Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner
ABSTRACT We examined the effects of mechanical forces on actin polymerization at focal adhesions (FAs). Actin polymerization at FAs was assessed by introducing fluorescence-labeled actin molecules into permeabilized fibroblasts cultured on fibronectin. When cell contractility was inhibited by the myosin-II inhibitor blebbistatin, actin polymerization at FAs was diminished, whereas alpha(5)beta(1) integrin remained accumulated at FAs. This suggests that actin polymerization at FAs depends on mechanical forces. To examine the action of mechanical forces more directly, the blebbistatin-treated cells were subjected to a sustained uniaxial stretch, which induced actin polymerization at FAs. These results demonstrate the novel role of mechanical forces in inducing actin polymerization at FAs. To reveal the molecular mechanism underlying the force-induced actin polymerization at FAs, we examined the distribution of zyxin, a postulated actin-regulatory protein. Actin-polymerizing activity was strong at zyxin-rich FAs. Accumulation of zyxin at FAs was diminished by blebbistatin, whereas uniaxial stretching of the cells induced zyxin accumulation. Displacing endogenous zyxin from FAs by expressing the FA-targeting region of zyxin decreased the force-induced actin polymerization at FAs. These results suggest that zyxin is involved in mechanical-force-dependent facilitation of actin polymerization at FAs.
- SourceAvailable from: Keiko Kawauchi
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- "Because the effects of Y-27632 on cell behavior can vary depending on experimental conditions (Nakayama et al., 2005; Olivero and Furcht, 1993), we tested various concentrations (0–20 mM) of Y-27632. Consistent with previous reports describing the dissociation of adhesion complexes upon inhibition of actomyosin contraction (Balaban et al., 2001; Hirata et al., 2008), treatment with 10 mM or 20 mM Y-27632 disrupted the distinct assembly of focal adhesions and actin cytoskeletons (supplementary material Fig. S1A,B). However, the shape of the focal adhesions and their connection to actin cytoskeletons were retained at a lower concentration (2 mM or 5 mM) of Y-27632 (supplementary material Fig. S1A,B). "
ABSTRACT: Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix. Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas), a mechanosensor at focal adhesions, correlates with the actin retrograde flow, and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. FRAP experiments reveals that CasSD phosphorylation, as opposed to its association with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, stabilization of Src-Cas binding, as well as myosin II inhibition, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent correlation with the actin cytoskeleton.Development 06/2014; DOI:10.1242/jcs.143438 · 6.27 Impact Factor
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- "Myosin proteins make the actin filaments slide past each other and confer on the stress fiber the ability to generate contractile force (Pellegrin and Mellor, 2007); in fact, it is well-established that a cell's traction force development is associated with stress fiber formation (Chrzanowska-Wodnicka and Burridge, 1996; Ingber, 2003). Besides, the kinetics of stress fiber formation and disassembly (Pollard and Borisy, 2003) appears itself to be modulated by force: traction force promotes the binding of stress fiber proteins (Colombelli et al., 2009; Hirata et al., 2008), which in turn enhances acto-myosin contractile activity, establishing a " feed-forward " process for stress fibers growth. Stress fibers are anchored to the extra-cellular matrix (ECM) through focal adhesions (Chrzanowska-Wodnicka and Burridge, 1996; Geiger et al., 2001). "
ABSTRACT: In this communication, we propose a model to study the non-equilibrium process by which actin stress fibers develop force in contractile cells. The emphasis here is on the non-equilibrium thermodynamics, which is necessary to address the mechanics as well as the chemistry of dynamic cell contractility. In this setting, we are able to develop a framework that relates (a) the dynamics of force generation within the cell and (b) the cell's response to external stimuli to the chemical processes occurring within the cell, as well as to the mechanics of linkage between the stress fibers, focal adhesions and extracellular matrix.Biomechanics and Modeling in Mechanobiology 05/2014; 14(1). DOI:10.1007/s10237-014-0588-2 · 3.25 Impact Factor
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- "Changes in intracellular mechanical properties also affect zyxin localization. For example, reduction of the internal tensile force on SFs by myosin II inhibition diminished zyxin accumulation and actin polymerization at FAs (Hirata et al., 2008). Furthermore, local reduction of tension on SFs induced by myosin II or artificial manipulation stimulated the translocation of zyxin to the damaged sites on SFs (Smith et al., 2010; Colombelli et al., 2009). "
ABSTRACT: Cells recognize and respond to changes in intra- and extracellular mechanical conditions to maintain their mechanical homeostasis. Linear contractile bundles of actin filaments and myosin II known as stress fibres (SFs) mediate mechanical signals. Mechanical cues such as excessive stress driven by myosin II and/or external force may damage SFs and induce the local transient accumulation of SF-repair complexes (zyxin and VASP) at the damaged sites. Using an atomic force microscope mounted on a fluorescence microscope, we applied mechanical damage to cells expressing fluorescently tagged cytoskeletal proteins and recorded the subsequent mobilization of SF-repair complexes. We found that a LIM protein, paxillin, transiently accumulated at the damaged sites earlier than zyxin, while paxillin knockdown did not affect the kinetics of zyxin translocation. The C-terminal half of paxillin, comprising four-tandem LIM domains, can still translocate to damaged sites on SFs, suggesting that the LIM domain is essential for the mechanosensory function of paxillin. Our findings demonstrate a crucial role of the LIM domain in mechanosensing LIM proteins.07/2013; 2(7):667-74. DOI:10.1242/bio.20134531