reflect polarity variations. Using nucleus imaging as a ref., CQPP was applied to report the increase in LD polarity and the homogenization of polarity between LDs and cytoplasm in the ferroptosis model. This LD/nucleus dual-targeted fluorescent probe shows the great potential of using fluorescence imaging to study ferroptosis and ferroptosis-related diseases.
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    34. 34
      Zhu, H.; Fan, J.; Mu, H.; Zhu, T.; Zhang, Z.; Du, J.; Peng, X. d-PET-controlled “off-on” Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes. Sci. Rep. 2016, 6, 35627  DOI: 10.1038/srep35627
      34
      d-PET-controlled "off-on" Polarity-sensitive Probes for Reporting Local Hydrophilicity within Lysosomes
      Zhu, Hao; Fan, Jiangli; Mu, Huiying; Zhu, Tao; Zhang, Zhen; Du, Jianjun; Peng, Xiaojun
      Scientific Reports (2016), 6 (), 35627CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Polarity-sensitive fluorescent probes are powerful chem. tools for studying biomol. structures and activities both in vitro and in vivo. However, the lack of "off-on" polarity-sensing probes has limited the accurate monitoring of biol. processes that involve an increase in local hydrophilicity. Here, we design and synthesize a series of "off-on" polarity-sensitive fluorescent probes BP series consisting of the difluoroboron dippyomethene (BODIPY) fluorophore connected to a quaternary ammonium moiety via different carbon linkers. All these probes showed low fluorescence quantum yields in nonpolar soln. but became highly fluorescent in polar media. BP-2, which contains a two-carbon linker and a tri-Me quaternary ammonium, displayed a fluorescence intensity and quantum yield that were both linearly correlated with solvent polarity. In addn., BP-2 exhibited high sensitivity and selectivity for polarity over other environmental factors and a variety of biol. relevant species. BP-2 can be synthesized readily via an unusual Mannich reaction followed by methylation. Using electrochem. combined with theor. calcns., we demonstrated that the "off-on" sensing behavior of BP-2 is primarily due to the polarity-dependent donor-excited photoinduced electron transfer (d-PET) effect. Live-cell imaging established that BP-2 enables the detection of local hydrophilicity within lysosomes under conditions of lysosomal dysfunction.
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    35. 35
      Hu, L.; Shi, D.; Li, X.; Zhu, J.; Mao, F.; Li, X.; Xia, C.; Jiang, B.; Guo, Y.; Li, J. Curcumin-based polarity fluorescent probes: Design strategy and biological applications. Dyes Pigm. 2020, 177, 108320  DOI: 10.1016/j.dyepig.2020.108320
      35
      Curcumin-based polarity fluorescent probes: Design strategy and biological applications
      Hu, Linghao; Shi, Donglei; Li, Xinming; Zhu, Jin; Mao, Fei; Li, Xiaokang; Xia, Conglong; Jiang, Bei; Guo, Yuan; Li, Jian
      Dyes and Pigments (2020), 177 (), 108320CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)
      Polarity is a crucial characteristic of indispensable organelles, which has been indicated to be closely related to many pathol. and biol. processes. Hence, a novel design strategy of curcumin-based polarity fluorescent probes was presented. Curcumin attracted much attention due to its environment-sensitivity and optical properties. The authors reformed the structure of curcumin by attaching a galactose moiety that was used to increase the water soly. and regulate the probe working mode. Furthermore, the authors also introduced a diethylamino group, which played a substantial role in adjusting the polarity specificity and sensitivity of the probes. The authors finally screened two polarity-specific fluorescent probes 0301 and 0302. Both probes exhibited remarkable changes in fluorescence from red to green upon increasing polarity of environment. Using 0301 in confocal imaging, the authors obtained a clear and exact view of lysosomal polarity in different cells, suggesting that the lysosomal polarity in normal cells is higher than that in cancer cells. Also, the authors use 0301 to monitor variations in lysosomal polarity in live cells induced by dimethylsulfoxide and sucrose, indicating that the authors' probe could specifically monitor the changes of polarity in live cells to elucidate polarity-related biol. processes.
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    36. 36
      Orte, A.; Crovetto, L.; Talavera, E. M.; Boens, N.; Alvarez-Pez, J. M. Absorption and Emission Study of 2′,7′-Difluorofluorescein and Its Excited-State Buffer-Mediated Proton Exchange Reactions. J. Phys. Chem. A 2005, 109, 734– 747,  DOI: 10.1021/jp046786v
      36
      Absorption and emission study of 2',7'-difluorofluorescein and its excited-state buffer-mediated proton exchange reactions
      Orte, Angel; Crovetto, Luis; Talavera, Eva M.; Boens, Noeel; Alvarez-Pez, Jose M.
      Journal of Physical Chemistry A (2005), 109 (5), 734-747CODEN: JPCAFH; ISSN:1089-5639. (American Chemical Society)
      We present steady-state emission evidence that acetate buffer species promote an excited-state proton transfer between neutral, monoanionic, and dianionic forms of 2',7'-difluorofluorescein. The time course of the excited species in this reaction was characterized through time-resolved fluorescence measurements, and the kinetics of the reaction were solved by using global compartmental anal. A previous identifiability study on the compartmental system set the conditions to design the fluorescence decay surface. This is the first exptl. system, studied within this kinetic model, solved under identifiability conditions through global compartmental anal. The recovered rate const. values for deactivation were 2.94 × 108 s-1 for the monoanion and 2.47 × 108 s-1 for the dianion, whereas the rate const. values of the buffer-mediated excited-state reaction were 9.70 × 108 and 1.79 × 108 M-1 s-1 for the deprotonation and protonation, resp. With these values, a pKa* = 4.02 was obtained. In this work, we addnl. provide an absorption study, including acid-base equil., detn. of ground-state pKa values (1.02, 3.61, and 4.69), and recovery of molar absorption coeffs. of every prototropic species, including absorption and NMR evidence for the existence of three tautomers in neutral species. Steady-state emission spectra of 2',7'-difluorofluorescein in aq. soln. are also described, where the strong photoacid behavior of the cation is noteworthy.
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    37. 37
      Li, J.; Xu, J.; Guo, W.; Zhong, W.; Li, Q.; Tan, L.; Shang, L. Ratiometric fluorescence sensors for heparin and heparinase based on enhanced excimer emission of perylene probe induced by cationic silver nanoparticles. Sens. Actuators, B 2020, 305, 127422  DOI: 10.1016/j.snb.2019.127422
      37
      Ratiometric fluorescence sensors for heparin and heparinase based on enhanced excimer emission of perylene probe induced by cationic silver nanoparticles
      Li, Juanmin; Xu, Jie; Guo, Wenfeng; Zhong, Wencheng; Li, Qiang; Tan, Lili; Shang, Li
      Sensors and Actuators, B: Chemical (2020), 305 (), 127422CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Herein, cationic MUTAB-Ag NPs of different sizes were synthesized and their interactions with a neg. charged perylene probe, N,N'-bis(6-caproic acid)-3,4:9,10-perylenediimide (PDI-COOH), were investigated. MUTAB-Ag NPs could result in significant monomer quenching and excimer formation by inducing probe aggregation due to the strong electrostatic attractive interactions between them. Moreover, significant excimer emission could be obsd. at extremely low probe concn. when induced by larger MUTAB-Ag NPs. A possible mechanism of metal-enhanced fluorescence was proposed, which is evidenced by the strong dependence between surface plasmon resonance absorption spectra of MUTAB-Ag NPs and the excimer emission spectra of PDI-COOH, together with the sharp decrease in fluorescence lifetime of excimer compared with that induced by poly(allylamine). Consequently, ratiometric fluorescence sensors for heparin and heparinase were developed based on the Ag NP-induced excimer emission of PDI-COOH. The adsorption of heparin on the surface of MUTAB-Ag NPs led to the release of monomer, while the degrdn. of heparin by heparinase facilitated the formation of PDI-COOH excimer. The monomer-excimer transition signal was harnessed for heparin and heparinase sensing, which exhibited good sensitivity and excellent selectivity. Finally, application of the present system for detection of heparin and heparinase in human serum sample was successfully demonstrated.
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    38. 38
      Das, S.; Sahana, A.; Banerjee, A.; Lohar, S.; Safin, D. A.; Babashkina, M. G.; Bolte, M.; Garcia, Y.; Hauli, I.; Mukhopadhyay, S. K.; Das, D. Ratiometric fluorescence sensing and intracellular imaging of Al3+ ions driven by an intramolecular excimer formation of a pyrimidine–pyrene scaffold. Dalton Trans. 2013, 42, 4757– 4763,  DOI: 10.1039/C3DT32908A
      38
      Ratiometric fluorescence sensing and intracellular imaging of Al3+ ions driven by an intramolecular excimer formation of a pyrimidine-pyrene scaffold
      Das, Sudipta; Sahana, Animesh; Banerjee, Arnab; Lohar, Sisir; Safin, Damir A.; Babashkina, Maria G.; Bolte, Michael; Garcia, Yann; Hauli, Ipsit; Mukhopadhyay, Subhra Kanti; Das, Debasis
      Dalton Transactions (2013), 42 (14), 4757-4763CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      An Al3+ selective ratiometric fluorescent probe (L) has been synthesized by condensation of 4,5-diaminopyrimidine with 1-pyrenecarboxaldehyde. The structure of L has been confirmed by single crystal X-ray anal. In the presence of Al3+, L exhibits a considerable excimer emission at 445 nm along with the decrease of its monomer emission at 368 nm in DMSO-H2O (4 : 1, vol./vol.). The lowest detection limit for Al3+ is 0.24 μM. Furthermore, L can permeate through the cell membrane and detects intracellular Al3+ ions under a fluorescence microscope.
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    39. 39
      Razi, S. S.; Ali, R.; Srivastava, P.; Misra, A. Smart excimer fluorescence probe for visual detection, cell imaging and extraction of Hg2+. RSC Adv. 2015, 5, 79538– 79547,  DOI: 10.1039/C5RA13021B
      39
      Smart excimer fluorescence probe for visual detection, cell imaging and extraction of Hg2+
      Razi, Syed S.; Ali, Rashid; Srivastava, Priyanka; Misra, Arvind
      RSC Advances (2015), 5 (97), 79538-79547CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Smart pyrene-based simple fluorescent probes 2 and 4 were designed, synthesized and characterized by different spectroscopic methods. The photophys. properties of the probes and their affinity towards different metal ions in phosphate buffer were investigated. Upon selective interaction with Hg2+, the mol. probes showed enhanced static excimer emission at 506 nm along with a naked-eye detectable chromo- and fluoro-genic response. Probe 2 sensitively showed a high limit of detection (3.4 pM) for Hg2+ in the soln. The pyrene silicate deriv., 4, was utilized to detect and ext. Hg2+ in soln. as well as in the solid state. The data obtained from NMR and ESI-MS spectroscopy supported the postulate that the mode of interaction of the probe with Hg2+ involves the N and O atoms of the -C=N and -OH functional groups to complex Hg2+ in 2 : 1 stoichiometry. Moreover, probe 2 exhibited excellent selectivity for Hg2+ in protein medium (BSA/HSA) and was used to detect Hg2+ in live HeLa cells, on test paper strips and in real contaminated water samples.
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    40. 40
      Upadhyay, Y.; Anand, T.; Babu, L. T.; Paira, P.; Crisponi, G.; Sk, A. K.; Kumar, R.; Sahoo, S. K. Three-in-one type fluorescent sensor based on a pyrene pyridoxal cascade for the selective detection of Zn(II), hydrogen phosphate and cysteine. Dalton Trans. 2018, 47, 742– 749,  DOI: 10.1039/C7DT04234E
      40
      Three-in-one type fluorescent sensor based on a pyrene pyridoxal cascade for the selective detection of Zn(II), hydrogen phosphate and cysteine
      Upadhyay, Yachana; Anand, Thangaraj; Babu, Lavanya Thilak; Paira, Priyankar; Crisponi, Guido; S K, Ashok Kumar; Kumar, Rajender; Sahoo, Suban K.
      Dalton Transactions (2018), 47 (3), 742-749CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      A novel fluorescent receptor L was synthesized by Schiff base condensation of 1-pyrenemethylamine with the vitamin B6 cofactor pyridoxal. The receptor L is highly selective and sensitive towards Zn2+ ions among other tested metal ions. Upon interaction with Zn2+, the receptor L showed a distinct fluorescence enhancement at 485 nm due to the excimer formation leading to the fluorescent color change from blue to bluish-green. Subsequently, when the in situ generated ZnL2 complex interacted with various anions and amino acids, the addn. of H2PO4- and cysteine reinstated the fluorescence of the receptor L due to the demetalation of Zn2+ from the ZnL2 complex. Accordingly, the receptor L was developed for the highly selective, specific and sensitive detection of three important bioactive analytes, i.e., Zn2+, H2PO4- and cysteine with a detection limit down to 2.3 × 10-6 M, 2.18 × 10-7 M and 1.59 × 10-7 M, resp. Addnl., the receptor L was applied to the detection of intracellular Zn2+ ions in live HeLa cells.
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    41. 41
      Gao, M.; Tang, B. Z. Fluorescent Sensors Based on Aggregation-Induced Emission: Recent Advances and Perspectives. ACS Sens. 2017, 2, 1382– 1399,  DOI: 10.1021/acssensors.7b00551
      41
      Fluorescent Sensors Based on Aggregation-Induced Emission: Recent Advances and Perspectives
      Gao, Meng; Tang, Ben Zhong
      ACS Sensors (2017), 2 (10), 1382-1399CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      A review. Fluorescent sensors with advantages of excellent sensitivity, rapid response, and easy operation are emerging as powerful tools in environmental monitoring, biol. research, and disease diagnosis. However, conventional fluorophores featured with π-planar structures usually suffer from serious self-quenching in the aggregated state, poor photostability, and small Stokes' shift. In contrast to conventional aggregation-caused quenching (ACQ) fluorophores, the newly emerged aggregation-induced emission fluorogens (AIEgens) are featured with high emission efficiency in the aggregated state, which provide unique opportunities for various sensing applications with advantages of high signal-to-noise ratio, strong photostability, and large Stokes' shift. In this review, the authors will first briefly give an introduction of the AIE concept and the turn-on sensing principles. Then, the authors will discuss the recent examples of AIE sensors according to types of analytes. Finally, the authors will give a perspective on the future developments of AIE sensors. The authors hope this review will inspire more endeavors to devote to this emerging world.
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    42. 42
      Zhang, Q.; Xiao, K.; Paredes, J. M.; Mamonova, T.; Sneddon, W. B.; Liu, H.; Wang, D.; Li, S.; McGarvey, J. C.; Uehling, D.; Al-awar, R.; Joseph, B.; Jean-Alphonse, F.; Orte, A.; Friedman, P. A. Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport. J. Biol. Chem. 2019, 294, 4546– 4571,  DOI: 10.1074/jbc.RA119.007421
      42
      Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport
      Zhang, Qiangmin; iao, Kunhong; Paredes, Jose M.; Mamonova, Tatyana; Sneddon, W. Bruce; Liu, Hongda; Wang, Dawei; Li, Sheng; McGarvey, Jennifer C.; Uehling, David; Al-awar, Rima; Joseph, Babu; Jean-Alphonse, Frederic; Orte, Angel; Friedman, Peter A.
      Journal of Biological Chemistry (2019), 294 (12), 4546-4571CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Na+-Hα exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium- phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS anal., we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290. Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we obsd. that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange M S revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochem. mechanisms underlying PTH control of phosphate transport.
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    43. 43
      Paredes, J. M.; Giron, M. D.; Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Salto, R.; Alvarez-Pez, J. M. Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM). J. Phys. Chem. B 2013, 117, 8143– 8149,  DOI: 10.1021/jp405041c
      43
      Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM)
      Paredes, Jose M.; Giron, Maria D.; Ruedas-Rama, Maria J.; Orte, Angel; Crovetto, Luis; Talavera, Eva M.; Salto, Rafael; Alvarez-Pez, Jose M.
      Journal of Physical Chemistry B (2013), 117 (27), 8143-8149CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Phosphate ions play important roles in signal transduction and energy storage in biol. systems. However, robust chem. sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein deriv. dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivs. at approx. physiol. pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concn. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodol. has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alk. phosphatase activity in the extracellular medium as a marker of the differentiation process.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.1c01206.

    • Detailed materials and methods; supporting results from quantum chemistry calculations (Chart S1, Figure S1, and Table S1); supporting spectroscopic results (Figures S2–S6); subcellular localization study (Figures S7–S10 and Table S2); additional DYNEX images and results (Figures S11–S15); comparison of different lipid droplet polarity probes (Table S3); and sucrose and chloroquine treatments (Figures S16–S19) (PDF)

    • Supporting Videos S1–S4 illustrating the DYNEX segmentation method (ZIP)


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