Polarized epithelia form barriers that separate two biological compartments and regulate ionic homeostasis by vectorial transport of ions and solutes between those compartments. Abnormalities in epithelial structure and function are characteristic of many disease states. Our long-term goal is to understand how membrane proteins come to reside in specific membrane domains, termed apical and basal-lateral, that face these different compartments. In our previous studies, we showed that Na/K-ATPase distribution is regulated by retention in the lateral membrane and not by sorting in the Golgi; we defined protein-protein interactions linking Na/K-ATPase, through the membrane-cytoskeleton, to E-cadherin; and, we identified a large protein complex (sec 6/8) that is recruited to cell-cell contacts and specifies the docking of basal- lateral transport vesicles. Based on these studies, we have developed the following working hypothesis; cell-cell adhesion generates initial structural and molecular asymmetry at the cell surface; the ensuring reorganization of the actin cytoskeleton drives a subset of membrane proteins to sites of adhesion; and, the concomitant recruitment of a targeting patch specifies the delivery of transport vesicles containing other basal-lateral membrane proteins to the site of adhesion. We propose to test specific tenets of our hypothesis: 1). Investigate regulation of actin cytoskeleton re-organization at sites of cadherin-mediated cell-cell adhesion. 2). Define roles of cell-adhesion complexes in specifying assembly of components of the basal-lateral transport vesicle targeting patch at sites of cell-cell adhesion. 3). Analyze protein-protein interactions that specify sec6/8 recruitment to the plasma membrane and assembly of the targeting patch. 4). Reconstitute functional assembly of vesicle docking/fusion complexes at sites of cell-cell adhesion. The significance of these studies is that they will identify molecular links between cell-cell adhesion, the membrane-cytoskeleton, and vesicle docking/fusion machinery, thereby elucidating mechanisms involved in regulating the generation and maintenance of epithelial cell polarity in normal and disease states.
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