Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner

Cell Mechanosensing Project, ICORP/SORST, Japan Science and Technology Agency, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan.
Journal of Cell Science (Impact Factor: 5.43). 10/2008; 121(Pt 17):2795-804. DOI: 10.1242/jcs.030320
Source: PubMed


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.

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    • "Myosin II activation is required for localisation of zyxin at force-bearing sites [54,57], whereas the three LIM domains of zyxin are necessary for its mechanosensitive function [54]. In addition, zyxin has been shown to allow actin polymerisation at adhesion sites [59] and to be required for SF maintenance and repair [60] . Zyxin also promotes VASP localisation at strain sites along the SFs, while interaction with VASP is required for zyxin to rescue SF repair defects in zyxin-null cells [60] and to induce SF thickening after cyclic stretch [61]. "
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    ABSTRACT: Cell and tissue stiffness have been known to contribute to both developmental and pathological signalling for some time, but the underlying mechanisms remain elusive. Integrins and their associated adhesion signalling complexes (IACs), which form a nexus between the cell cytoskeleton and the extracellular matrix, act as a key force sensing and transducing unit in cells. Accordingly, there has been much interest in obtaining a systems-level understanding of IAC composition. Proteomic approaches have revealed the complexity of IACs and identified a large number of components that are regulated by cytoskeletal force. Here we review the function of the consensus adhesome, an assembly of core IAC proteins that emerged from a meta-analysis of multiple proteomic datasets, in the context of mechanosensing. As IAC components have been linked to a variety of diseases involved with rigidity sensing, the field is now in a position to define the mechanosensing function of individual IAC proteins and elucidate their mechanisms of action.
    No preview · Article · Oct 2015 · Experimental Cell Research
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    • "Integrins can either be part of focal complexes located in the posterior region of a lamellipodia or in focal adhesions (FAs) [2], which are formed upon the suspension of advancement or retraction of the cell's leading edge. Here, ß3 integrin particularly in conjunction with zyxin has been reported during the transition from focal complexes to definitive FAs, also called mature or late stage FAs ([3] [4] [5], for review see [6] [7]). With respect to FAs, integrins involved in their formation belong to the ß3 and/or ß1 families [1], the latter also constituting fibrillar adhesions in 2D cell cultures [8]. "
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    ABSTRACT: Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jun 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
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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). "
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    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.
    Full-text · Article · Jun 2014 · Development
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