Effects of substrate stiffness and actomyosin contractility on coupling between force transmission and vinculin–paxillin recruitment at single focal adhesions

Focal adhesions (FAs) regulate force transfer between the cytoskeleton and ECM–integrin complexes. We previously showed that vinculin regulates force transmission at FAs. Vinculin residence time in FAs correlated with applied force, supporting a mechanosensitive model in which forces stabilize vinculin’s active conformation to promote force transfer. In the present study, we examined the relationship between traction force and vinculin–paxillin localization to single FAs in the context of substrate stiffness and actomyosin contractility. We found that vinculin and paxillin FA area did not correlate with traction force magnitudes at single FAs, and this was consistent across different ECM stiffness and cytoskeletal tension states. However, vinculin residence time at FAs varied linearly with applied force for stiff substrates, and this was disrupted on soft substrates and after contractility inhibition. In contrast, paxillin residence time at FAs was independent of local applied force and substrate stiffness. Paxillin recruitment and residence time at FAs, however, were dependent on cytoskeletal contractility on lower substrate stiffness values. Finally, substrate stiffness and cytoskeletal contractility regulated whether vinculin and paxillin turnover dynamics are correlated to each other at single FAs. This analysis sheds new insights on the coupling among force, substrate stiffness, and FA dynamics.

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Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy

PLoS ONE ◽

10.1371/journal.pone.0251411 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0251411

Author(s):

Lorena Sigaut ◽

Micaela Bianchi ◽

Catalina von Bilderling ◽

Lía Isabel Pietrasanta

Keyword(s):

Extracellular Matrix ◽

Residence Time ◽

Focal Adhesion ◽

Focal Adhesions ◽

Traction Force ◽

Correlation Spectroscopy ◽

Substrate Stiffness ◽

Dissociation Kinetics ◽

Traction Forces ◽

Cellular Division

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.

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TRIP6 is required for tension at adherens junctions

Journal of Cell Science ◽

10.1242/jcs.247866 ◽

2021 ◽

Vol 134 (6) ◽

pp. jcs247866

Author(s):

Srividya Venkatramanan ◽

Consuelo Ibar ◽

Kenneth D. Irvine

Keyword(s):

Adherens Junctions ◽

Hippo Pathway ◽

Focal Adhesions ◽

Stress Fibers ◽

Hippo Signaling ◽

Organ Growth ◽

Dependent Inhibition ◽

Cytoskeletal Tension ◽

The Hippo Pathway ◽

The Relationship

ABSTRACTHippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

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Actin flow-dependent and -independent force transmission through integrins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010292117 ◽

2020 ◽

Vol 117 (51) ◽

pp. 32413-32422

Author(s):

Tristan P. Driscoll ◽

Sang Joon Ahn ◽

Billy Huang ◽

Abhishek Kumar ◽

Martin A. Schwartz

Keyword(s):

Binding Site ◽

Focal Adhesion ◽

Protein Interactions ◽

Flow Speed ◽

Substrate Stiffness ◽

Actin Binding ◽

Force Transmission ◽

Force Transfer ◽

Independent Mechanism ◽

Actin Binding Site

Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the “focal adhesion clutch,” in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we measured force on talin together with actin flow speed. While force on talin in small lamellipodial adhesions correlated with actin flow, talin tension in large adhesions further from the cell edge was mainly flow-independent. Stiff substrates shifted force transfer toward the flow-independent mechanism. Flow-dependent force transfer required talin’s C-terminal actin binding site, ABS3, but not vinculin. Flow-independent force transfer initially required vinculin and at later times the central actin binding site, ABS2. Force transfer through integrins thus occurs not through a continuous clutch but through a series of discrete states mediated by distinct protein interactions, with their ratio modulated by substrate stiffness.

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Traction Forces During Cell Migration Predicted by the Multiphysics Model

Volume 8: Mechanics of Solids, Structures and Fluids; Vibration, Acoustics and Wave Propagation ◽

10.1115/imece2011-63843 ◽

2011 ◽

Author(s):

Sangyoon J. Han ◽

Nathan J. Sniadecki

Keyword(s):

Computational Models ◽

Temporal Dynamics ◽

Focal Adhesions ◽

Traction Force ◽

Leading Edge ◽

Traction Forces ◽

Spatial And Temporal Dynamics ◽

Trailing Edges ◽

Cytoskeletal Tension ◽

Biochemical Activation

Cells rely on traction forces in order to crawl across a substrate. These traction forces come from dynamic changes in focal adhesions, cytoskeletal structures, and chemical and mechanical signals from the extracellular matrix. Several computational models have been developed that help explain the trajectory or accumulation of cells during migration, but little attention has been placed on traction forces during this process. Here, we investigated the spatial and temporal dynamics of traction forces by using a multiphysics model that describes the cycle of steps for a migrating cell on an array of posts. The migration cycle includes extension of the leading edge, formation of new adhesions at the front, contraction of the cytoskeleton, and the release of adhesions at the rear. In the model, an activation signal triggers the assembly of actin and myosin into a stress fiber, which generates a cytoskeletal tension in a manner similar to Hill’s muscle model. In addition, the role that adhesion dynamics has in regulating cytoskeletal tension has been added to the model. The multiphysics model was simulated in Matlab for 1-D simulations, and in Comsol for 2-D simulations. The model was able to predict the spatial distribution of traction forces observed with previous experiments in which large forces were seen at the leading and trailing edges. The large traction force at the trailing edge during the extension phase likely contributes to detachment of the focal adhesion by overcoming its adhesion strength with the post. Moreover, the model found that the mechanical work of a migrating cell underwent a cyclic relationship that rose with the formation of a new adhesion and fell with the release of an adhesion at its rear. We applied a third activation signal at the time of release and found it helped to maintain a more consistent level of work during migration. Therefore, the results from both our 1-D and 2-D migration simulations strongly suggest that cells use biochemical activation to supplement the loss in cytoskeletal tension upon adhesion release.

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Does cellular adaptation to force loading rate determine the biphasic vs monotonic response of actin retrograde flow with substrate rigidity?

10.1101/2021.04.23.441062 ◽

2021 ◽

Author(s):

Partho Sakha De ◽

Rumi De

Keyword(s):

Cancer Progression ◽

Loading Rate ◽

Focal Adhesions ◽

Traction Force ◽

Leading Edge ◽

Substrate Stiffness ◽

Retrograde Flow ◽

Force Loading ◽

Substrate Rigidity ◽

Cell Traction

AbstractThe transmission of cytoskeletal forces to the extracellular matrix through focal adhesion complexes is essential for a multitude of biological processes such as cell migration, differentiation, tissue development, cancer progression, among others. During migration, focal adhesions arrest the actin retrograde flow towards the cell interior, allowing the cell front to move forward. Here, we address a puzzling observation of the existence of two distinct phenomena: a biphasic relationship of the retrograde flow and cell traction force with increasing substrate rigidity, with maximum traction force and minimum retrograde flow velocity being present at an optimal substrate stiffness; in contrast, a monotonic relationship between them where the retrograde flow decreases and traction force increases with substrate stiffness. We propose a theoretical model for cell-matrix adhesions at the leading edge of a migrating cell, incorporating a novel approach in force loading rate sensitive binding and reinforcement of focal adhesions assembly and the subsequent force-induced slowing down of actin flow. Our model unravels both biphasic and monotonic responses of the retrograde flow and cell traction force with increasing substrate rigidity, owing to the cell’s ability to sense and adapt to the fast-growing forces. Moreover, we also elucidate how the viscoelastic properties of the substrate regulate these nonlinear responses and alter cellular behaviours.

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Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

10.1101/2021.08.15.456175 ◽

2021 ◽

Author(s):

Yukako Nishimura ◽

Shidong Shi ◽

Qingsen Li ◽

Alexander D. Bershadsky ◽

Virgile Viasnoff

Keyword(s):

Contractile Activity ◽

Myosin Ii ◽

Focal Adhesions ◽

Traction Force ◽

Stress Fiber ◽

Force Generation ◽

Stress Fibers ◽

Force Microscopy ◽

Fiber Dynamics ◽

The Relationship

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation.

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Cell cycle–dependent force transmission in cancer cells

Molecular Biology of the Cell ◽

10.1091/mbc.e17-12-0726 ◽

2018 ◽

Vol 29 (21) ◽

pp. 2528-2539 ◽

Cited By ~ 10

Author(s):

Magdalini Panagiotakopoulou ◽

Tobias Lendenmann ◽

Francesca Michela Pramotton ◽

Costanza Giampietro ◽

Georgios Stefopoulos ◽

...

Keyword(s):

Cell Cycle ◽

Cancer Cells ◽

Cell Cycle Progression ◽

Focal Adhesions ◽

Traction Force ◽

Periodic Variation ◽

Cycle Phase ◽

Force Transmission ◽

Functional Link ◽

Cycle Dependent

The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.

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The relationship between force and focal complex development

The Journal of Cell Biology ◽

10.1083/jcb.200204153 ◽

2002 ◽

Vol 159 (4) ◽

pp. 695-705 ◽

Cited By ~ 629

Author(s):

Catherine G. Galbraith ◽

Kenneth M. Yamada ◽

Michael P. Sheetz

Keyword(s):

Extracellular Matrix ◽

Complex Formation ◽

Focal Adhesions ◽

Mechanical Force ◽

Force Transmission ◽

Integrin Receptors ◽

Focal Complex ◽

Complex Development ◽

The Relationship

To adhere and migrate, cells must be capable of applying cytoskeletal force to the extracellular matrix (ECM) through integrin receptors. However, it is unclear if connections between integrins and the ECM are immediately capable of transducing cytoskeletal contraction into migration force, or whether engagement of force transmission requires maturation of the adhesion. Here, we show that initial integrin–ECM adhesions become capable of exerting migration force with the recruitment of vinculin, a marker for focal complexes, which are precursors of focal adhesions. We are able to induce the development of focal complexes by the application of mechanical force to fibronectin receptors from inside or outside the cell, and we are able to extend focal complex formation to vitronectin receptors by the removal of c-Src. These results indicate that cells use mechanical force as a signal to strengthen initial integrin–ECM adhesions into focal complexes and regulate the amount of migration force applied to individual adhesions at localized regions of the advancing lamella.

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Cell Response to Cyclic Strain at Focal Adhesions

Volume 3: Biomedical and Biotechnology Engineering ◽

10.1115/imece2016-66843 ◽

2016 ◽

Author(s):

Toshihiko Shiraishi ◽

Tomohiro Fukuno

Keyword(s):

Focal Adhesion ◽

Mechanical Vibration ◽

Focal Adhesions ◽

Traction Force ◽

Cyclic Strain ◽

Macromolecular Assemblies ◽

Migration Direction ◽

A Cell ◽

Sense And Respond ◽

The Relationship

Cells are known to sense and respond to mechanical stimulations. The fact shows that there are some cellular mechanosensors for mechanical stimulations. One of the candidates of the mechanosensors is focal adhesions which are large macromolecular assemblies via which mechanical force and regulatory signals may be transmitted between the extracellular matrix and an interacting cell. Although it is quite important to clarify the mechanism of sensing and responding to the mechanical vibration via focal adhesions, there was no micro device applying time-varied mechanical loading to a single focal adhesion of the order of a micrometer. In order to solve the challenging issue, we developed a magnetic micropillar substrate which is able to apply cyclic strain to focal adhesions of a cell. Using the substrate, we investigated how a single osteoblast-like cell changed the direction of migration on micropillars cyclically deflected at 5 Hz and revealed the relationship between the cell migration and the traction force. The experimental results indicate that a cell may sense the cyclic strain and reduce the traction force which is not enough to move the cell body forward leading to changing the migration direction toward the place without cyclic strain.

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Stick-Slip Dynamics of Migrating Cells on Viscoelastic Substrates

10.1101/712059 ◽

2019 ◽

Author(s):

Partho Sakha De ◽

Rumi De

Keyword(s):

Focal Adhesions ◽

Traction Force ◽

Reaction Diffusion ◽

Substrate Stiffness ◽

Retrograde Flow ◽

Stick Slip ◽

Cell Substrate ◽

Elastic Substrates ◽

Individual Bond ◽

Stick Slip Motion

Stick-slip motion, a common phenomenon observed during crawling of cells, is found to be strongly sensitive to the substrate stiffness. Stick-slip behaviours have previously been investigated typically using purely elastic substrates. For a more realistic understanding of this phenomenon, we propose a theoretical model to study the dynamics on a viscoelastic substrate. Our model based on a reaction-diffusion framework, incorporates known important interactions such as retrograde flow of actin, myosin contractility, force dependent assembly and disassembly of focal adhesions coupled with cell-substrate interaction. We show that consideration of a viscoelastic substrate not only captures the usually observed stick-slip jumps, but also predicts the existence of an optimal substrate viscosity corresponding to maximum traction force and minimum retrograde flow which was hitherto unexplored. Moreover, our theory predicts the time evolution of individual bond force that characterizes the stick-slip patterns on soft versus stiff substrates. Our analysis also elucidates how the duration of the stick-slip cycles are affected by various cellular parameters.

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