Recent studies indicate that the extracellular matrix of the nervous system is influential in the control of neuronal development and regeneration. We propose to use defined culture substrates containing known molecular components of the matrix, primarily fibronectin (Fn) and glycosaminoglycans (GAGs), but including collagens and laminin, for controlled studies of the molecular and cellular interactions of growing nerve fibers with the extracellular matrix. Our initial studies showed that substrates prepared from 2-hydroxyethylmetherylate (HEMA) supported attachment of neurons but little nerve fiber growth. Incorporation of Fn or collagen, but few other proteins, into HEMA rendered it an excellent substrate for fiber growth. GAGs, profoundly inhibited nerve fiber growth on Fn substrates. First, we will prepare HEMA substrates incorporating proteolytic fragments that retain one or more of the functional domains of the multifunctional Fn molecule. With a variety of fragments we will evaluate which domains of Fn support nerve fiber growth and which mediate the inhibition produced by GAGs. In complementary studies with monoclonal antibodies (Mabs) to functional sites on Fn we will block sites on intact Fn to evaluate their contribution to fiber growth. In addition Mabs may prove valuable probes for detecting changes in the functional sites of Fn produced by GAGs. The second section of this proposal is aimed at describing which, of a complex series of cellular events, is stimulated by Fn and results in adhesion of neurons to substrates. Focusing on these relatively rapid events we will treat neurons with variety of pharmacological agents to help sort out the intracellular mechanisms stimulated by Fn to produce these changes. Finally, with antibodies to Fn, collagens (I, III, IV) laminin, heparan sulfate proteoglycan, and chondroitin sulfate proteoglycan we will probe the distribution of these molecules on DRG cells growing in culture as well as in developing and hatched chicks. We are especially interested in whether Fn or other matrix proteins collect at sites of adhesion of cells to each other or to their substrates and whether proteoglycans collect at sites where adhesions turnover or nerve fiber growth is restricted.
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