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.

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Abstract. The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell-binding sequence (RGD-) or the first type III repeats (FNAIIII_7) including the IIIx FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FNAIIII_7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino-terminal 70 kD fragment of FN, showing that FNAIIII_7 follows an assembly pathway similar to FN. The progression of FNAIIII_7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD-recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the 1111_7 modules. The altered rate of FNAIIII_7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation. 1989;Hynes, 1990). From within the matrix, FN interacts with cells to control cell adhesion, cytoskeletal organization, and intracellular signaling. It also forms a structural framework for cell migration, differentiation, cell--cell interactions, and deposition of other matrix proteins. The importance of FN has been underscored by recent results with an FN-null mutation which result in embryonic lethality in mice (George et al., 1993). Moreover, while the loss of an FN matrix is characteristic of many tumorigenic cells, restoration of this matrix can suppress the transformed phenotype (Giancotti and Ruoslahti, 1990).Models for assembly of FN into a fibrillar matrix propose a stepwise process initiated by binding to cell surface receptors followed by assembly and reorganization into fibrils (for reviews see McDonald, 1988;Mosher et al., 1992;Mosher, 1993). As increasing amounts of FN bind to cells, dimeric FN is converted into a complex network of