Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Abstract

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

“…Our results suggest that integrins may (1) mediate cell adhesion in such a way as to control display of FN assembly sites, (2) interact with conformationally changed FN to enable fibril progression and (3) transduce force to the elongated and assembled FN to stretch the molecules and open individual type III modules, thus allowing FN-FN interactions that result in the SDS-non-dissociable complexes (Baneyx et al, 2002;Ohashi et al, 2002;Zhong et al, 1998). In agreement with these ideas, a FN construct lacking the RGD sequence was found to bind in "short stitches" on FN−/− cells cultured on collagen in 72-h assays (Sottile et al, 2000), indicating that binding to integrins via the RGD region is not essential for binding of full length FN to cell surfaces but is important for fibril progression.…”

“…Bound 70K co-localizes with preexisting FN fibrils (Chernousov et al, 1985), albeit incompletely (Zhang et al, 1994). 70K also binds to FN that is adsorbed to surfaces and subjected to tension (Zhong et al, 1998). Thus, the N-terminal region of FN has been proposed to mediate binding to stretched or otherwise conformationally altered FN molecules (Hocking et al, 1994(Hocking et al, , 1996WierzbickaPatynowski and Schwarzbauer, 2003;Zhong et al, 1998).…”

“…70K also binds to FN that is adsorbed to surfaces and subjected to tension (Zhong et al, 1998). Thus, the N-terminal region of FN has been proposed to mediate binding to stretched or otherwise conformationally altered FN molecules (Hocking et al, 1994(Hocking et al, , 1996WierzbickaPatynowski and Schwarzbauer, 2003;Zhong et al, 1998). This proposed interaction is incorporated into a model in which FN fibrillogenesis begins by binding of an integrin recognition sequence in FN, e.g., RGD in repeat III 10 , to an integrin, e.g., α 5β 1 (Mao and Schwarzbauer, 2005;Wierzbicka-Patynowski and Schwarzbauer, 2003).…”

Transforming growth factor β induces fibroblast fibrillin‐1 matrix formation

“…Type I collagen assembles passively into matrix by self-polymerization (32), although certain proteins, including other collagen types, fibromodulin, lumican, decorin, and tenascin, can modify this process (33)(34)(35). In contrast, fibronectin matrix assembly is an active process and requires several conditions: an intact intracellular cytoskeleton, an activated fibronectin-binding integrin receptor, and tension across the developing fibrillar structure (36,37). Investigators in our group showed previously that TGF␤ and PDGF stimulate fibronectin matrix assembly, possibly through changes in integrin receptor activity (29).…”

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

“…The conformations Fn assumes on synthetic surfaces, however, are rather distinct from those of native Fn in soluble and fibrillar forms (reviewed in Antia et al, 2006;Baugh and Vogel, 2004;Halter et al, 2005). Fn polymerization into fibers is thought to proceed as cryptic self-assembly sites are exposed by cell contractility (Baneyx and Vogel, 1999;Mao and Schwarzbauer, 2005;Ohashi et al, 1999;Zhong et al, 1998).…”