In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5β1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.

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3270of assembly, where it binds to FN and colocalizes with clustered α5β1 integrin (Dzamba et al., 1994; WierzbickaPatynowski and Schwarzbauer, 2002). The existence of one essential FN-binding site enhances control of the assembly process since all interactions depend on accessibility to this single site. The fact that there are multiple partners for this site suggests that the alignment of FN dimers within fibrils varies depending on which partners are available for assembly domain binding. Variable alignment would place the other binding domains (for heparin, cells, collagen, etc.) into different molecular contexts and close to different 'near-neighbors' on adjacent dimers. In this way, dimer alignment would have a significant impact on fibril complexity. In most instances, FN assembly is initiated by integrins that recognize the RGD and synergy sequences. Surprisingly, the specific location of the cell-binding site within FN is not critical. Placement of repeats III 9-10 more N-terminal in place of III4-5 (Fig. 1) generated a recombinant FN that assembled normally (Sechler et al., 2001). An RGD-independent mechanism acts through binding of α4β1 integrin to the CS1 site † within the alternatively spliced V region near the C-terminus (Sechler et al., 2000). Clearly, the integrin-binding site does not need to be centrally located for initiation and propagation of FN fibril formation.At early stages of de novo assembly, FN fibrils are short and usually extend between adjacent cells or from the cell to nearby substrate (Fig. 2). These fibrils are soluble in buffers containing 2% deoxycholate detergent. As more FN accumulates at the cell surface, fibrils are gradually converted into a detergentinsoluble form, and a significant proportion of these exist as high-molecular-weight multimers (McKeown-Longo and Mosher, 1983). Insolubility and multimerization might involve intermolecular disulfide bonding catalyzed by the intrinsic protein disulfide isomerase activity of FN (Langenbach and Sottile, 1999) or might result from highly stable protein-protein interactions (Chen and Mosher, 1996). Partial unfolding of the III9 module of FN promotes formation of amyloid-like fibrils in vitro (Litvinovich et al., 1998); so perhaps a similar process of β-strand exchange contributes to the detergent insolubility of the FN matrix. Further investigation of this and other potential mechanisms is needed to decipher the process by which FN fibrils become insoluble. Fibronectin activation by conformational changeA key feature of the matrix assembly model is a conformational change that converts soluble FN into an activated dimer. In vitro manipulation of soluble FN has provided compelling evidence for conformational changes that take FN from a compact to an extended form. Changes in pH or ionic strength, addition of mild denaturants, as well as interactions with heparin or collagen fragments, can induce conformational changes as measured by a variety of biophysical, biochemical and microscopic approaches (Bushuev et al., 1985;Erickson an...

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.