The objective of these studies is to increase our understanding of how epidermal cells interact with the extracellular matrix (ECM) during wound closure. The behavior of adult newt (Notophthalmus viridescens) epidermal cells in wounded limbs will be analyzed as the cells migrate over the natural wound bed or over implants coated with various ECM proteins, fragments of ECM proteins, or synthetic peptides modeled after potentially important regions of ECM proteins. These studies will be supplemented by adhesion and spreading assays using single cell suspensions of freshly isolated epidermal cells seeded onto plastic coated with many of the same substrates as in implant experiments. More specifically: Novel Fibrinogen (FGN) fragments, cyanogen bromide (CB) peptides of the alpha chain, and synthetic peptides mimicking the RGD-containing regions of the alpha chain will be tested as migration substrates to better localize the migration-supporting active sites in FGN. The effects of pretreating FGN and FGN fragment-coated implants with monoclonal antibodies against known regions of the alpha chain and against the platelet binding site in the gamma chain will be determined. The ability of the previously mentioned synthetic peptides to act as competitive inhibitors of migration on FGN and FGN fragments will also be assessed. To better localize the active site(s) controlling adhesion, spreading, and migration on fibronectin (FN), recombinant DNA-generated FN fragments with known amino acid deletions in domains III and IV will be tested as migration substrates. Adhesion and spreading assays will be performed on these same fragments as well as on a heparin-binding fragment that contains most of the III CS region. To localize epidermal recognition site(s) in type IV collagen, adhesion, spreading, and migration assays will be conducted using as substrates: isolated type IV structural domains, isolated alpha chains, pepsin and CB-generated alpha chain fragments, and synthetic peptides modeled after certain regions of the alpha chains. To determine the epidermal interactive potential of laminin, entactin, and heparan sulfate proteoglycan, each protein will be tested as a substrate in adhesion, spreading, and migration assays. Freshly wounded limbs will be treated with polyclonal antibodies to collagen, FGN and FN to determine the relative involvement of each protein in wound closure in vivo. To characterize the ECM receptors on epidermal cells, polyclonal antibodies against the beta subunit of known integrins will be used for immunoprecipitation and immunoblot analysis of epidermal cell extracts and to inhibit adhesion, spreading, and migration on various ECM proteins. Migration-inhibiting polyclonal antibodies against newt epidermal cell membranes will be used to identify cell surface proteins involved in ECM interaction. FN receptors will be purified from epidermal extracts by affinity chromatography on the alpha chymotrypsin 120 kD cell-binding fragment of FN conjugated to Sepharose.
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