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

ArticleinJournal of Cell Science 121(Pt 17):2795-804 · October 2008with33 Reads
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.
    • "Central FAs are connected by short actin stress fibers that go under the nucleus, whereas peripheral FAs are connected by long stress fibers that often go over the nucleus (Fig. S3A; Kim et al., 2012; Li et al., 2014 ). Previous studies found that FA localization of zyxin is tension dependent, as is phosphorylation of paxillin on tyrosines 31 and 118 (Wodnicka and Burridge, 1996; Lele et al., 2006; Hirata et al., 2008; Pasapera et al., 2010). Indeed, these markers showed lower levels in central FAs (Fig. S3, B–D), whereas total paxillin (Fig. S3 E)and integrin β1 were similar (Fig. S3, F and G). "
    [Show abstract] [Hide abstract] ABSTRACT: Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We find that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing.
    Full-text · Article · May 2016
    • "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]. "
    [Show abstract] [Hide abstract] 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.
    Article · Apr 2016
    • "The immunofluorescence staining was performed as described previously (Hirata et al., 2008). The samples were viewed with a confocal microscope (A1R, Nikon) equipped with oil-immersion (NA 1.40, 100×; Plan Apochromat VC, Nikon) and air (NA 0.95, 40×; Plan Apochromat, Nikon) objectives. "
    [Show abstract] [Hide abstract] ABSTRACT: Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Casin vitroin a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration.
    Full-text · Article · Mar 2016
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