The goal of this project is to understand the role of extracellular matrix in the development of the peripheral nervous system. We have focused our studies on the basement membrane produced by Schwann cells (SCs) and its effects on SC terminal differentiation. Previous experiments have shown that 1) SCs are the major, if not sole, source of the structural components of this matrix; these include collagen (primarily types I and IV), laminin and heparan sulfate proteoglycan; 2) SC terminal differentiation (i.e., ensheathment and myelination) is dependent upon contact with this matrix; and 3) SC matrix assembly is controlled by nerve cells. The work proposed for the next grant period is aimed at elucidating the molecular mechanisms involved in regulating matrix production and the developmental effect of the matrix on SCs. As steps toward achieving these goals we have outlined the following objectives. 1) To identify and characterize the basement membrane and SC proteins which mediate the cell-matrix interaction; we will utilize an immunological approach to exploit the observation that simple, reconstituted basement membrane induces normal differentiation in cultured SCs; specific antibodies will be prepared against this matrix and SC membrane proteins and tested for their ability to block the SC spreading induced by contact with the matrix. 2) To charcterize the structure and functional properties of a SC plasma membrane proteoglycan that is anchored to the cytoskeleton; we will attempt to delineate the domain structure of this molecule, and to study, using in vitro binding assays, its mode of binding to basement membrane and the cytoskeleton; we will also at tempt to purify this molecule and produce specific antibodies. 3) To elucidate the mechanism of regulation by nerve cells of SC basement membrane formation; we will determine whether nerve cell contact or a soluble factor released by nerve cells mediate this phenomenon; we will also investigate whether nerve cells, which modulate the levels of specific SC matrix proteins, do this through changes in the rates of synthesis, degradation or utilization of these proteins. 4) To describe the basement membrane induced alterations in the SC cytoskeleton that accompany the morphological changes which are required for terminal differentiation; we will investigate changes in cytoskeletal protein metabolsim (rates of synthesis, degradation, post-translational modification, and polymerization state) by biochemical methods; structural alterations in cytoskeletal filaments will be studied by immunofluorescence with specific antibodies and electron microscopy.
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