Actin flow-dependent and -independent force transmission through integrins.
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ABSTRACT:
SUBMITTER: Driscoll TP
PROVIDER: S-EPMC7768777 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable β-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study elucidates a force transmission mechanism, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.
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Project description:When cells move using integrin-based focal adhesions, they pull in the direction of motion with large, ∼100 Pa, stresses that contract the substrate. Integrin-mediated adhesions, however, are not required for in vivo confined migration. During focal adhesion-free migration, the transmission of propelling forces, and their magnitude and orientation, are not understood. Here, we combine theory and experiments to investigate the forces involved in adhesion-free migration. Using a non-adherent blebbing cell line as a model, we show that actin cortex flows drive cell movement through nonspecific substrate friction. Strikingly, the forces propelling the cell forward are several orders of magnitude lower than during focal-adhesion-based motility. Moreover, the force distribution in adhesion-free migration is inverted: it acts to expand, rather than contract, the substrate in the direction of motion. This fundamentally different mode of force transmission may have implications for cell-cell and cell-substrate interactions during migration in vivo.
| S-EPMC6485532 | biostudies-literature
Project description:Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.
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Project description:Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.
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Project description:Full T cell activation requires coordination of signals from multiple receptor-ligand pairs that interact in parallel at a specialized cell-cell contact site termed the immunological synapse (IS). Signaling at the IS is intimately associated with actin dynamics; T cell receptor (TCR) engagement induces centripetal flow of the T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins, such as talin, vinculin, and paxillin, are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the IS.
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Project description:The generation of neurons by neural stem cells is a highly choreographed process that requires extensive and dynamic remodelling of the cytoskeleton at each step of the process. The atypical RhoGTPase Rnd3 is expressed by progenitors in the embryonic brain but its role in early steps of neurogenesis has not been addressed. Here we show that silencing Rnd3 in the embryonic cerebral cortex interferes with the interkinetic nuclear migration of radial glial stem cells, disrupts their apical attachment and modifies the orientation of their cleavage plane. These defects are rescued by co-expression of a constitutively active form of cofilin, demonstrating that Rnd3-mediated disassembly of actin filaments coordinates the cellular behaviour of radial glial. Rnd3 also limits the divisions of basal progenitors via a distinct mechanism involving the suppression of cyclin D1 translation. Interestingly, although Rnd3 expression is controlled transcriptionally by Ascl1, this proneural factor is itself required in radial glial progenitors only for proper orientation of cell divisions.
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Project description:Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.
| S-EPMC7384861 | biostudies-literature
Project description: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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Project description:In this study, we present a new technique called correlative atomic force and transmission electron microscopy (correlative AFM/TEM) in which a targeted region of a sample can be observed under AFM and TEM. The ultimate goal of developing this new technique is to provide a technical platform to expand the fields of AFM application to complex biological systems such as cell extracts. Recent advances in the time resolution of AFM have enabled detailed observation of the dynamic nature of biomolecules. However, specifying molecular species, by AFM alone, remains a challenge. Here, we demonstrate correlative AFM/TEM, using actin filaments as a test sample, and further show that immuno-electron microscopy (immuno-EM), to specify molecules, can be integrated into this technique. Therefore, it is now possible to specify molecules, captured under AFM, by subsequent observation using immuno-EM. In conclusion, correlative AFM/TEM can be a versatile method to investigate complex biological systems at the molecular level.
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Project description:Skeletal alpha-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5'-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/micrometer(2)) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca(2+)-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase.
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