The specific goal of the proposed work is to further our understanding of the molecular basis for the role of the plasma membrane and the cytoskeleton in mediating cell adhesion to biological substrata. The problem will be approached by isolating the dorsal region of the plasma membrane adjacent to the culture medium separately from the ventral region attached to the culture dish. The isolation will be done so as to keep the isolated membrane in an open sheets so we can directly probe the cytoplasmic side. These regions are analogous to the abluminal and luminal surfaces of the plasma membrane for many types of cells. For example, the endothelium lining blood and lymphatic vessels, and the epithelium lining renal tubules and bile ductules. The relative amounts and temporal changes of the proteins and the associated cytoskeleton will be determined in the dorsal and the ventral regions as cells attach and spread on collagen. HeLa cells will be used as the model so we can focus on the plasma membrane. The cells do not make an extracellular matrix, they grow in suspension and can be made to synchronously attach and spread in-vitro with well defined kinetics. The ventral region will also be subdivided into two domains, that which weakly binds the substratum and that which strongly binds. The degree of plasma membrane protein self-association and asssociation with others including the cytoskeleton will be determined by chemical cross-linking experiments. Small protein clusters will be isolated by conventional two-dimensional gel electrophoresis and aggregates will be separated based upon their ability to interact with an antibody to the cytoskeletal protein, actin . This will test the hypothesis that cell adhesion is regulated in part by the formation of membrane protein clusters as a consequence of substrate binding, the result of such clustering is an increased affinity for the cytoskeleton. Similar clustering phenomena could be the basis for transmembrane signalling in endocytosis and cell-cell adhesion as well.
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