Assembly and degradation of extracellular matrices are dynamic processes that occur during wound healing, embryogenesis, and metastasis. Fibronectin circulates at a high concentration as a soluble dimeric molecule in plasma and various other body fluids, and exists in an insoluble, multimeric form in the fibrillar network in the extracellular matrix of connective tissue, granulation tissue, basement membranes, and many embryonic structures. This multimeric form of fibronectin is thought to be the primary functional form of the molecule, mediating various adhesive and migratory events associated with wound repair, cell migration, embryogenesis, and neovascularization. Therefore, understanding the fundamental process of how fibronectin is assembled into the extracellular matrix is essential to our knowledge of how cells interact with the matrix during these adhesive and migratory events. Transformed cells exhibit a variety of alterations in their interactions with the extracellular matrix, including reduced adherence, and reduced assembly of fibronectin- containing extracellular matrices. Altered deposition of fibronectin has also been associated with atherosclerosis and fibrosis. Thus, understanding how cells assemble fibronectin into the extracellular matrix will also yield insights into the importance of altered fibronectin matrix assembly to various pathological processes. The broad aim of this proposal is to elucidate the mechanisms involved in the polymerization and subsequent assembly of soluble fibronectin into the extracellular matrix. Deposition of fibronectin in the extracellular matrix is a cell-mediated process that is initiated by the binding of soluble fibronectin to specific sites on the cell surface. This initial binding of fibronectin to cell surfaces is mediated by the amino-terminal 70kDa region of fibronectin. Following the binding of soluble fibronectin to the cell surface, fibronectin-fibronectin interactions are involved in accumulation of fibronectin into disulfide-stabilized aggregates in the extracellular matrix. The goals of this project are to define the structural features of fibronectin that are important for fibronectin matrix assembly, and to determine how specific regions of fibronectin participate in the cell-mediated polymerization of fibronectin into the extracellular matrix. These studies will use variant recombinant fibronectin molecules to investigate which regions of fibronectin are required for matrix assembly, and what role these regions play in this process. Previous data indicate that the sequences necessary for de novo establishment of nucleation sites for matrix assembly are distinct from sequences important for polymerization of fibronectin into preformed matrices. The hypothesis to be tested in this grant is that the first type III module of fibronectin and the integrin- binding site in the 10th type III module of fibronectin are required for the de novo establishment of matrix assembly sites, and that once these sites are established, exogenous fibronectin lacking these sequences can assemble into matrix.
