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J. Cell Biol.,
Volume 141, Number 2, April 20, 1998 539-551
Department of Cell Biology and Anatomy, and Lineberger Comprehensive Cancer Center, University of North Carolina,
Chapel Hill, North Carolina
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.
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