Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and CellsClick to copy article linkArticle link copied!
- Kevin JahnkeKevin JahnkeBiophysical Engineering Group, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyDepartment of Physics and Astronomy, Heidelberg University, D-69120 Heidelberg, GermanyMore by Kevin Jahnke
- Stefan J. MaurerStefan J. MaurerBiophysical Engineering Group, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyDepartment of Physics and Astronomy, Heidelberg University, D-69120 Heidelberg, GermanyMore by Stefan J. Maurer
- Cornelia WeberCornelia WeberDepartment of Cellular Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyMore by Cornelia Weber
- Jochen Estebano Hernandez BücherJochen Estebano Hernandez BücherDepartment of Cellular Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyMore by Jochen Estebano Hernandez Bücher
- Andreas SchoenitAndreas SchoenitBiophysical Engineering Group, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyMore by Andreas Schoenit
- Elisa D’EsteElisa D’EsteOptical Microscopy Facility, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyMore by Elisa D’Este
- Elisabetta Ada Cavalcanti-AdamElisabetta Ada Cavalcanti-AdamDepartment of Cellular Biophysics, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyMore by Elisabetta Ada Cavalcanti-Adam
- Kerstin Göpfrich*Kerstin Göpfrich*Email: [email protected]Biophysical Engineering Group, Max Planck Institute for Medical Research, Jahnstraße 29, D-69120 Heidelberg, GermanyDepartment of Physics and Astronomy, Heidelberg University, D-69120 Heidelberg, GermanyMore by Kerstin Göpfrich
Abstract
Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.
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Molecular motors govern various cellular processes from intracellular transport to contraction and cell division. While the development of artificial macroscale motors flourishes, many biophysical goals benefit from the engineering of motors on the nanoscale. (1,2) In particular, man-made machines like optical tweezers or atomic force microscopes have long been employed to probe cellular properties like membrane-to-cortex adhesion, (3) yet the use of molecular machines for this purpose remains largely unexplored. To date, natural motor proteins have been used to deform giant vesicles, (4−7) assemble contractile systems, (8−10) or transport cargo. (11−15) Moreover, noteworthy efforts have been made to build synthetic nanoscale motors with DNA nanotechnology as transporters, (16,17) rotors (18) or sliders. (19) However, due to their comparably low processivity and force generation compared to natural motors, the amount of suitable applications for synthetic motors is still limited. On the other hand, recent progress has been made using a minimal system of vesicles and natural motor proteins to elucidate the complex interplay of membrane tubulation of synthetic vesicles (20,21) and membrane dynamics. (22) Still, it remains elusive and uncertain how these minimal systems perform in more complex environments of natural cells and if they can possibly provide direct evidence of a cell’s biophysical properties or even guide cell functions.
Here, we develop a minimal system to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs) and natural cells. Lipid nanotubes are membrane-enclosed tubes with nanoscale diameters that can guide the transfer of vesicles and organelles between cells. (23) We analyze the network length per cell and find that it critically depends on the cell type. This indicates that our minimal motor-based system could be used as a straightforward method to probe membrane-to-cortex attachment as a crucial biophysical indicator for the cell state. (3)
First, we set out to establish a motor-based force-generating system that can be used to actively pull lipid nanotubes. This requires a mechanism for directional force generation. For this purpose, we engineer two variants of an in vitro actin motility assay as illustrated in Figure 1a. First, the substrate is functionalized with a truncated version of myosin consisting of only the functional headgroup of myosin II. This so-called heavy meromyosin (HMM) is capable of performing a power stroke like myosin II but is still easily soluble in water at physiological conditions. We purify the HMM and actin from rabbit skeletal muscle and verify the successful purification with denaturing polyacrylamide gel electrophoresis (SDS-PAGE, Supporting Information (SI) Figure S1). The HMM is immobilized on the substrate where it binds prepolymerized actin filaments to the substrate and translocates them in the presence of adenosine triphosphate (ATP) like in a conventional motility assay. (24) At low actin concentrations from 0.5 to 20 nM, we observe the attachment and random movement of individual actin filaments, termed random filaments, with confocal microscopy (Figure 1b, SI Video S1). In order to make the movement of actin filaments directional, we induce the nematic ordering of actin filaments by adding a high concentration (24 μM) of unlabeled F-actin to the motility assay. We thereby surpass the critical filament density ρc of 5 filaments/μm2 above which filament ordering takes place. (25) This causes the alignment and movement of actin filaments on parallel tracks (Figure 1b, SI Video S2). With particle image velocimetry (PIV), we obtain the velocity vector field which yields a correlation length of 6.7 ± 3.9 μm for random filaments and 29.1 ± 12.5 μm for aligned filaments (Figure 1c). (26) From the confocal images (Figure 1b) and the corresponding velocity vector field (Figure 1c), we verify the alignment of actin filaments within equidistant filament streams with a spacing of 3.6 ± 1.4 μm. We analyze the orientation, that is, the direction of the velocity vector, of individual actin filaments over time and find that in the standard in vitro actin motility assay the filaments move in random directions (Figure 1d), whereas they move along one axis with a strong bias to one direction when the actin was nematically aligned. For the aligned condition, some filaments move in the 180° opposing direction and swirls and vortices can occur inducing a global change in the direction of the bias (SI Figure S2 and Video S3). (27) To probe the effect that the alignment has on the actin filament velocity, we quantified the average filament velocity for both, random and aligned actin at room temperature (RT) and 30 °C (Figure 1e), which is closer to the optimum temperature for HMM activity. (28) The velocity for random filaments at RT is significantly smaller than at 30 °C. Additionally, the alignment significantly enhances the average velocity of actin filaments from 0.9 ± 0.9 to 2.0 ± 1.0 μm s–1 (p ≤ 0.0001). This might be due to the existence of defined tracks for aligned actin that allow for a higher motor processivity compared to when actin filaments are randomly distributed. Additionally, dysfunctional HMM may be blocked by unlabeled actin filaments increasing the overall actin filament velocity. (29)
Next, we test if we can pull lipid nanotubes from GUVs using the directional force of gliding actin filaments. In order to bind actin filaments to the lipid membrane of GUVs as illustrated in Figure 2a, we prepare filaments with 10% biotinylated actin monomers and verify the successful functionalization with SDS-PAGE (SI Figure S1). Additionally, we form GUVs containing 20 mol % biotinylated lipids. We observe that actin filaments bind to the GUVs in the presence of streptavidin forming an actin exoskeleton on the GUV membrane which links the GUVs to the HMM substrate. A few minutes after addition of the actin-coated GUVs to the in vitro motility assay, we observe the formation of lipid nanotubes on the HMM substrate at the bottom of the GUVs (Figure 2b). Note that the GUVs remain intact as proven by the images taken at the equatorial plane (Figure 2b, top). Some GUV clustering is expected due to the use of biotinylated lipids in the presence of streptavidin. We verify the tubular structure of the lipid nanotubes pulled from GUVs using 3D stimulated emission depletion (3D-STED) microscopy (Figure 2c). (30) The 3D-STED reveals a typical lipid nanotube diameter of around 200 nm (Figure 2d, SI Figure S3). Importantly, the lipid nanotubes are continuously pulled out of the GUVs due to the motor activity, and we can observe the lipid nanotube networks grow over time. Within 37.5 s, more than 30 μm of nanotubes are pulled from a single GUV (Figure 2e). Remarkably, the lipid nanotube networks undergo further remodeling after being pulled from the GUV leading to the emergence of nanotube networks composed of multiple GUVs (SI Video S4). Next, we quantify the network length per GUV for random and aligned actin filaments and GUVs containing 0 or 20 mol % biotinylated lipids (Figure 2f,g). In absence of biotinylated lipids and for random actin filaments, the actin filaments do not bind to the GUVs. Hence, no lipid nanotubes are formed, whereas the network length per GUV and the number of lipid nanotube branches (SI Figure S4) significantly increases in the presence of 20 mol % biotinylated lipids. In accordance with the increased actin filament velocity and directionality, the network length per GUV increases further for aligned actin filaments. Interestingly, even though the trend is the same for random and aligned filaments, we observe pulling of lipid nanotubes even in the absence of biotinylated lipids. We hypothesize that this is due to the high amount of unlabeled F-actin present in the assay to induce the alignment which promotes electrostatic interactions of actin filaments with the GUV membrane (SI Figure S5). This might even be enhanced by the presence of divalent ions in the final buffer. (31) However, we still observe the longest networks in the presence of 20 mol % biotinylated lipids and for aligned actin filaments. We also find that different lipid compositions can be used to form lipid nanotubes (SI Figure S6). Notably, when we encapsulated a membrane impermeable dye inside the GUV compartment, we find that it can permeate from the GUV lumen into the lipid nanotubes confirming the formation of defect-free hollow nanotubes (SI Figure S7). In summary, it is possible to exploit natural motors to engineer distinct vesicle morphologies which visually resemble neurons. (32)
As a next step, we translate the nanotube pulling assay from GUVs to living cells, where the membrane is attached to the underlying cytoskeletal cortex. We first probe whether our motor system can pull lipid nanotubes from T-lymphocyte (Jurkat) cells. We verify that cholesterol self-assembles into the cell plasma membrane (SI Figure S8). This allows us to functionalize the Jurkat cells with biotinylated cholesterol. Like for the GUVs, this enables biotinylated actin filaments to bind to the cell in the presence of streptavidin. We observe that the Jurkat cells, despite being nonadherent suspension cells, adhere to the HMM-functionalized substrate due to the artificial linkage established via the actin filaments. Notably, they exhibit many lipid nanotubes at the cell–substrate interface (SI Video S5). By tracking individual cells over time, we find that the pulling of lipid nanotubes mediated by motile actin filaments sets in after about 5 min after the attachment of cells to the HMM (Figure 3a). Note that the cells remain near-stationary during the nanotube pulling process (SI Figure S9 and Videos S6 and S7). Tens of micrometer-long lipid nanotubes are pulled out of each cell over the course of minutes (SI Videos S5, S8, and S9). In the absence of biotinylated cholesterol (0 μM), Jurkat cells do not bind to the substrate and maintain their spherical morphology without the formation of any lipid nanotubes for random as well as aligned filaments (Figure 3b, SI Videos S10 and S11). The absence of unspecific interactions for cells compared to GUVs may be due to their dense glycocalyx, a more complex lipid composition or increased membrane-to-cortex adhesion. By quantifying the network length per cell, we find that whereas the network length for random filaments with 25 μM biotinylated cholesterol is similar to the network length previously determined for GUVs (23 ± 33 μm), the length for aligned filaments exceeds it by an order of magnitude (131 ± 116 μm, Figure 3c). This can partially be explained by the smaller size of the GUVs compared to the Jurkat cells. Since actin-mediated structures provide support of the cell shape and are linked to the cell membrane forming the actin cortex, we focused on the intracellular actin filament organization and dynamics in proximity of lipid nanotubes. Therefore, we stained the intracellular actin with SiR-actin (Figure 3d) and performed live cell imaging. Interestingly, the cellular actin is indeed remodeled and actin filaments are found to extend into the lipid nanotubes, although not along their full length (SI Figure S10). More specifically, we find that no actin filament reaches further than 6 μm into the lipid nanotubes and that they are on average only present within less than half of the nanotube length (SI Figure S11 and Videos S12 and S13). We hypothesize that this is due to the membrane-to-cortex attachment of cellular actin to the cell membrane which causes the actin filaments to be dragged along the membrane during the pulling process. However, the fact that cellular actin filaments only occur at the beginning of nanotubes suggests that the membrane-to-cortex attachment is disrupted at elevated distances and forces of the outer filaments pulling on the cell membrane. Beyond actin, we also stained the mitochondria and the lysosomes of Jurkat cells, as it has been shown that these organelles can be present within tunneling nanotubes of living cells. (23) However, we do not find any evidence for their presence in the lipid nanotubes pulled from Jurkat cells (SI Figure S12).
Having shown that molecular motors can pull lipid nanotubes from Jurkat cells, we test if we can expand our assay to a range of different cell types to probe membrane-to-cortex attachment depending on the cells’ adhesive interaction with the surface. We choose keratinocytes (HaCaTs) and fibroblasts (NIH 3T3) as adherent cells and compare them to semiadherent macrophages (J774A.1) and nonadherent Jurkat cells. In order to obtain the best nanotube pulling efficiency, we use aligned actin at 30 °C. As shown in the confocal images in Figure 4a, we observe the pulling of lipid nanotubes for Jurkat cells and macrophages, whereas we do not observe nanotubes for HaCaTs and fibroblasts. We quantify the network length per cell (Figure 4b) and find that Jurkat cells form significantly bigger networks than macrophages (136 ± 116 μm vs 60 ± 58 μm). Noteworthy, the cell size is not the dominant factor that determines the network size per cell as Jurkat cells are smaller than macrophages, keratinocytes, and fibroblasts. (33) This indicates that the nanotube length is cell type dependent. We hypothesize that no lipid nanotubes form for HaCaTs (SI Figure S13) and fibroblasts due to their high membrane-to-cortex attachment and stiffness compared to nonadherent cells. (3) The pulling force is therefore likely not sufficient to transiently disrupt the membrane-to-cortex attachment, so that a nanotube can form and actin remodeling can take place. To test this hypothesis, we treat fibroblasts with the actin polymerization inhibitor Latrunculin A (LatA) and perform our lipid nanotube pulling assay. Strikingly, under these conditions we observe the formation of lipid nanotubes (Figure 4c) confirming that the network length per cell is influenced by the membrane-to-cortex attachment of the respective cell type. Importantly, the network length per cell for fibroblasts treated with LatA increased to 312 ± 152 μm and thus exceeds the one for Jurkat cells (136 ± 116 μm, Figure 4d). Possibly, this can be explained by the bigger cell size of fibroblasts or due to the complete absence of any attachment sites for fibroblasts treated with LatA compared to untreated Jurkat cells which still possess membrane-cortex attachment sites. (34) To summarize, we have shown that our minimal motor-based system can successfully be transferred to natural cells and be used to probe cell type dependent membrane properties.
The question of how mechanical properties of cell membranes and their underlying cortex regulate cell function and behavior is pivotal for a quantitative understanding of force transduction, cell motility, and cell morphology. Here, we developed a minimal system consisting of natural motor proteins that induce membrane deformation and lipid nanotube extraction from GUVs. In the context of bottom-up synthetic biology, this allows one to establish and explore different vesicle morphologies, in particular morphologies that resemble neurons. Moving toward more immediate biological questions, we translate our findings from GUVs to cells, demonstrating how simplified model membrane systems can allow the development of biological assays. By pulling lipid nanotubes from different cell types, we find cell type specific differences in the lipid nanotube length, whereby nonadherent cells exhibit longer nanotubes compared to adherent cells. This pinpoints toward their different membrane mechanics and the level of membrane-to-cortex attachment. Unlike studies using atomic force microscopy or optical tweezers, we are able to screen the mechanical properties on a single-cell level at very high throughput since nanotubes are extracted from multiple cells simultaneously. Moreover, the use of fluorescence microscopy as a readout of cell morphology also allows for the investigation of other cellular processes in real time in parallel. It will be especially exciting to combine this assay with novel fluorescent membrane-tension probes to enhance our understanding of membrane-to-cortex attachment. In general, it will be exciting to witness a conceptual shift from man-made macroscale machines to molecular machines as biophysical tools in the biosciences.
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.1c04254.
Additional experimental details and control experiments including further quantification, confocal images and (Experimental Methods 1.1 to 1.18); Supporting Figures S1–S13 with an SDS-PAGE of the purified proteins, a confocal image of actin swirls, 3D-STED images of lipid nanotubes, quantification of the number of lipid nanotube branches pulled from GUVs, images showing unspecific interactions of actin and the GUV membrane, the number of lipid nanotubes pulled from GUVs with different lipid compositions, confocal images of dye permeation into lipid nanotubes of GUVs, the verification of the self-assembly of cholesterol-PEG into cell membranes, confocal images of the GUV and cell displacement over time, confocal images and quantification of cellular actin inside lipid nanotubes, confocal images of stained mitochondria and lysosomes after pulling of lipid nanotubes, and a confocal image of HaCaT cells after the pulling assay (PDF)
Video of time series of random actin filaments (MP4)
Video of time series of aligned actin filaments (MP4)
Video of time series of aligned actin filament patterns (MP4)
Video of time series of lipid nanotube dynamics after pulling from GUVs (MP4)
Video of time series of lipid nanotube pulling from Jurkat cells (MP4)
Video of displacement over time of a Jurkat cell during lipid nanotube pulling (MP4)
Video of displacement over time of a GUV during lipid nanotube pulling (MP4)
Video of 3D projection of Jurkat cells with random actin and biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with aligned actin and biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with random actin and no biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with aligned actin and no biotinylated cholesterol (MP4)
Video of actin filament dynamics during lipid nanotube pulling of a Jurkat cell (DOPE-Atto488) (MP4)
Video of actin filament dynamics during lipid nanotube pulling of a Jurkat cell (SiR-actin) (MP4)
Terms & Conditions
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Acknowledgments
The authors thank V. Levario Diaz for providing fibroblasts and D. Missirlis for providing Latrunculin A. Furthermore, they thank I. Platzman, J. P. Spatz, and S. W. Hell for their support. K.G. received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy via the Excellence Cluster 3D Matter Made to Order (EXC-2082/1-390761711) and the Max Planck Society as well as the optical microscopy facility. K.J. thanks the Carl Zeiss Foundation and the Joachim Herz Foundation for financial support. E.A.C.-A. acknowledges support from the DFG (SFB1129 P15) and the Baden-Württemberg Stiftung (3D MOSAIC). The Max Planck Society is appreciated for its general support.
References
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- 5Vutukuri, H. R.; Hoore, M.; Abaurrea-Velasco, C.; van Buren, L.; Dutto, A.; Auth, T.; Fedosov, D. A.; Gompper, G.; Vermant, J. Active particles induce large shape deformations in giant lipid vesicles. Nature 2020, 586, 52– 56, DOI: 10.1038/s41586-020-2730-xGoogle Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFKnt7bI&md5=f88e609922be71afd5f385fc48a39fafActive particles induce large shape deformations in giant lipid vesiclesVutukuri, Hanumantha Rao; Hoore, Masoud; Abaurrea-Velasco, Clara; van Buren, Lennard; Dutto, Alessandro; Auth, Thorsten; Fedosov, Dmitry A.; Gompper, Gerhard; Vermant, JanNature (London, United Kingdom) (2020), 586 (7827), 52-56CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Biol. cells generate intricate structures by sculpting their membrane from within to actively sense and respond to external stimuli or to explore their environment1-4. Several pathogenic bacteria also provide examples of how localized forces strongly deform cell membranes from inside, leading to the invasion of neighboring healthy mammalian cells5. Giant unilamellar vesicles have been successfully used as a minimal model system with which to mimic biol. cells6-11, but the realization of a minimal system with localized active internal forces that can strongly deform lipid membranes from within and lead to dramatic shape changes remains challenging. Here we present a combined exptl. and simulation study that demonstrates how self-propelled particles enclosed in giant unilamellar vesicles can induce a plethora of non-equil. shapes and active membrane fluctuations. Using confocal microscopy, in the expts. we explore the membrane response to local forces exerted by self-phoretic Janus microswimmers. To quantify dynamic membrane changes, we perform Langevin dynamics simulations of active Brownian particles enclosed in thin membrane shells modelled by dynamically triangulated surfaces. The most pronounced shape changes are obsd. at low and moderate particle loadings, with the formation of tether-like protrusions and highly branched, dendritic structures, whereas at high vol. fractions globally deformed vesicle shapes are obsd. The resulting state diagram predicts the conditions under which local internal forces generate various membrane shapes. A controlled realization of such distorted vesicle morphologies could improve the design of artificial systems such as small-scale soft robots and synthetic cells.
- 6Shaklee, P. M.; Idema, T.; Koster, G.; Storm, C.; Schmidt, T.; Dogterom, M. Bidirectional membrane tube dynamics driven by nonprocessive motors. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7993– 7997, DOI: 10.1073/pnas.0709677105Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXns1Sqt78%253D&md5=223793dabcbc5dd5f47034e479d00d42Bidirectional membrane tube dynamics driven by nonprocessive motorsShaklee, Paige M.; Idema, Timon; Koster, Gerbrand; Storm, Cornelis; Schmidt, Thomas; Dogterom, MarileenProceedings of the National Academy of Sciences of the United States of America (2008), 105 (23), 7993-7997, s7993/1-s7993/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In cells, membrane tubes are extd. by mol. motors. Although individual motors cannot provide enough force to pull a tube, clusters of such motors can. Here, we investigate, using a minimal in vitro model system, how the tube pulling process depends on fundamental properties of the motor species involved. Previously, it has been shown that processive motors can pull tubes by dynamic assocn. at the tube tip. We demonstrate that, remarkably, nonprocessive motors can also cooperatively ext. tubes. Moreover, the tubes pulled by nonprocessive motors exhibit rich dynamics as compared to those pulled by their processive counterparts. We report distinct phases of persistent growth, retraction, and an intermediate regime characterized by highly dynamic switching between the two. We interpret the different phases in the context of a single-species model. The model assumes only a simple motor clustering mechanism along the length of the entire tube and the presence of a length-dependent tube tension. The resulting dynamic distribution of motor clusters acts as both a velocity and distance regulator for the tube. We show the switching phase to be an attractor of the dynamics of this model, suggesting that the switching obsd. exptl. is a robust characteristic of nonprocessive motors. A similar system could regulate in vivo biol. membrane networks.
- 7Oriola, D.; Roth, S.; Dogterom, M.; Casademunt, J. Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A. Nat. Commun. 2015, 6, 8025, DOI: 10.1038/ncomms9025Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVKhtbfI&md5=245b4b56dc27c43570d8218bd746dc47Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1AOriola, David; Roth, Sophie; Dogterom, Marileen; Casademunt, JaumeNature Communications (2015), 6 (), 8025CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochem. cycle. However, recent theor. studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to ext. membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to ext. tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helixes. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvring capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.
- 8Jahnke, K.; Weiss, M.; Weber, C.; Platzman, I.; Göpfrich, K.; Spatz, J. P. Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology. Advanced Biosystems 2020, 4, 2000102, DOI: 10.1002/adbi.202000102Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslygu7rL&md5=6e593089817c68bb826f6a601e65ca40Engineering Light-Responsive Contractile Actomyosin Networks with DNA NanotechnologyJahnke, Kevin; Weiss, Marian; Weber, Cornelia; Platzman, Ilia; Goepfrich, Kerstin; Spatz, Joachim P.Advanced Biosystems (2020), 4 (9), 2000102CODEN: ABDIHL; ISSN:2366-7478. (Wiley-VCH Verlag GmbH & Co. KGaA)External control and precise manipulation is key for the bottom-up engineering of complex synthetic cells. Minimal actomyosin networks have been reconstituted into synthetic cells; however, their light-triggered symmetry breaking contraction has not yet been demonstrated. Here, light-activated directional contractility of a minimal synthetic actomyosin network inside microfluidic cell-sized compartments is engineered. Actin filaments, heavy-meromyosin-coated beads, and caged ATP are co-encapsulated into water-in-oil droplets. ATP is released upon illumination, leading to a myosin-generated force which results in a motion of the beads along the filaments and hence a contraction of the network. Symmetry breaking is achieved using DNA nanotechnol. to establish a link between the network and the compartment periphery. It is demonstrated that the DNA-linked actin filaments contract to one side of the compartment forming actin asters and quantify the dynamics of this process. This work exemplifies that an engineering approach to bottom-up synthetic biol., combining biol. and artificial elements, can circumvent challenges related to active multi-component systems and thereby greatly enrich the complexity of synthetic cellular systems.
- 9Juniper, M. P. N.; Weiss, M.; Platzman, I.; Spatz, J. P.; Surrey, T. Spherical network contraction forms microtubule asters in confinement. Soft Matter 2018, 14, 901– 909, DOI: 10.1039/C7SM01718AGoogle Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFKrtrk%253D&md5=252eacca88618134515a78215f563d4fSpherical network contraction forms microtubule asters in confinementJuniper, Michael P. N.; Weiss, Marian; Platzman, Ilia; Spatz, Joachim P.; Surrey, ThomasSoft Matter (2018), 14 (6), 901-909CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)Microtubules and motor proteins form active filament networks that are crit. for a variety of functions in living cells. Network topol. and dynamics are the result of a self-organization process that takes place within the boundaries of the cell. Previous biochem. in vitro studies with biomimetic systems consisting of purified motors and microtubules have demonstrated that confinement has an important effect on the outcome of the self-organization process. However, the pathway of motor/microtubule self-organization under confinement and its effects on network morphol. are still poorly understood. Here, we have investigated how minus-end directed microtubule crosslinking kinesins organize microtubules inside polymer-stabilized microfluidic droplets of well-controlled size. We find that confinement can impose a novel pathway of microtubule aster formation proceeding via the constriction of an initially spherical motor/microtubule network. This mechanism illustrates the close relationship between confinement, network contraction, and aster formation. The spherical constriction pathway robustly produces single, well-centered asters with remarkable reproducibility across thousands of droplets. These results show that the addnl. constraint of well-defined confinement can improve the robustness of active network self-organization, providing insight into the design principles of self-organizing active networks in micro-scale confinement.
- 10Litschel, T.; Kelley, C. F.; Holz, D.; Koudehi, M. A.; Vogel, S. K.; Burbaum, L.; Mizuno, N.; Vavylonis, D.; Schwille, P. Reconstitution of contractile actomyosin rings in vesicles. Nat. Commun. 2021, 12, 2254, DOI: 10.1038/s41467-021-22422-7Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXptFOqsL8%253D&md5=5f2aaa315f2c39163e5b7e59c7b2aacbReconstitution of contractile actomyosin rings in vesiclesLitschel, Thomas; Kelley, Charlotte F.; Holz, Danielle; Adeli Koudehi, Maral; Vogel, Sven K.; Burbaum, Laura; Mizuno, Naoko; Vavylonis, Dimitrios; Schwille, PetraNature Communications (2021), 12 (1), 2254CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)One of the grand challenges of bottom-up synthetic biol. is the development of minimal machineries for cell division. The mech. transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the mol. scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theor. modeling. By changing few key parameters, actin polymn. can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theor. considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.
- 11Persson, M.; Gullberg, M.; Tolf, C.; Lindberg, A. M.; Månsson, A.; Kocer, A. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments. PLoS One 2013, 8, e55931, DOI: 10.1371/journal.pone.0055931Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs12gtbk%253D&md5=b8a57799780799ae6525badd40dfe2aaTransportation of nanoscale cargoes by myosin propelled actin filamentsPersson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Maansson, Alf; Kocer, ArmaganPLoS One (2013), 8 (2), e55931CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addn., actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different no. of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 μm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing no. of side-attached cargoes, a redn. that occurred within a range of 10-50 streptavidin mols., 1-10 quantum dots or with just 1 liposome. However, as obsd. by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomech. energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in mol. diagnostics and other nanotechnol. applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.
- 12Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Light-Controlled Molecular Shuttles Made from Motor Proteins Carrying Cargo on Engineered Surfaces. Nano Lett. 2001, 1, 235– 239, DOI: 10.1021/nl015521eGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXivVyhs7c%253D&md5=75d0eff58da6d35936c82e3a4b27ee89Light-Controlled Molecular Shuttles Made from Motor Proteins Carrying Cargo on Engineered SurfacesHess, Henry; Clemmens, John; Qin, Dong; Howard, Jonathon; Vogel, ViolaNano Letters (2001), 1 (5), 235-239CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Mol. shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymic ATP degrdn. by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize mol. motors for the construction of nanoscale assembly lines.
- 13Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Control of actin moving trajectory by patterned poly(methylmethacrylate) tracks. Biophys. J. 1997, 72, 1997– 2001, DOI: 10.1016/S0006-3495(97)78844-1Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivVOjs7k%253D&md5=2b2e24c854f3580d2049f7d5671f98f8Control of actin moving trajectory by patterned poly(methylmethacrylate) tracksSuzuki, Hitoshi; Yamada, Akira; Oiwa, Kazuhiro; Nakayama, Haruto; Mashiko, ShinroBiophysical Journal (1997), 72 (5), 1997-2001CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Poly(methylmethacrylate) (PMMA), a photoresist polymer, was found to be useful for immobilizing heavy meromyosin (HMM) mols. while retaining their abilities to support the movement of actin filaments. PMMA substrate was spin-coated on a coverslip, and various shapes of PMMA tracks, such as straight lines, concentric circles, and alphabetical letters, were fabricated by UV photolithog. An observation by a Tapping mode at. force microscope (AFM) shows that the typical circular tracks were 1-2 μm wide and about 200 nm high. In in vitro motility assay, a soln. of HMM mols. was applied to immobilize the mols. on the tracks by adsorption, and movement of actin filaments labeled with tetramethylrhodamine-phalloidin were obsd. in the presence of ATP by using an epifluorescence microscope and an image-intensified CCD camera. Actin filaments were seen to move precisely only on the PMMA tracks, and their traces drew the exact shapes of the tracks. The mean velocity of actin movement on the PMMA was 4.5 mm/s at 25°C, and it was comparable to that on a conventionally used nitrocellulose film.
- 14Herold, C.; Leduc, C.; Stock, R.; Diez, S.; Schwille, P. Long-Range Transport of Giant Vesicles along Microtubule Networks. ChemPhysChem 2012, 13, 1001– 1006, DOI: 10.1002/cphc.201100669Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1yrtr%252FJ&md5=70ee88c812c31839aa44bf9e83f4f717Long-Range Transport of Giant Vesicles along Microtubule NetworksHerold, Christoph; Leduc, Cecile; Stock, Robert; Diez, Stefan; Schwille, PetraChemPhysChem (2012), 13 (4), 1001-1006CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diams. of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, the authors were able to est. the no. of GFP-labeled motors involved in the transport of a single vesicle. The vesicles were transported by the cooperative activity of typically 5-10 motor mols. The presented assay is expected to open up further applications in the field of synthetic biol., aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnol., where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.
- 15Diez, S.; Reuther, C.; Dinu, C.; Seidel, R.; Mertig, M.; Pompe, W.; Howard, J. Stretching and Transporting DNA Molecules Using Motor Proteins. Nano Lett. 2003, 3, 1251– 1254, DOI: 10.1021/nl034504hGoogle Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmsFOitrs%253D&md5=ad2aa1cc0e797351aecff8ce163857b4Stretching and transporting DNA molecules using motor proteinsDiez, Stefan; Reuther, Cordula; Dinu, Cerasela; Seidel, Ralf; Mertig, Michael; Pompe, Wolfgang; Howard, JonathonNano Letters (2003), 3 (9), 1251-1254CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Inside cells, motor proteins perform a variety of complex tasks including the transport of vesicles and the sepn. of chromosomes. The authors demonstrate a novel use of such biol. machines for the mech. manipulation of nanostructures in a cell-free environment. Specifically, the authors show that purified kinesin motors in combination with chem. modified microtubules can transport and stretch individual λ-phage DNA mols. across a surface. This technique, in contrast to existing ones, enables the parallel yet individual manipulation of many mols. and may offer an efficient mechanism for assembling multidimensional DNA structures.
- 16Cha, T.-G.; Pan, J.; Chen, H.; Salgado, J.; Li, X.; Mao, C.; Choi, J. H. A synthetic DNA motor that transports nanoparticles along carbon nanotubes. Nat. Nanotechnol. 2014, 9, 39– 43, DOI: 10.1038/nnano.2013.257Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvV2hsLvO&md5=2c36fd998e86a3dd62090e64d57cd7b1A synthetic DNA motor that transports nanoparticles along carbon nanotubesCha, Tae-Gon; Pan, Jing; Chen, Haorong; Salgado, Janette; Li, Xiang; Mao, Chengde; Choi, Jong HyunNature Nanotechnology (2014), 9 (1), 39-43CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Intracellular protein motors have evolved to perform specific tasks crit. to the function of cells such as intracellular trafficking and cell division. Kinesin and dynein motors, for example, transport cargoes in living cells by walking along microtubules powered by ATP hydrolysis. These motors can make discrete 8 nm center-of-mass steps and can travel over 1 μm by changing their conformations during the course of ATP binding, hydrolysis and product release. Inspired by such biol. machines, synthetic analogs have been developed including self-assembled DNA walkers that can make stepwise movements on RNA/DNA substrates or can function as programmable assembly lines. Here, we show that motors based on RNA-cleaving DNA enzymes can transport nanoparticle cargoes-CdS nanocrystals in this case-along single-walled carbon nanotubes. Our motors ext. chem. energy from RNA mols. decorated on the nanotubes and use that energy to fuel autonomous, processive walking through a series of conformational changes along the one-dimensional track. The walking is controllable and adapts to changes in the local environment, which allows us to remotely direct 'go' and 'stop' actions. The translocation of individual motors can be visualized in real time using the visible fluorescence of the cargo nanoparticle and the near-infared emission of the carbon-nanotube track. We obsd. unidirectional movements of the mol. motors over 3 μm with a translocation velocity on the order of 1 nm min-1 under our exptl. conditions.
- 17Wickham, S. F. J.; Bath, J.; Katsuda, Y.; Endo, M.; Hidaka, K.; Sugiyama, H.; Turberfield, A. J. A DNA-based molecular motor that can navigate a network of tracks. Nat. Nanotechnol. 2012, 7, 169– 173, DOI: 10.1038/nnano.2011.253Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqu74%253D&md5=27698363e23db94f4ad32d96b4e501b2A DNA-based molecular motor that can navigate a network of tracksWickham, Shelley F. J.; Bath, Jonathan; Katsuda, Yousuke; Endo, Masayuki; Hidaka, Kumi; Sugiyama, Hiroshi; Turberfield, Andrew J.Nature Nanotechnology (2012), 7 (3), 169-173CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Synthetic mol. motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been obsd. on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks contg. four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, mol. systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands.
- 18Ketterer, P.; Willner, E. M.; Dietz, H. Nanoscale rotary apparatus formed from tight-fitting 3D DNA components. Science Advances 2016, 2, e1501209, DOI: 10.1126/sciadv.1501209Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVWku7w%253D&md5=b84b80d00436c4f1c95556819bc2f6ddNanoscale rotary apparatus formed from tight-fitting 3D DNA componentsKetterer, Philip; Willner, Elena M.; Dietz, HendrikScience Advances (2016), 2 (2), e1501209/1-e1501209/9CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)We report a nanoscale rotary mechanism that reproduces some of the dynamic properties of biol. rotary motors in the absence of an energy source, such as random walks on a circle with dwells at docking sites. Our mechanism is built modularly from tight-fitting components that were self-assembled using multilayer DNA origami. The app. has greater structural complexity than previous mech. interlocked objects and features a well-defined angular degree of freedom without restricting the range of rotation. We studied the dynamics of our mechanism using single-particle expts. analogous to those performed previously with actin-labeled ATP synthases. In our mechanism, rotor mobility, the no. of docking sites, and the dwell times at these sites may be controlled through rational design. Our prototype thus realizes a working platform toward creating synthetic nanoscale rotary motors. Our methods will support creating other complex nanoscale mechanisms based on tightly fitting, sterically constrained, but mobile, DNA components.
- 19Urban, M. J.; Both, S.; Zhou, C.; Kuzyk, A.; Lindfors, K.; Weiss, T.; Liu, N. Gold nanocrystal-mediated sliding of doublet DNA origami filaments. Nat. Commun. 2018, 9, 1454, DOI: 10.1038/s41467-018-03882-wGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjisF2rsQ%253D%253D&md5=ee6d55cac42f27291d967f088661f025Gold nanocrystal-mediated sliding of doublet DNA origami filamentsUrban Maximilian J; Zhou Chao; Kuzyk Anton; Liu Na; Urban Maximilian J; Zhou Chao; Liu Na; Both Steffen; Weiss Thomas; Kuzyk Anton; Lindfors KlasNature communications (2018), 9 (1), 1454 ISSN:.Sliding is one of the fundamental mechanical movements in machinery. In macroscopic systems, double-rack pinion machines employ gears to slide two linear tracks along opposite directions. In microscopic systems, kinesin-5 proteins crosslink and slide apart antiparallel microtubules, promoting spindle bipolarity and elongation during mitosis. Here we demonstrate an artificial nanoscopic analog, in which gold nanocrystals can mediate coordinated sliding of two antiparallel DNA origami filaments powered by DNA fuels. Stepwise and reversible sliding along opposite directions is in situ monitored and confirmed using fluorescence spectroscopy. A theoretical model including different energy transfer mechanisms is developed to understand the observed fluorescence dynamics. We further show that such sliding can also take place in the presence of multiple DNA sidelocks that are introduced to inhibit the relative movements. Our work enriches the toolbox of DNA-based nanomachinery, taking one step further toward the vision of molecular nanofactories.
- 20Roux, A.; Cappello, G.; Cartaud, J.; Prost, J.; Goud, B.; Bassereau, P. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5394– 5399, DOI: 10.1073/pnas.082107299Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKlsLs%253D&md5=c43c4d4592dc899963d857aae7f22ad8A minimal system allowing tubulation with molecular motors pulling on giant liposomesRoux, Aurelien; Cappello, Giovanni; Cartaud, Jean; Prost, Jacques; Goud, Bruno; Bassereau, PatriciaProceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5394-5399CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The elucidation of phys. and mol. mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that mol. motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin mols. have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was obsd. when kinesins were directly bound to the GUV membrane, suggesting that it is crit. to distribute the load on both lipids and motors by means of beads. A kinetic anal. shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled expts. aimed at the understanding of basic mechanisms in intracellular transport.
- 21Leduc, C.; Campas, O.; Zeldovich, K. B.; Roux, A.; Jolimaitre, P.; Bourel-Bonnet, L.; Goud, B.; Joanny, J.-F.; Bassereau, P.; Prost, J. Cooperative extraction of membrane nanotubes by molecular motors. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17096– 17101, DOI: 10.1073/pnas.0406598101Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFagu7nL&md5=2e1e9570ab476c6f81f67cb179a0db94Cooperative extraction of membrane nanotubes by molecular motorsLeduc, Cecile; Campas, Otger; Zeldovich, Konstantin B.; Roux, Aurelien; Jolimaitre, Pascale; Bourel-Bonnet, Line; Goud, Bruno; Joanny, Jean-Francois; Bassereau, Patricia; Prost, JacquesProceedings of the National Academy of Sciences of the United States of America (2004), 101 (49), 17096-17101CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In eukaryotic cells, nanotubes represent a substantial fraction of transport intermediates between organelles. They are extd. from membranes by mol. motors walking along microtubules. We previously showed that kinesins fixed on giant unilamellar vesicles in contact with microtubules are sufficient to form nanotubes in vitro. Motors were attached to the membrane through beads, thus facilitating cooperative effects. Koster et al. [Koster, G., VanDuijn, M., Hofs, B. & Dogterom, M. (2003) Proc. Natl. Acad. Sci. USA 100, 15583-15588] proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. We demonstrate, in a recently designed exptl. system, the existence of an accumulation of motors allowing tube extn. We det. the motor d. along a tube by using fluorescence intensity measurements. We also perform a theor. anal. describing the dynamics of motors and tube growth. The only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 ± 2.4 s-1. In addn., we quant. det., for a given membrane tension, the existence of a threshold in motor d. on the vesicle above which nanotubes can be formed. We find that the no. of motors pulling a tube can range from four at threshold to a few tens away from it. The threshold in motor d. (or in membrane tension at const. motor d.) could be important for the understanding of membrane traffic regulation in cells.
- 22Campillo, C.; Sens, P.; Köster, D.; Pontani, L.-L.; Lévy, D.; Bassereau, P.; Nassoy, P.; Sykes, C. Unexpected Membrane Dynamics Unveiled by Membrane Nanotube Extrusion. Biophys. J. 2013, 104, 1248– 1256, DOI: 10.1016/j.bpj.2013.01.051Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlOgtbc%253D&md5=a668a303d051288b75b509824dc3406aUnexpected Membrane Dynamics Unveiled by Membrane Nanotube ExtrusionCampillo, Clement; Sens, Pierre; Koster, Darius; Pontani, Lea-Laetitia; Levy, Daniel; Bassereau, Patricia; Nassoy, Pierre; Sykes, CecileBiophysical Journal (2013), 104 (6), 1248-1256CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)In cell mechanics, distinguishing the resp. roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular and pure lipid membranes is usually attributed to the presence of the cytoskeleton as explored by membrane nanotube extrusion. Here we revisit this prevalent picture by unveiling unexpected force responses of plasma membrane spheres devoid of cytoskeleton and synthetic liposomes. We show that a tiny variation in the content of synthetic membranes does not affect their static mech. properties, but is enough to reproduce the dynamic behavior of their cellular counterparts. This effect is attributed to an amplified intramembrane friction. Reconstituted actin cortices inside liposomes induce an addnl., but not dominant, contribution to the effective membrane friction. Our work underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheol. in addn. to the role of the cytoskeleton.
- 23Rustom, A.; Saffrich, R.; Markovic, I.; Walther, P.; Gerdes, H.-H. Nanotubular Highways for Intercellular Organelle Transport. Science 2004, 303, 1007– 1010, DOI: 10.1126/science.1093133Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlWnu7w%253D&md5=e5bf1883daee6fc0f07f00afeff004e0Nanotubular Highways for Intercellular Organelle TransportRustom, Amin; Saffrich, Rainer; Markovic, Ivanka; Walther, Paul; Gerdes, Hans-HermannScience (Washington, DC, United States) (2004), 303 (5660), 1007-1010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chem. synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small mols. Accordingly, we propose a novel biol. principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.
- 24Kron, S. J.; Spudich, J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 6272– 6276, DOI: 10.1073/pnas.83.17.6272Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XlvVGqurc%253D&md5=ac4102efdaa2d11d67e8a60763dab2a9Fluorescent actin filaments move on myosin fixed to a glass surfaceKron, Stephen J.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1986), 83 (17), 6272-6CODEN: PNASA6; ISSN:0027-8424.Single actin filaments stabilized with fluorescent phalloidin exhibit ATP-dependent movement on myosin filaments fixed to a surface. At pH 7.4 and 24°, the rates of movement av. 3-4 μm/s with skeletal muscle myosin and 1-2 μm/s with Dictyostelium myosin. These rates are very similar to those measured in previous myosin movement assays. The rates of movement are relatively independent of the type of actin used. The filament velocity shows a broad pH optimum of 7.0-9.0, and the concn. of ATP required for half-maximal velocity is 50 μM. Apparently, movement of actin over myosin requires at most the no. of heads in a single thick filament. This system provides a practical, quant. myosin-movement assay with purified proteins.
- 25Iwase, T.; Sasaki, Y.; Hatori, K. Alignment of actin filament streams driven by myosin motors in crowded environments. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861, 2717– 2725, DOI: 10.1016/j.bbagen.2017.07.016Google ScholarThere is no corresponding record for this reference.
- 26Das, T.; Safferling, K.; Rausch, S.; Grabe, N.; Boehm, H.; Spatz, J. P. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat. Cell Biol. 2015, 17, 276– 287, DOI: 10.1038/ncb3115Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFemtrg%253D&md5=7b9531bf4c004cbe23c5051e52b8ba0cA molecular mechanotransduction pathway regulates collective migration of epithelial cellsDas, Tamal; Safferling, Kai; Rausch, Sebastian; Grabe, Niels; Boehm, Heike; Spatz, Joachim P.Nature Cell Biology (2015), 17 (3), 276-287CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Collective movement of epithelial cells drives essential multicellular organization during various fundamental physiol. processes encompassing embryonic morphogenesis, cancer and wound healing. Yet the mol. mechanism that ensures the coordinated movement of many cells remains elusive. Here we show that a tumor suppressor protein, merlin, coordinates collective migration of tens of cells, by acting as a mechanochem. transducer. In a stationary epithelial monolayer and also in three-dimensional human skin, merlin localizes to cortical cell-cell junctions. During migration initiation, a fraction of cortical merlin relocalizes to the cytoplasm. This relocalization is triggered by the intercellular pulling force of the leading cell and depends on the actomyosin-based cell contractility. Then in migrating cells, taking its cue from the intercellular pulling forces, which show long-distance ordering, merlin coordinates polarized Rac1 activation and lamellipodium formation on the multicellular length scale. Together, these results provide a distinct mol. mechanism linking intercellular forces to collective cell movements in migrating epithelia.
- 27Schaller, V.; Weber, C.; Semmrich, C.; Frey, E.; Bausch, A. R. Polar patterns of driven filaments. Nature 2010, 467, 73– 77, DOI: 10.1038/nature09312Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFaqtbbM&md5=961fbd27f738fc9be43702dc4e49e206Polar patterns of driven filamentsSchaller, Volker; Weber, Christoph; Semmrich, Christine; Frey, Erwin; Bausch, Andreas R.Nature (London, United Kingdom) (2010), 467 (7311), 73-77CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The emergence of collective motion exhibited by systems ranging from flocks of animals to self-propelled microorganisms to the cytoskeleton is a ubiquitous and fascinating self-organization phenomenon. Similarities between these systems, such as the inherent polarity of the constituents, a d.-dependent transition to ordered phases or the existence of very large d. fluctuations, suggest universal principles underlying pattern formation. This idea is followed by theor. models at all levels of description: micro- or mesoscopic models directly map local forces and interactions using only a few, preferably simple, interaction rules, and more macroscopic approaches in the hydrodynamic limit rely on the systems' generic symmetries. All these models characteristically have a broad parameter space with a manifold of possible patterns, most of which have not yet been exptl. verified. The complexity of interactions and the limited parameter control of existing exptl. systems are major obstacles to our understanding of the underlying ordering principles. Here we demonstrate the emergence of collective motion in a high-d. motility assay that consists of highly concd. actin filaments propelled by immobilized mol. motors in a planar geometry. Above a crit. d., the filaments self-organize to form coherently moving structures with persistent d. modulations, such as clusters, swirls and interconnected bands. These polar nematic structures are long lived and can span length scales orders of magnitudes larger than their constituents. Our exptl. approach, which offers control of all relevant system parameters, complemented by agent-based simulations, allows backtracking of the assembly and disassembly pathways to the underlying local interactions. We identify weak and local alignment interactions to be essential for the obsd. formation of patterns and their dynamics. The presented minimal polar-pattern-forming system may thus provide new insight into emerging order in the broad class of active fluids and self-propelled particles.
- 28Homsher, E.; Wang, F.; Sellers, J. R. Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin. American Journal of Physiology-Cell Physiology 1992, 262, C714– C723, DOI: 10.1152/ajpcell.1992.262.3.C714Google ScholarThere is no corresponding record for this reference.
- 29Salhotra, A.; Zhu, J.; Surendiran, P.; Meinecke, C. R.; Lyttleton, R.; Ušaj, M.; Lindberg, F. W.; Norrby, M.; Linke, H.; Månsson, A. Prolonged function and optimization of actomyosin motility for upscaled network-based biocomputation. New J. Phys. 2021, 23, 085005, DOI: 10.1088/1367-2630/ac1809Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVaktbfP&md5=2e180bcf4cdb72184589dee348057dcaProlonged function and optimization of actomyosin motility for upscaled network-based biocomputationSalhotra, Aseem; Zhu, Jingyuan; Surendiran, Pradheebha; Meinecke, Christoph Robert; Lyttleton, Roman; Usaj, Marko; Lindberg, Frida W.; Norrby, Marlene; Linke, Heiner; Maansson, AlfNew Journal of Physics (2021), 23 (Aug.), 085005CODEN: NJOPFM; ISSN:1367-2630. (IOP Publishing Ltd.)Significant advancements have been made towards exploitation of naturally available mol. motors and their assocd. cytoskeletal filaments in nanotechnol. applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biol. system. Here, we systematically investigated potentially crit. factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay soln. compn. and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addn., we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chem. oxygen scavenger system (glucose oxidase-glucose-catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted com. available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at -80°C for more than 10 years before use for nanotechnol. purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.
- 30Roy, D.; Steinkühler, J.; Zhao, Z.; Lipowsky, R.; Dimova, R. Mechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced Nanotubes. Nano Lett. 2020, 20, 3185– 3191, DOI: 10.1021/acs.nanolett.9b05232Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslehtLk%253D&md5=1b80c36662eb76a13a99e3704d75caaeMechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced NanotubesRoy, Debjit; Steinkuehler, Jan; Zhao, Ziliang; Lipowsky, Reinhard; Dimova, RumianaNano Letters (2020), 20 (5), 3185-3191CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Membrane tension modulates the morphol. of plasma-membrane tubular protrusions in cells but is difficult to measure. Here, the authors propose to use microscopy imaging to assess the membrane tension. The authors report direct measurement of membrane nanotube diams. with unprecedented resoln. using stimulated emission depletion (STED) microscopy. For this purpose, the authors integrated an optical tweezers setup in a com. microscope equipped for STED imaging and established micropipette aspiration of giant vesicles. Membrane nanotubes were pulled from the vesicles at specific membrane tension imposed by the aspiration pipet. Tube diams. calcd. from the applied tension using the membrane curvature elasticity model are in excellent agreement with data measured directly with STED. The approach can be extended to cellular membranes and will then allow us to est. the mech. membrane tension within the force-induced nanotubes.
- 31Schroer, C. F. E.; Baldauf, L.; van Buren, L.; Wassenaar, T. A.; Melo, M. N.; Koenderink, G. H.; Marrink, S. J. Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 5861– 5872, DOI: 10.1073/pnas.1914884117Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Wlu7g%253D&md5=0eedf86d33cf3a45a5b7af0cc7044c78Charge-dependent interactions of monomeric and filamentous actin with lipid bilayersSchroer, Carsten F. E.; Baldauf, Lucia; van Buren, Lennard; Wassenaar, Tsjerk A.; Melo, Manuel N.; Koenderink, Gijsje H.; Marrink, Siewert J.Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (11), 5861-5872CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The cytoskeletal protein actin polymerizes into filaments that are essential for the mech. stability of mammalian cells. In vitro expts. showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biol. design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained mol. dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly det. the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated exptl. by reconstitution assays with actin on lipid bilayer membranes and provide a mol.-level understanding of the actin-membrane interaction.
- 32Schubert, P. J.; Dorkenwald, S.; Januszewski, M.; Jain, V.; Kornfeld, J. Learning cellular morphology with neural networks. Nat. Commun. 2019, 10, 2736, DOI: 10.1038/s41467-019-10836-3Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M3psF2hug%253D%253D&md5=b749ceee569e0cb32de82ff20a23e158Learning cellular morphology with neural networksSchubert Philipp J; Dorkenwald Sven; Kornfeld Joergen; Januszewski Michal; Jain VirenNature communications (2019), 10 (1), 2736 ISSN:.Reconstruction and annotation of volume electron microscopy data sets of brain tissue is challenging but can reveal invaluable information about neuronal circuits. Significant progress has recently been made in automated neuron reconstruction as well as automated detection of synapses. However, methods for automating the morphological analysis of nanometer-resolution reconstructions are less established, despite the diversity of possible applications. Here, we introduce cellular morphology neural networks (CMNs), based on multi-view projections sampled from automatically reconstructed cellular fragments of arbitrary size and shape. Using unsupervised training, we infer morphology embeddings (Neuron2vec) of neuron reconstructions and train CMNs to identify glia cells in a supervised classification paradigm, which are then used to resolve neuron reconstruction errors. Finally, we demonstrate that CMNs can be used to identify subcellular compartments and the cell types of neuron reconstructions.
- 33Milo, R.; Jorgensen, P.; Moran, U.; Weber, G.; Springer, M. BioNumbers─the database of key numbers in molecular and cell biology. Nucleic Acids Res. 2010, 38, D750– D753, DOI: 10.1093/nar/gkp889Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktl2nsQ%253D%253D&md5=6c311e7fa440e5a22d6028907ca1e221BioNumbers-the database of key numbers in molecular and cell biologyMilo, Ron; Jorgensen, Paul; Moran, Uri; Weber, Griffin; Springer, MichaelNucleic Acids Research (2010), 38 (Database Iss), D750-D753CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)BioNumbers (http://www.bionumbers.hms.harvard.edu) is a database of key nos. in mol. and cell biol.-the quant. properties of biol. systems of interest to computational, systems and mol. cell biologists. Contents of the database range from cell sizes to metabolite concns., from reaction rates to generation times, from genome sizes to the no. of mitochondria in a cell. While always of importance to biologists, having nos. in hand is becoming increasingly crit. for experimenting, modeling, and analyzing biol. systems. BioNumbers was motivated by an appreciation of how long it can take to find even the simplest no. in the vast biol. literature. All nos. are taken directly from a literature source and that ref. is provided with the no. BioNumbers is designed to be highly searchable and queries can be performed by keywords or browsed by menus. BioNumbers is a collaborative community platform where registered users can add content and make comments on existing data. All new entries and commentary are curated to maintain high quality. Here we describe the database characteristics and implementation, demonstrate its use, and discuss future directions for its development.
- 34Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M. Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy. J. Phys. D: Appl. Phys. 2017, 50, 064002, DOI: 10.1088/1361-6463/aa52a1Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFKqs7k%253D&md5=aacea4a00fe7f3031301ff15773fa536Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopyClausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.Journal of Physics D: Applied Physics (2017), 50 (6), 064002/1-064002/11CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphol., mechanics, and function. Measuring this distance has been a key challenge in cell biol. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resoln. is insufficient to quantify distances on the nanoscale. Using dual-color super-resoln. STED (stimulated emission-depletion) microscopy, we here overcome this challenge and accurately measure the d. distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asym. cortical actin d. distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the max. d. peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, resp. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a max. of 20 nm in others.
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This article references 34 other publications.
- 1van den Heuvel, M. G. L.; Dekker, C. Motor Proteins at Work for Nanotechnology. Science 2007, 317, 333– 336, DOI: 10.1126/science.11395701https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslGrs7Y%253D&md5=96877d4426278f133b82783e7e0ac9c7Motor Proteins at Work for Nanotechnologyvan den Heuvel, Martin G. L.; Dekker, CeesScience (Washington, DC, United States) (2007), 317 (5836), 333-336CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. The biol. cell is equipped with a variety of mol. machines that perform complex mech. tasks such as cell division or intracellular transport. One can envision employing these biol. motors in artificial environments. The authors review the progress that has been made in using motor proteins for powering or manipulating nanoscale components. In particular, kinesin and myosin biomotors that move along linear biofilaments have been widely explored as active components. Currently realized applications are merely proof-of-principle demonstrations. Yet, the sheer availability of an entire ready-to-use toolbox of nanosized biol. motors is a great opportunity that calls for exploration.
- 2Saper, G.; Hess, H. Synthetic Systems Powered by Biological Molecular Motors. Chem. Rev. 2020, 120, 288– 309, DOI: 10.1021/acs.chemrev.9b002492https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslektr%252FN&md5=e9b4499656ef83ef272964d21edb71dcSynthetic Systems Powered by Biological Molecular MotorsSaper, Gadiel; Hess, HenryChemical Reviews (Washington, DC, United States) (2020), 120 (1), 288-309CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Biol. mol. motors (or biomol. motors for short) are nature's soln. to the efficient conversion of chem. energy to mech. movement. In biol. systems, these fascinating mols. are responsible for movement of mols., organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomol. motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their assocd. cytoskeletal filaments, we review recent work aiming for the integration of these biomol. motors into actuators, sensors, and computing devices. In some systems, the creation of mech. work and the processing of information become intertwined at the mol. scale, creating a fascinating type of "active matter". We discuss efforts to optimize biomol. motor performance, construct new motors combining artificial and biol. components, and contrast biomol. motors with current artificial mol. motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomol. motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomol. motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomol. motors and try to ascertain if there are theor. performance limits. Engineering with biomol. motors has the potential to yield com. viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biol. and potentially also for medicine.
- 3Sitarska, E.; Diz-Muñoz, A. Pay attention to membrane tension: Mechanobiology of the cell surface. Curr. Opin. Cell Biol. 2020, 66, 11– 18, DOI: 10.1016/j.ceb.2020.04.0013https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXos1Smsr4%253D&md5=32f9d02a4bbb99c5926e0ac0f1dea91cPay attention to membrane tension: Mechanobiology of the cell surfaceSitarska, Ewa; Diz-Munoz, AlbaCurrent Opinion in Cell Biology (2020), 66 (), 11-18CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)A review. The cell surface is a mechanobiol. unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could det. whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.
- 4Leduc, C.; Campás, O.; Joanny, J.-F.; Prost, J.; Bassereau, P. Mechanism of membrane nanotube formation by molecular motors. Biochimica et Biophysica Acta (BBA) - Biomembranes 2010, 1798, 1418– 1426, DOI: 10.1016/j.bbamem.2009.11.012There is no corresponding record for this reference.
- 5Vutukuri, H. R.; Hoore, M.; Abaurrea-Velasco, C.; van Buren, L.; Dutto, A.; Auth, T.; Fedosov, D. A.; Gompper, G.; Vermant, J. Active particles induce large shape deformations in giant lipid vesicles. Nature 2020, 586, 52– 56, DOI: 10.1038/s41586-020-2730-x5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFKnt7bI&md5=f88e609922be71afd5f385fc48a39fafActive particles induce large shape deformations in giant lipid vesiclesVutukuri, Hanumantha Rao; Hoore, Masoud; Abaurrea-Velasco, Clara; van Buren, Lennard; Dutto, Alessandro; Auth, Thorsten; Fedosov, Dmitry A.; Gompper, Gerhard; Vermant, JanNature (London, United Kingdom) (2020), 586 (7827), 52-56CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Biol. cells generate intricate structures by sculpting their membrane from within to actively sense and respond to external stimuli or to explore their environment1-4. Several pathogenic bacteria also provide examples of how localized forces strongly deform cell membranes from inside, leading to the invasion of neighboring healthy mammalian cells5. Giant unilamellar vesicles have been successfully used as a minimal model system with which to mimic biol. cells6-11, but the realization of a minimal system with localized active internal forces that can strongly deform lipid membranes from within and lead to dramatic shape changes remains challenging. Here we present a combined exptl. and simulation study that demonstrates how self-propelled particles enclosed in giant unilamellar vesicles can induce a plethora of non-equil. shapes and active membrane fluctuations. Using confocal microscopy, in the expts. we explore the membrane response to local forces exerted by self-phoretic Janus microswimmers. To quantify dynamic membrane changes, we perform Langevin dynamics simulations of active Brownian particles enclosed in thin membrane shells modelled by dynamically triangulated surfaces. The most pronounced shape changes are obsd. at low and moderate particle loadings, with the formation of tether-like protrusions and highly branched, dendritic structures, whereas at high vol. fractions globally deformed vesicle shapes are obsd. The resulting state diagram predicts the conditions under which local internal forces generate various membrane shapes. A controlled realization of such distorted vesicle morphologies could improve the design of artificial systems such as small-scale soft robots and synthetic cells.
- 6Shaklee, P. M.; Idema, T.; Koster, G.; Storm, C.; Schmidt, T.; Dogterom, M. Bidirectional membrane tube dynamics driven by nonprocessive motors. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7993– 7997, DOI: 10.1073/pnas.07096771056https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXns1Sqt78%253D&md5=223793dabcbc5dd5f47034e479d00d42Bidirectional membrane tube dynamics driven by nonprocessive motorsShaklee, Paige M.; Idema, Timon; Koster, Gerbrand; Storm, Cornelis; Schmidt, Thomas; Dogterom, MarileenProceedings of the National Academy of Sciences of the United States of America (2008), 105 (23), 7993-7997, s7993/1-s7993/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In cells, membrane tubes are extd. by mol. motors. Although individual motors cannot provide enough force to pull a tube, clusters of such motors can. Here, we investigate, using a minimal in vitro model system, how the tube pulling process depends on fundamental properties of the motor species involved. Previously, it has been shown that processive motors can pull tubes by dynamic assocn. at the tube tip. We demonstrate that, remarkably, nonprocessive motors can also cooperatively ext. tubes. Moreover, the tubes pulled by nonprocessive motors exhibit rich dynamics as compared to those pulled by their processive counterparts. We report distinct phases of persistent growth, retraction, and an intermediate regime characterized by highly dynamic switching between the two. We interpret the different phases in the context of a single-species model. The model assumes only a simple motor clustering mechanism along the length of the entire tube and the presence of a length-dependent tube tension. The resulting dynamic distribution of motor clusters acts as both a velocity and distance regulator for the tube. We show the switching phase to be an attractor of the dynamics of this model, suggesting that the switching obsd. exptl. is a robust characteristic of nonprocessive motors. A similar system could regulate in vivo biol. membrane networks.
- 7Oriola, D.; Roth, S.; Dogterom, M.; Casademunt, J. Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A. Nat. Commun. 2015, 6, 8025, DOI: 10.1038/ncomms90257https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVKhtbfI&md5=245b4b56dc27c43570d8218bd746dc47Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1AOriola, David; Roth, Sophie; Dogterom, Marileen; Casademunt, JaumeNature Communications (2015), 6 (), 8025CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochem. cycle. However, recent theor. studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to ext. membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to ext. tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helixes. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvring capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.
- 8Jahnke, K.; Weiss, M.; Weber, C.; Platzman, I.; Göpfrich, K.; Spatz, J. P. Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology. Advanced Biosystems 2020, 4, 2000102, DOI: 10.1002/adbi.2020001028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslygu7rL&md5=6e593089817c68bb826f6a601e65ca40Engineering Light-Responsive Contractile Actomyosin Networks with DNA NanotechnologyJahnke, Kevin; Weiss, Marian; Weber, Cornelia; Platzman, Ilia; Goepfrich, Kerstin; Spatz, Joachim P.Advanced Biosystems (2020), 4 (9), 2000102CODEN: ABDIHL; ISSN:2366-7478. (Wiley-VCH Verlag GmbH & Co. KGaA)External control and precise manipulation is key for the bottom-up engineering of complex synthetic cells. Minimal actomyosin networks have been reconstituted into synthetic cells; however, their light-triggered symmetry breaking contraction has not yet been demonstrated. Here, light-activated directional contractility of a minimal synthetic actomyosin network inside microfluidic cell-sized compartments is engineered. Actin filaments, heavy-meromyosin-coated beads, and caged ATP are co-encapsulated into water-in-oil droplets. ATP is released upon illumination, leading to a myosin-generated force which results in a motion of the beads along the filaments and hence a contraction of the network. Symmetry breaking is achieved using DNA nanotechnol. to establish a link between the network and the compartment periphery. It is demonstrated that the DNA-linked actin filaments contract to one side of the compartment forming actin asters and quantify the dynamics of this process. This work exemplifies that an engineering approach to bottom-up synthetic biol., combining biol. and artificial elements, can circumvent challenges related to active multi-component systems and thereby greatly enrich the complexity of synthetic cellular systems.
- 9Juniper, M. P. N.; Weiss, M.; Platzman, I.; Spatz, J. P.; Surrey, T. Spherical network contraction forms microtubule asters in confinement. Soft Matter 2018, 14, 901– 909, DOI: 10.1039/C7SM01718A9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsFKrtrk%253D&md5=252eacca88618134515a78215f563d4fSpherical network contraction forms microtubule asters in confinementJuniper, Michael P. N.; Weiss, Marian; Platzman, Ilia; Spatz, Joachim P.; Surrey, ThomasSoft Matter (2018), 14 (6), 901-909CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)Microtubules and motor proteins form active filament networks that are crit. for a variety of functions in living cells. Network topol. and dynamics are the result of a self-organization process that takes place within the boundaries of the cell. Previous biochem. in vitro studies with biomimetic systems consisting of purified motors and microtubules have demonstrated that confinement has an important effect on the outcome of the self-organization process. However, the pathway of motor/microtubule self-organization under confinement and its effects on network morphol. are still poorly understood. Here, we have investigated how minus-end directed microtubule crosslinking kinesins organize microtubules inside polymer-stabilized microfluidic droplets of well-controlled size. We find that confinement can impose a novel pathway of microtubule aster formation proceeding via the constriction of an initially spherical motor/microtubule network. This mechanism illustrates the close relationship between confinement, network contraction, and aster formation. The spherical constriction pathway robustly produces single, well-centered asters with remarkable reproducibility across thousands of droplets. These results show that the addnl. constraint of well-defined confinement can improve the robustness of active network self-organization, providing insight into the design principles of self-organizing active networks in micro-scale confinement.
- 10Litschel, T.; Kelley, C. F.; Holz, D.; Koudehi, M. A.; Vogel, S. K.; Burbaum, L.; Mizuno, N.; Vavylonis, D.; Schwille, P. Reconstitution of contractile actomyosin rings in vesicles. Nat. Commun. 2021, 12, 2254, DOI: 10.1038/s41467-021-22422-710https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXptFOqsL8%253D&md5=5f2aaa315f2c39163e5b7e59c7b2aacbReconstitution of contractile actomyosin rings in vesiclesLitschel, Thomas; Kelley, Charlotte F.; Holz, Danielle; Adeli Koudehi, Maral; Vogel, Sven K.; Burbaum, Laura; Mizuno, Naoko; Vavylonis, Dimitrios; Schwille, PetraNature Communications (2021), 12 (1), 2254CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)One of the grand challenges of bottom-up synthetic biol. is the development of minimal machineries for cell division. The mech. transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the mol. scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theor. modeling. By changing few key parameters, actin polymn. can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theor. considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.
- 11Persson, M.; Gullberg, M.; Tolf, C.; Lindberg, A. M.; Månsson, A.; Kocer, A. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments. PLoS One 2013, 8, e55931, DOI: 10.1371/journal.pone.005593111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs12gtbk%253D&md5=b8a57799780799ae6525badd40dfe2aaTransportation of nanoscale cargoes by myosin propelled actin filamentsPersson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Maansson, Alf; Kocer, ArmaganPLoS One (2013), 8 (2), e55931CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addn., actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different no. of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 μm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing no. of side-attached cargoes, a redn. that occurred within a range of 10-50 streptavidin mols., 1-10 quantum dots or with just 1 liposome. However, as obsd. by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomech. energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in mol. diagnostics and other nanotechnol. applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.
- 12Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Light-Controlled Molecular Shuttles Made from Motor Proteins Carrying Cargo on Engineered Surfaces. Nano Lett. 2001, 1, 235– 239, DOI: 10.1021/nl015521e12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXivVyhs7c%253D&md5=75d0eff58da6d35936c82e3a4b27ee89Light-Controlled Molecular Shuttles Made from Motor Proteins Carrying Cargo on Engineered SurfacesHess, Henry; Clemmens, John; Qin, Dong; Howard, Jonathon; Vogel, ViolaNano Letters (2001), 1 (5), 235-239CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Mol. shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymic ATP degrdn. by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize mol. motors for the construction of nanoscale assembly lines.
- 13Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Control of actin moving trajectory by patterned poly(methylmethacrylate) tracks. Biophys. J. 1997, 72, 1997– 2001, DOI: 10.1016/S0006-3495(97)78844-113https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivVOjs7k%253D&md5=2b2e24c854f3580d2049f7d5671f98f8Control of actin moving trajectory by patterned poly(methylmethacrylate) tracksSuzuki, Hitoshi; Yamada, Akira; Oiwa, Kazuhiro; Nakayama, Haruto; Mashiko, ShinroBiophysical Journal (1997), 72 (5), 1997-2001CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)Poly(methylmethacrylate) (PMMA), a photoresist polymer, was found to be useful for immobilizing heavy meromyosin (HMM) mols. while retaining their abilities to support the movement of actin filaments. PMMA substrate was spin-coated on a coverslip, and various shapes of PMMA tracks, such as straight lines, concentric circles, and alphabetical letters, were fabricated by UV photolithog. An observation by a Tapping mode at. force microscope (AFM) shows that the typical circular tracks were 1-2 μm wide and about 200 nm high. In in vitro motility assay, a soln. of HMM mols. was applied to immobilize the mols. on the tracks by adsorption, and movement of actin filaments labeled with tetramethylrhodamine-phalloidin were obsd. in the presence of ATP by using an epifluorescence microscope and an image-intensified CCD camera. Actin filaments were seen to move precisely only on the PMMA tracks, and their traces drew the exact shapes of the tracks. The mean velocity of actin movement on the PMMA was 4.5 mm/s at 25°C, and it was comparable to that on a conventionally used nitrocellulose film.
- 14Herold, C.; Leduc, C.; Stock, R.; Diez, S.; Schwille, P. Long-Range Transport of Giant Vesicles along Microtubule Networks. ChemPhysChem 2012, 13, 1001– 1006, DOI: 10.1002/cphc.20110066914https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1yrtr%252FJ&md5=70ee88c812c31839aa44bf9e83f4f717Long-Range Transport of Giant Vesicles along Microtubule NetworksHerold, Christoph; Leduc, Cecile; Stock, Robert; Diez, Stefan; Schwille, PetraChemPhysChem (2012), 13 (4), 1001-1006CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diams. of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, the authors were able to est. the no. of GFP-labeled motors involved in the transport of a single vesicle. The vesicles were transported by the cooperative activity of typically 5-10 motor mols. The presented assay is expected to open up further applications in the field of synthetic biol., aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnol., where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.
- 15Diez, S.; Reuther, C.; Dinu, C.; Seidel, R.; Mertig, M.; Pompe, W.; Howard, J. Stretching and Transporting DNA Molecules Using Motor Proteins. Nano Lett. 2003, 3, 1251– 1254, DOI: 10.1021/nl034504h15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmsFOitrs%253D&md5=ad2aa1cc0e797351aecff8ce163857b4Stretching and transporting DNA molecules using motor proteinsDiez, Stefan; Reuther, Cordula; Dinu, Cerasela; Seidel, Ralf; Mertig, Michael; Pompe, Wolfgang; Howard, JonathonNano Letters (2003), 3 (9), 1251-1254CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Inside cells, motor proteins perform a variety of complex tasks including the transport of vesicles and the sepn. of chromosomes. The authors demonstrate a novel use of such biol. machines for the mech. manipulation of nanostructures in a cell-free environment. Specifically, the authors show that purified kinesin motors in combination with chem. modified microtubules can transport and stretch individual λ-phage DNA mols. across a surface. This technique, in contrast to existing ones, enables the parallel yet individual manipulation of many mols. and may offer an efficient mechanism for assembling multidimensional DNA structures.
- 16Cha, T.-G.; Pan, J.; Chen, H.; Salgado, J.; Li, X.; Mao, C.; Choi, J. H. A synthetic DNA motor that transports nanoparticles along carbon nanotubes. Nat. Nanotechnol. 2014, 9, 39– 43, DOI: 10.1038/nnano.2013.25716https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvV2hsLvO&md5=2c36fd998e86a3dd62090e64d57cd7b1A synthetic DNA motor that transports nanoparticles along carbon nanotubesCha, Tae-Gon; Pan, Jing; Chen, Haorong; Salgado, Janette; Li, Xiang; Mao, Chengde; Choi, Jong HyunNature Nanotechnology (2014), 9 (1), 39-43CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Intracellular protein motors have evolved to perform specific tasks crit. to the function of cells such as intracellular trafficking and cell division. Kinesin and dynein motors, for example, transport cargoes in living cells by walking along microtubules powered by ATP hydrolysis. These motors can make discrete 8 nm center-of-mass steps and can travel over 1 μm by changing their conformations during the course of ATP binding, hydrolysis and product release. Inspired by such biol. machines, synthetic analogs have been developed including self-assembled DNA walkers that can make stepwise movements on RNA/DNA substrates or can function as programmable assembly lines. Here, we show that motors based on RNA-cleaving DNA enzymes can transport nanoparticle cargoes-CdS nanocrystals in this case-along single-walled carbon nanotubes. Our motors ext. chem. energy from RNA mols. decorated on the nanotubes and use that energy to fuel autonomous, processive walking through a series of conformational changes along the one-dimensional track. The walking is controllable and adapts to changes in the local environment, which allows us to remotely direct 'go' and 'stop' actions. The translocation of individual motors can be visualized in real time using the visible fluorescence of the cargo nanoparticle and the near-infared emission of the carbon-nanotube track. We obsd. unidirectional movements of the mol. motors over 3 μm with a translocation velocity on the order of 1 nm min-1 under our exptl. conditions.
- 17Wickham, S. F. J.; Bath, J.; Katsuda, Y.; Endo, M.; Hidaka, K.; Sugiyama, H.; Turberfield, A. J. A DNA-based molecular motor that can navigate a network of tracks. Nat. Nanotechnol. 2012, 7, 169– 173, DOI: 10.1038/nnano.2011.25317https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqu74%253D&md5=27698363e23db94f4ad32d96b4e501b2A DNA-based molecular motor that can navigate a network of tracksWickham, Shelley F. J.; Bath, Jonathan; Katsuda, Yousuke; Endo, Masayuki; Hidaka, Kumi; Sugiyama, Hiroshi; Turberfield, Andrew J.Nature Nanotechnology (2012), 7 (3), 169-173CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)Synthetic mol. motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been obsd. on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks contg. four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, mol. systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands.
- 18Ketterer, P.; Willner, E. M.; Dietz, H. Nanoscale rotary apparatus formed from tight-fitting 3D DNA components. Science Advances 2016, 2, e1501209, DOI: 10.1126/sciadv.150120918https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlvVWku7w%253D&md5=b84b80d00436c4f1c95556819bc2f6ddNanoscale rotary apparatus formed from tight-fitting 3D DNA componentsKetterer, Philip; Willner, Elena M.; Dietz, HendrikScience Advances (2016), 2 (2), e1501209/1-e1501209/9CODEN: SACDAF; ISSN:2375-2548. (American Association for the Advancement of Science)We report a nanoscale rotary mechanism that reproduces some of the dynamic properties of biol. rotary motors in the absence of an energy source, such as random walks on a circle with dwells at docking sites. Our mechanism is built modularly from tight-fitting components that were self-assembled using multilayer DNA origami. The app. has greater structural complexity than previous mech. interlocked objects and features a well-defined angular degree of freedom without restricting the range of rotation. We studied the dynamics of our mechanism using single-particle expts. analogous to those performed previously with actin-labeled ATP synthases. In our mechanism, rotor mobility, the no. of docking sites, and the dwell times at these sites may be controlled through rational design. Our prototype thus realizes a working platform toward creating synthetic nanoscale rotary motors. Our methods will support creating other complex nanoscale mechanisms based on tightly fitting, sterically constrained, but mobile, DNA components.
- 19Urban, M. J.; Both, S.; Zhou, C.; Kuzyk, A.; Lindfors, K.; Weiss, T.; Liu, N. Gold nanocrystal-mediated sliding of doublet DNA origami filaments. Nat. Commun. 2018, 9, 1454, DOI: 10.1038/s41467-018-03882-w19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjisF2rsQ%253D%253D&md5=ee6d55cac42f27291d967f088661f025Gold nanocrystal-mediated sliding of doublet DNA origami filamentsUrban Maximilian J; Zhou Chao; Kuzyk Anton; Liu Na; Urban Maximilian J; Zhou Chao; Liu Na; Both Steffen; Weiss Thomas; Kuzyk Anton; Lindfors KlasNature communications (2018), 9 (1), 1454 ISSN:.Sliding is one of the fundamental mechanical movements in machinery. In macroscopic systems, double-rack pinion machines employ gears to slide two linear tracks along opposite directions. In microscopic systems, kinesin-5 proteins crosslink and slide apart antiparallel microtubules, promoting spindle bipolarity and elongation during mitosis. Here we demonstrate an artificial nanoscopic analog, in which gold nanocrystals can mediate coordinated sliding of two antiparallel DNA origami filaments powered by DNA fuels. Stepwise and reversible sliding along opposite directions is in situ monitored and confirmed using fluorescence spectroscopy. A theoretical model including different energy transfer mechanisms is developed to understand the observed fluorescence dynamics. We further show that such sliding can also take place in the presence of multiple DNA sidelocks that are introduced to inhibit the relative movements. Our work enriches the toolbox of DNA-based nanomachinery, taking one step further toward the vision of molecular nanofactories.
- 20Roux, A.; Cappello, G.; Cartaud, J.; Prost, J.; Goud, B.; Bassereau, P. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5394– 5399, DOI: 10.1073/pnas.08210729920https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKlsLs%253D&md5=c43c4d4592dc899963d857aae7f22ad8A minimal system allowing tubulation with molecular motors pulling on giant liposomesRoux, Aurelien; Cappello, Giovanni; Cartaud, Jean; Prost, Jacques; Goud, Bruno; Bassereau, PatriciaProceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5394-5399CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The elucidation of phys. and mol. mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that mol. motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin mols. have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was obsd. when kinesins were directly bound to the GUV membrane, suggesting that it is crit. to distribute the load on both lipids and motors by means of beads. A kinetic anal. shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled expts. aimed at the understanding of basic mechanisms in intracellular transport.
- 21Leduc, C.; Campas, O.; Zeldovich, K. B.; Roux, A.; Jolimaitre, P.; Bourel-Bonnet, L.; Goud, B.; Joanny, J.-F.; Bassereau, P.; Prost, J. Cooperative extraction of membrane nanotubes by molecular motors. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17096– 17101, DOI: 10.1073/pnas.040659810121https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFagu7nL&md5=2e1e9570ab476c6f81f67cb179a0db94Cooperative extraction of membrane nanotubes by molecular motorsLeduc, Cecile; Campas, Otger; Zeldovich, Konstantin B.; Roux, Aurelien; Jolimaitre, Pascale; Bourel-Bonnet, Line; Goud, Bruno; Joanny, Jean-Francois; Bassereau, Patricia; Prost, JacquesProceedings of the National Academy of Sciences of the United States of America (2004), 101 (49), 17096-17101CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)In eukaryotic cells, nanotubes represent a substantial fraction of transport intermediates between organelles. They are extd. from membranes by mol. motors walking along microtubules. We previously showed that kinesins fixed on giant unilamellar vesicles in contact with microtubules are sufficient to form nanotubes in vitro. Motors were attached to the membrane through beads, thus facilitating cooperative effects. Koster et al. [Koster, G., VanDuijn, M., Hofs, B. & Dogterom, M. (2003) Proc. Natl. Acad. Sci. USA 100, 15583-15588] proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. We demonstrate, in a recently designed exptl. system, the existence of an accumulation of motors allowing tube extn. We det. the motor d. along a tube by using fluorescence intensity measurements. We also perform a theor. anal. describing the dynamics of motors and tube growth. The only adjustable parameter is the motor binding rate onto microtubules, which we measure to be 4.7 ± 2.4 s-1. In addn., we quant. det., for a given membrane tension, the existence of a threshold in motor d. on the vesicle above which nanotubes can be formed. We find that the no. of motors pulling a tube can range from four at threshold to a few tens away from it. The threshold in motor d. (or in membrane tension at const. motor d.) could be important for the understanding of membrane traffic regulation in cells.
- 22Campillo, C.; Sens, P.; Köster, D.; Pontani, L.-L.; Lévy, D.; Bassereau, P.; Nassoy, P.; Sykes, C. Unexpected Membrane Dynamics Unveiled by Membrane Nanotube Extrusion. Biophys. J. 2013, 104, 1248– 1256, DOI: 10.1016/j.bpj.2013.01.05122https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXktlOgtbc%253D&md5=a668a303d051288b75b509824dc3406aUnexpected Membrane Dynamics Unveiled by Membrane Nanotube ExtrusionCampillo, Clement; Sens, Pierre; Koster, Darius; Pontani, Lea-Laetitia; Levy, Daniel; Bassereau, Patricia; Nassoy, Pierre; Sykes, CecileBiophysical Journal (2013), 104 (6), 1248-1256CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)In cell mechanics, distinguishing the resp. roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular and pure lipid membranes is usually attributed to the presence of the cytoskeleton as explored by membrane nanotube extrusion. Here we revisit this prevalent picture by unveiling unexpected force responses of plasma membrane spheres devoid of cytoskeleton and synthetic liposomes. We show that a tiny variation in the content of synthetic membranes does not affect their static mech. properties, but is enough to reproduce the dynamic behavior of their cellular counterparts. This effect is attributed to an amplified intramembrane friction. Reconstituted actin cortices inside liposomes induce an addnl., but not dominant, contribution to the effective membrane friction. Our work underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheol. in addn. to the role of the cytoskeleton.
- 23Rustom, A.; Saffrich, R.; Markovic, I.; Walther, P.; Gerdes, H.-H. Nanotubular Highways for Intercellular Organelle Transport. Science 2004, 303, 1007– 1010, DOI: 10.1126/science.109313323https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtlWnu7w%253D&md5=e5bf1883daee6fc0f07f00afeff004e0Nanotubular Highways for Intercellular Organelle TransportRustom, Amin; Saffrich, Rainer; Markovic, Ivanka; Walther, Paul; Gerdes, Hans-HermannScience (Washington, DC, United States) (2004), 303 (5660), 1007-1010CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chem. synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small mols. Accordingly, we propose a novel biol. principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.
- 24Kron, S. J.; Spudich, J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 6272– 6276, DOI: 10.1073/pnas.83.17.627224https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XlvVGqurc%253D&md5=ac4102efdaa2d11d67e8a60763dab2a9Fluorescent actin filaments move on myosin fixed to a glass surfaceKron, Stephen J.; Spudich, James A.Proceedings of the National Academy of Sciences of the United States of America (1986), 83 (17), 6272-6CODEN: PNASA6; ISSN:0027-8424.Single actin filaments stabilized with fluorescent phalloidin exhibit ATP-dependent movement on myosin filaments fixed to a surface. At pH 7.4 and 24°, the rates of movement av. 3-4 μm/s with skeletal muscle myosin and 1-2 μm/s with Dictyostelium myosin. These rates are very similar to those measured in previous myosin movement assays. The rates of movement are relatively independent of the type of actin used. The filament velocity shows a broad pH optimum of 7.0-9.0, and the concn. of ATP required for half-maximal velocity is 50 μM. Apparently, movement of actin over myosin requires at most the no. of heads in a single thick filament. This system provides a practical, quant. myosin-movement assay with purified proteins.
- 25Iwase, T.; Sasaki, Y.; Hatori, K. Alignment of actin filament streams driven by myosin motors in crowded environments. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861, 2717– 2725, DOI: 10.1016/j.bbagen.2017.07.016There is no corresponding record for this reference.
- 26Das, T.; Safferling, K.; Rausch, S.; Grabe, N.; Boehm, H.; Spatz, J. P. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat. Cell Biol. 2015, 17, 276– 287, DOI: 10.1038/ncb311526https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFemtrg%253D&md5=7b9531bf4c004cbe23c5051e52b8ba0cA molecular mechanotransduction pathway regulates collective migration of epithelial cellsDas, Tamal; Safferling, Kai; Rausch, Sebastian; Grabe, Niels; Boehm, Heike; Spatz, Joachim P.Nature Cell Biology (2015), 17 (3), 276-287CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)Collective movement of epithelial cells drives essential multicellular organization during various fundamental physiol. processes encompassing embryonic morphogenesis, cancer and wound healing. Yet the mol. mechanism that ensures the coordinated movement of many cells remains elusive. Here we show that a tumor suppressor protein, merlin, coordinates collective migration of tens of cells, by acting as a mechanochem. transducer. In a stationary epithelial monolayer and also in three-dimensional human skin, merlin localizes to cortical cell-cell junctions. During migration initiation, a fraction of cortical merlin relocalizes to the cytoplasm. This relocalization is triggered by the intercellular pulling force of the leading cell and depends on the actomyosin-based cell contractility. Then in migrating cells, taking its cue from the intercellular pulling forces, which show long-distance ordering, merlin coordinates polarized Rac1 activation and lamellipodium formation on the multicellular length scale. Together, these results provide a distinct mol. mechanism linking intercellular forces to collective cell movements in migrating epithelia.
- 27Schaller, V.; Weber, C.; Semmrich, C.; Frey, E.; Bausch, A. R. Polar patterns of driven filaments. Nature 2010, 467, 73– 77, DOI: 10.1038/nature0931227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFaqtbbM&md5=961fbd27f738fc9be43702dc4e49e206Polar patterns of driven filamentsSchaller, Volker; Weber, Christoph; Semmrich, Christine; Frey, Erwin; Bausch, Andreas R.Nature (London, United Kingdom) (2010), 467 (7311), 73-77CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The emergence of collective motion exhibited by systems ranging from flocks of animals to self-propelled microorganisms to the cytoskeleton is a ubiquitous and fascinating self-organization phenomenon. Similarities between these systems, such as the inherent polarity of the constituents, a d.-dependent transition to ordered phases or the existence of very large d. fluctuations, suggest universal principles underlying pattern formation. This idea is followed by theor. models at all levels of description: micro- or mesoscopic models directly map local forces and interactions using only a few, preferably simple, interaction rules, and more macroscopic approaches in the hydrodynamic limit rely on the systems' generic symmetries. All these models characteristically have a broad parameter space with a manifold of possible patterns, most of which have not yet been exptl. verified. The complexity of interactions and the limited parameter control of existing exptl. systems are major obstacles to our understanding of the underlying ordering principles. Here we demonstrate the emergence of collective motion in a high-d. motility assay that consists of highly concd. actin filaments propelled by immobilized mol. motors in a planar geometry. Above a crit. d., the filaments self-organize to form coherently moving structures with persistent d. modulations, such as clusters, swirls and interconnected bands. These polar nematic structures are long lived and can span length scales orders of magnitudes larger than their constituents. Our exptl. approach, which offers control of all relevant system parameters, complemented by agent-based simulations, allows backtracking of the assembly and disassembly pathways to the underlying local interactions. We identify weak and local alignment interactions to be essential for the obsd. formation of patterns and their dynamics. The presented minimal polar-pattern-forming system may thus provide new insight into emerging order in the broad class of active fluids and self-propelled particles.
- 28Homsher, E.; Wang, F.; Sellers, J. R. Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin. American Journal of Physiology-Cell Physiology 1992, 262, C714– C723, DOI: 10.1152/ajpcell.1992.262.3.C714There is no corresponding record for this reference.
- 29Salhotra, A.; Zhu, J.; Surendiran, P.; Meinecke, C. R.; Lyttleton, R.; Ušaj, M.; Lindberg, F. W.; Norrby, M.; Linke, H.; Månsson, A. Prolonged function and optimization of actomyosin motility for upscaled network-based biocomputation. New J. Phys. 2021, 23, 085005, DOI: 10.1088/1367-2630/ac180929https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVaktbfP&md5=2e180bcf4cdb72184589dee348057dcaProlonged function and optimization of actomyosin motility for upscaled network-based biocomputationSalhotra, Aseem; Zhu, Jingyuan; Surendiran, Pradheebha; Meinecke, Christoph Robert; Lyttleton, Roman; Usaj, Marko; Lindberg, Frida W.; Norrby, Marlene; Linke, Heiner; Maansson, AlfNew Journal of Physics (2021), 23 (Aug.), 085005CODEN: NJOPFM; ISSN:1367-2630. (IOP Publishing Ltd.)Significant advancements have been made towards exploitation of naturally available mol. motors and their assocd. cytoskeletal filaments in nanotechnol. applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biol. system. Here, we systematically investigated potentially crit. factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay soln. compn. and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addn., we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chem. oxygen scavenger system (glucose oxidase-glucose-catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted com. available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at -80°C for more than 10 years before use for nanotechnol. purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.
- 30Roy, D.; Steinkühler, J.; Zhao, Z.; Lipowsky, R.; Dimova, R. Mechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced Nanotubes. Nano Lett. 2020, 20, 3185– 3191, DOI: 10.1021/acs.nanolett.9b0523230https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslehtLk%253D&md5=1b80c36662eb76a13a99e3704d75caaeMechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced NanotubesRoy, Debjit; Steinkuehler, Jan; Zhao, Ziliang; Lipowsky, Reinhard; Dimova, RumianaNano Letters (2020), 20 (5), 3185-3191CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Membrane tension modulates the morphol. of plasma-membrane tubular protrusions in cells but is difficult to measure. Here, the authors propose to use microscopy imaging to assess the membrane tension. The authors report direct measurement of membrane nanotube diams. with unprecedented resoln. using stimulated emission depletion (STED) microscopy. For this purpose, the authors integrated an optical tweezers setup in a com. microscope equipped for STED imaging and established micropipette aspiration of giant vesicles. Membrane nanotubes were pulled from the vesicles at specific membrane tension imposed by the aspiration pipet. Tube diams. calcd. from the applied tension using the membrane curvature elasticity model are in excellent agreement with data measured directly with STED. The approach can be extended to cellular membranes and will then allow us to est. the mech. membrane tension within the force-induced nanotubes.
- 31Schroer, C. F. E.; Baldauf, L.; van Buren, L.; Wassenaar, T. A.; Melo, M. N.; Koenderink, G. H.; Marrink, S. J. Charge-dependent interactions of monomeric and filamentous actin with lipid bilayers. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 5861– 5872, DOI: 10.1073/pnas.191488411731https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Wlu7g%253D&md5=0eedf86d33cf3a45a5b7af0cc7044c78Charge-dependent interactions of monomeric and filamentous actin with lipid bilayersSchroer, Carsten F. E.; Baldauf, Lucia; van Buren, Lennard; Wassenaar, Tsjerk A.; Melo, Manuel N.; Koenderink, Gijsje H.; Marrink, Siewert J.Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (11), 5861-5872CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The cytoskeletal protein actin polymerizes into filaments that are essential for the mech. stability of mammalian cells. In vitro expts. showed that direct interactions between actin filaments and lipid bilayers are possible and that the net charge of the bilayer as well as the presence of divalent ions in the buffer play an important role. In vivo, colocalization of actin filaments and divalent ions are suppressed, and cells rely on linker proteins to connect the plasma membrane to the actin network. Little is known, however, about why this is the case and what microscopic interactions are important. A deeper understanding is highly beneficial, first, to obtain understanding in the biol. design of cells and, second, as a possible basis for the building of artificial cortices for the stabilization of synthetic cells. Here, we report the results of coarse-grained mol. dynamics simulations of monomeric and filamentous actin in the vicinity of differently charged lipid bilayers. We observe that charges on the lipid head groups strongly det. the ability of actin to adsorb to the bilayer. The inclusion of divalent ions leads to a reversal of the binding affinity. Our in silico results are validated exptl. by reconstitution assays with actin on lipid bilayer membranes and provide a mol.-level understanding of the actin-membrane interaction.
- 32Schubert, P. J.; Dorkenwald, S.; Januszewski, M.; Jain, V.; Kornfeld, J. Learning cellular morphology with neural networks. Nat. Commun. 2019, 10, 2736, DOI: 10.1038/s41467-019-10836-332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M3psF2hug%253D%253D&md5=b749ceee569e0cb32de82ff20a23e158Learning cellular morphology with neural networksSchubert Philipp J; Dorkenwald Sven; Kornfeld Joergen; Januszewski Michal; Jain VirenNature communications (2019), 10 (1), 2736 ISSN:.Reconstruction and annotation of volume electron microscopy data sets of brain tissue is challenging but can reveal invaluable information about neuronal circuits. Significant progress has recently been made in automated neuron reconstruction as well as automated detection of synapses. However, methods for automating the morphological analysis of nanometer-resolution reconstructions are less established, despite the diversity of possible applications. Here, we introduce cellular morphology neural networks (CMNs), based on multi-view projections sampled from automatically reconstructed cellular fragments of arbitrary size and shape. Using unsupervised training, we infer morphology embeddings (Neuron2vec) of neuron reconstructions and train CMNs to identify glia cells in a supervised classification paradigm, which are then used to resolve neuron reconstruction errors. Finally, we demonstrate that CMNs can be used to identify subcellular compartments and the cell types of neuron reconstructions.
- 33Milo, R.; Jorgensen, P.; Moran, U.; Weber, G.; Springer, M. BioNumbers─the database of key numbers in molecular and cell biology. Nucleic Acids Res. 2010, 38, D750– D753, DOI: 10.1093/nar/gkp88933https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXktl2nsQ%253D%253D&md5=6c311e7fa440e5a22d6028907ca1e221BioNumbers-the database of key numbers in molecular and cell biologyMilo, Ron; Jorgensen, Paul; Moran, Uri; Weber, Griffin; Springer, MichaelNucleic Acids Research (2010), 38 (Database Iss), D750-D753CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)BioNumbers (http://www.bionumbers.hms.harvard.edu) is a database of key nos. in mol. and cell biol.-the quant. properties of biol. systems of interest to computational, systems and mol. cell biologists. Contents of the database range from cell sizes to metabolite concns., from reaction rates to generation times, from genome sizes to the no. of mitochondria in a cell. While always of importance to biologists, having nos. in hand is becoming increasingly crit. for experimenting, modeling, and analyzing biol. systems. BioNumbers was motivated by an appreciation of how long it can take to find even the simplest no. in the vast biol. literature. All nos. are taken directly from a literature source and that ref. is provided with the no. BioNumbers is designed to be highly searchable and queries can be performed by keywords or browsed by menus. BioNumbers is a collaborative community platform where registered users can add content and make comments on existing data. All new entries and commentary are curated to maintain high quality. Here we describe the database characteristics and implementation, demonstrate its use, and discuss future directions for its development.
- 34Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M. Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy. J. Phys. D: Appl. Phys. 2017, 50, 064002, DOI: 10.1088/1361-6463/aa52a134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFKqs7k%253D&md5=aacea4a00fe7f3031301ff15773fa536Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopyClausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.Journal of Physics D: Applied Physics (2017), 50 (6), 064002/1-064002/11CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphol., mechanics, and function. Measuring this distance has been a key challenge in cell biol. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resoln. is insufficient to quantify distances on the nanoscale. Using dual-color super-resoln. STED (stimulated emission-depletion) microscopy, we here overcome this challenge and accurately measure the d. distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asym. cortical actin d. distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the max. d. peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, resp. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a max. of 20 nm in others.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.1c04254.
Additional experimental details and control experiments including further quantification, confocal images and (Experimental Methods 1.1 to 1.18); Supporting Figures S1–S13 with an SDS-PAGE of the purified proteins, a confocal image of actin swirls, 3D-STED images of lipid nanotubes, quantification of the number of lipid nanotube branches pulled from GUVs, images showing unspecific interactions of actin and the GUV membrane, the number of lipid nanotubes pulled from GUVs with different lipid compositions, confocal images of dye permeation into lipid nanotubes of GUVs, the verification of the self-assembly of cholesterol-PEG into cell membranes, confocal images of the GUV and cell displacement over time, confocal images and quantification of cellular actin inside lipid nanotubes, confocal images of stained mitochondria and lysosomes after pulling of lipid nanotubes, and a confocal image of HaCaT cells after the pulling assay (PDF)
Video of time series of random actin filaments (MP4)
Video of time series of aligned actin filaments (MP4)
Video of time series of aligned actin filament patterns (MP4)
Video of time series of lipid nanotube dynamics after pulling from GUVs (MP4)
Video of time series of lipid nanotube pulling from Jurkat cells (MP4)
Video of displacement over time of a Jurkat cell during lipid nanotube pulling (MP4)
Video of displacement over time of a GUV during lipid nanotube pulling (MP4)
Video of 3D projection of Jurkat cells with random actin and biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with aligned actin and biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with random actin and no biotinylated cholesterol (MP4)
Video of 3D projection of Jurkat cells with aligned actin and no biotinylated cholesterol (MP4)
Video of actin filament dynamics during lipid nanotube pulling of a Jurkat cell (DOPE-Atto488) (MP4)
Video of actin filament dynamics during lipid nanotube pulling of a Jurkat cell (SiR-actin) (MP4)
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