The major objective of these studies is to increase our understanding of how epidermal cells interact with the extracellular matrix (ECM) during wound closure. Thus, the behavior of adult newt (Notophthalmus viridescens) epidermal cells in wounded limbs will be analyzed as the cells migrate over plastic or nitrocellulose implants coated with collagen, laminin, fibrinogen (FGN), or fibronectin (FN). To learn more about the molecular features that epidermal cells recognize in FGN and FN, proteolytic fragments of each molecule will be tested as migration substrates and/or soluble inhibitors of migration over the intact molecule. In the case of FN, monoclonal antibodies will also be tested as inhibitors of migration over the intact molecule to determine if epidermal cells utilize the fibroblast binding site of FN. To begin characterizing the cell surface receptors which might be involved in recognition of ECM molecules, binding studies will be conducted using latex beads coated with various ECM proteins to determine if the receptors on the upper surface of migrating cells vary in relation to the substrate under the cells. Lectins and glycosidases will be tested as migration substrates to determine if bonds between cell surface saccharides and the substrate could be involved in migration. In addition, adhesion assays will be conducted using dissociated cells in an attempt to demonstrate interaction of epidermal cells with migration-promoting ECM proteins in a more defined environment than a wound. To analyze certain consequences of substrate recognition, immunohistochemical techniques will be used to study the expression of basement membrane zone proteins as epidermal cells migrate over implants coated with various ECM molecules. Finally, experiments will be conducted to determine if epidermal cells lose their dependence on the pre-existing ECM during migration.
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