RHO-MEDIATED CONTRACTILITY EXPOSES A CRYPTIC SITE IN FIBRONECTIN AND INDUCES FIBRONECTIN MATRIX ASSEMBLY

Many factors influence the assembly of fibronectin into an insoluble f ibrillar extracellular matrix. Previous work demonstrated that one com ponent in serum that promotes the assembly of fibronectin is lysophosp hatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, a nd 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 p rotein Rho, blocks the binding of fibronectin and the 70-kD NH2-termin al fibronectin fragment to cells and blocks the assembly of fibronecti n into matrix induced by serum or lysophosphatidic acid. Microinjectio n of recombinant, constitutively active Rho into quiescent Swiss 3T3 c ells promotes fibronectin matrix assembly by the injected cells. Inves tigating the mechanism by which Rho promotes fibronectin polymerizatio n, we have used C3 to determine whether integrin activation is involve d. Under conditions where C3 decreases fibronectin assembly we have on ly detected small changes in the state of integrin activation. However , several inhibitors of cellular contractility, that differ in their m ode of action, inhibit cell binding of fibronectin and the 70-kD NH2-t erminal 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 assem bly of fibronectin into a fibrillar matrix. One mechanism by which con tractility could enhance fibronectin assembly is by tension exposing c ryptic 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 slate of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Prin tseva, and V.E. Koteliansky, 1987. FEES (Fed. Eur. Biochem. Sec.) Lett . 217:124-128). When it is used to stain normal cultures that are deve loping tension, it reveals a matrix indistinguishable from that reveal ed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by LS is reduced relative to the polyclonal anti body when the contractility of cells is inhibited by C3. We have inves tigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30-35 % stretch to immobilized fibronect in 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.