Focal adhesion size controls tension-dependent recruitment of α-smooth muscle actin to stress fibers

Abstract

Expression of α-smooth muscle actin (α-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify α-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8–30-μm-long “supermature” focal adhesions (suFAs), which exert a stress approximately fourfold higher (∼12 nN/μm2) on micropatterned deformable substrates than 2–6-μm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of ≤11 kPa and on rigid micropatterns of 6-μm-long classical FA islets confines α-SMA to the cytosol. Reincorporation of α-SMA into stress fibers is established by stretching 6-μm-long classical FAs to 8.1-μm-long suFA islets on extendable membranes; the same stretch producing 5.4-μm-long classical FAs from initially 4-μm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

“…The morphologies of the stamps used for patterning were shown in Fig. 1a-c. Pattern L20 (12,6) consisting of rectangular islets of size 20 · 2 mm was designed to induce elongated FA according to a previous report [18]. We optimized the vertical spacing by changing it from 6 to 12 mm to guide cell growth along the direction of a higher ECM density, that is, denser islet to form elongated FA.…”

“…However, there are limited studies using FA modulation to induce stem cell differentiation. Among these, Goffin et al employed micropatterning to modulate FA development in myofibroblasts and found that the FA length controls the transition of smooth muscle actin (SMA) into stress fibers in a tension-dependant manner [18]. Seo et al found that FA maturation and actin polymerization were promoted via the RhoA pathway when MSCs were cultured on microtopographical substrates [19].…”

“…To create contraction in the cytoskeleton for myogenic differentiation, it is well known that myosin lightchain kinase (MLCK) is needed to phosphorylate myosin light chain (MLC) and actomyosin II [21]. It has also been shown that elongated FA would increase the expression of MLCK and cellular tension [18]. Based on the literature and above cues, we hypothesize that by generating elongated FA in hMSCs cultured on compliant surfaces, myogenic differentiation could be induced.…”

Cellular contractility and substrate elasticity: a numerical investigation of the actin cytoskeleton and cell adhesion

“…Changes in substrate stiffness have been shown to significantly alter the actin cytoskeleton and focal adhesions (FAs) of a range of cell types, including fibroblasts, mesenchymal stem cells, endothelial cells, and chondrocytes (Goffin et al 2006;Engler et al 2006;Byfield et al 2009;Schuh et al 2010). Different ranges of substrate stiffness that influence cytoskeletal remodelling have been reported for different cell phenotypes; particularly, more contractile cells, such as myoblasts, are most sensitive up to ~400 kPa (Ren et al 2008), but less contractile cells, such as fibroblasts, are sensitive up to ~20 kPa (Yeung et al 2005).…”

“…Different ranges of substrate stiffness that influence cytoskeletal remodelling have been reported for different cell phenotypes; particularly, more contractile cells, such as myoblasts, are most sensitive up to ~400 kPa (Ren et al 2008), but less contractile cells, such as fibroblasts, are sensitive up to ~20 kPa (Yeung et al 2005). Previous in-vitro studies have quantified the effect of different substrate stiffness on stress fibre (SF) formation (Solon et al 2007), FA area (Goffin et al 2006), cell traction (Califano and Reinhart-King 2010), and cell shape (Yeung et al 2005). However, the cellular mechanisms underlying these phenomena are poorly understood.…”