Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly.
Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly.
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DOI：
10.1083/jcb.141.2.539
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发表时间：
1998-04-20
期刊：
影响因子：
--
通讯作者：
Burridge K
中科院分区：
文献类型：
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作者：
Zhong C;Chrzanowska-Wodnicka M;Brown J;Shaub A;Belkin AM;Burridge K
许多因素影响纤连蛋白组装成不溶性纤维状细胞外基质。先前的工作证明，血清中促进纤连蛋白组装的一种组分是溶血磷脂酸（Zhang，Q.，W.J. Jovovich，D.M. Peters，R.M. Albrecht和D.F.莫舍1994. 127：1447-1459）。在这里，我们表明，C3转移酶，低分子量的GTP结合蛋白Rho的抑制剂，阻断纤连蛋白和70 kD的NH 2-末端纤连蛋白片段的细胞的结合，并阻止组装成基质诱导的血清或溶血磷脂酸的纤连蛋白。显微注射重组，组成型活性Rho到静止的瑞士3 T3细胞促进纤连蛋白基质组装的注射细胞。研究Rho促进纤连蛋白聚合的机制，我们使用C3来确定是否涉及整合素活化。在C3降低纤连蛋白组装的条件下，我们仅检测到整合素活化状态的微小变化。然而，细胞收缩性的几种抑制剂，其作用方式不同，抑制细胞结合的纤连蛋白和70 kD的NH 2-末端纤连蛋白片段，减少纤连蛋白掺入脱氧胆酸盐不溶性基质，并防止纤连蛋白的组装成纤维细胞表面上。由于Rho刺激收缩性，这些结果表明Rho介导的收缩性促进纤连蛋白组装成纤维基质。收缩性可以增强纤连蛋白组装的一种机制是通过张力暴露被拉伸的纤连蛋白内的隐蔽自组装位点。探索这种可能性，我们已经发现了一种单克隆抗体，L 8，染色纤连蛋白基质差异取决于状态的细胞收缩。先前显示L 8抑制纤连蛋白基质组装（Chernousov，M.A.，A.I. Faerman，M.G. Frid，O.Y. Printseva和V.E.科特连斯基1987. FEBS（美联储）EUR.（Biochem. Soc.）Lett. 217：124-128）。当它被用来染色正常的文化，正在发展的紧张局势，它揭示了一个矩阵无法区分的多克隆抗纤连蛋白抗体。然而，当细胞的收缩性被C3抑制时，相对于多克隆抗体，L 8对纤连蛋白基质的染色减少。我们已经研究了在没有细胞的情况下机械拉伸纤连蛋白的后果。将30-35%的拉伸施加到固定的纤连蛋白诱导可溶性纤连蛋白、70-kD纤连蛋白片段和L 8单克隆抗体的结合。总之，这些结果提供了证据，纤连蛋白内的自组装位点暴露的张力。
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.