The long-term goal is to understand the in vivo functions of the fibronectin matrix. The fibronectin matrix plays important roles in development, wound healing and tumorigenesis. Although much work in the field has focused on fibronectin matrix assembly, the mechanisms of the assembly process are still poorly understood. Moreover, much of the work in the field has focused on the interaction of cells with dimeric fibronectin, rather than the more relevant fibrillar matrix form of the molecule. Recently the investigator was able to drive fibronectin to form disulfide crosslinked fibrils in vitro by treating it with a recombinant fragment of fibronectin that can bind to the intact molecule. The fibronectin fibrils produced in vitro are functionally distinct from dimeric fibronectin and are highly adhesive to cells, suggesting that cells can sense the difference between these two forms of fibronectin. The hypothesis is that fibronectin matrix produced in vitro mimics the natural fibronectin matrix, and as such serves as a model for fibronectin matrix assembly. The hypothesis will be tested in several ways. First, the effects of known inhibitors of in vivo matrix assembly will be tested on in vitro fibril formation. Second, they will test whether in vitro matrix assembly requires that fibronectin be a dimer, as is the case with in vivo matrix assembly. Third, to learn more about the mechanisms of in vitro matrix assembly, they will construct fibronectin-fibronectin binding site mutants of the fibril-inducing proteins and test their abilities to induce fibril formation in vitro. Fourth, these mutants will be recreated in intact fibronectin in order to test the effects of the mutations on in vitro matrix assembly. Fifth, because the in vitro matrix assembly system indicates that the 1st and 11th type III modules in fibronectin are important for fibril formation, recombinant fibronectin molecules will be produced that either certain or lack these modules in order to test their role in natural matrix assembly. The mechanisms by which cells can distinguish between dimeric and fibrillar fibronectin will be studied by determining; 1) the repertoire of integrins that recognize the enhanced adhesiveness of fibrillar fibronectin, and 2) the effects of dimeric fibronectin and fibrillar fibronectin on protein tyrosine phosphorylation.
