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
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.
Available from: Thorsten Steinberg
- "Integrins can either be part of focal complexes located in the posterior region of a lamellipodia or in focal adhesions (FAs) , 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 (  , for review see  ). With respect to FAs, integrins involved in their formation belong to the ß3 and/or ß1 families , the latter also constituting fibrillar adhesions in 2D cell cultures . "
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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.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 06/2015; DOI:10.1016/j.bbamcr.2015.06.004 · 5.02 Impact Factor
Available from: Keiko Kawauchi
- "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.
Development 06/2014; DOI:10.1242/jcs.143438 · 6.46 Impact Factor
Available from: Krishna Garikipati
- "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). "
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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 · 3.15 Impact Factor
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