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.
Highlights
Adhesive interactions of a cell with neighboring cells and with extracellular matrices (ECMs) are essential for cellular morphogenesis, migration, proliferation and differentiation
Actin polymerization at zyxin-rich focal adhesions (FAs) Human skin fibroblasts grown on fibronectin (FN) developed many FAs, which contained α5 integrin (Fig. 1A)
The difference in the amount of actin that was incorporated in different regions did not seem to arise from the difference in accessibility of artificially introduced molecules to FAs depending on their location; when the mixture of Alexa568actin and anti-α5-integrin cytoplasmic-domain antibody was applied, the antibody was associated with FAs in both peripheral and central regions, whereas Alexa568-actin was incorporated preferentially at peripheral FAs
Summary
Adhesive interactions of a cell with neighboring cells and with extracellular matrices (ECMs) are essential for cellular morphogenesis, migration, proliferation and differentiation. Adherent cells, including fibroblasts, epithelial cells and endothelial cells, develop specialized sites for adhesive interactions with the ECM, called focal adhesions (FAs). Heterodimeric transmembrane receptors for ECM proteins, are clustered at FAs, where a variety of cytoplasmic proteins, including vinculin, talin and α-actinin, are accumulated. At a FA, integrins are linked to the actin cytoskeleton via a plaque of the cytoplasmic proteins, anchoring the actin cytoskeleton to the ECM (Burridge and Chrzanowska-Wodnicka, 1996; Geiger et al, 2001). Actomyosin-based contractile forces are transmitted from cells to the ECM at FAs (Harris et al, 1980; Chrzanowska-Wodnicka and Burridge, 1996; Balaban et al, 2001). Stimulation of contractility drives the development of FAs (ChrzanowskaWodnicka and Burridge, 1996). Inhibition of the contractile forces leads to the disassembly of FAs (Chrzanowska-Wodnicka and Burridge, 1996; Balaban et al, 2001). Mechanical forces are crucial for the regulation of FAs
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