CYTOSKELETON-MEMBRANE INTERACTIONS

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A complete understanding of phenomena such as morphogenesis, wound healing, and viral transformation is contingent upon an understanding of fundamental cell processes such as motility, the maintenance and change of cell shape, and cell-cell and cell-substrate attachment. At the core of our ignorance about these basic life processes is a lack of knowledge about the molecular details of transmembrane interactions between the cytoskeleton and the cell surface. The ameboid stage of the cellular slime mold, Dictyostelium discoideum is easily grown in large quantities, is motile, and exhibits cell-cell as well as cell-substrate attachment. Using D. discoideum as a model for cells in general, I am investigating the interactions between this cell's plasma membrane and its cytoplasmic cytoskeletal proteins. In the past three years we have isolated cell-cell contact regions from aggregated amebae, and have developed two new low-speed sedimentation binding assays to monitor associations between actin and membranes. We find that integral proteins mediate a specific, saturable binding of plasma membranes to the sides of actin filaments and that plasma membranes can catalyze the polymerization of actin at the membrane surface even at actin concentrations well below the normal critical concentration for polymerization. Using detergent solubilization and F-actin affinity chromatography, we have identified an integral actin-binding protein with an apparent molecular weight of 17,000 daltons. The immediate goals are to characterize and reconstitute the 17kd protein, to use chemical crosslinking to identify detergent-sensitive actin-binding proteins missed by F-actin affinity chromatography, to prepare monospecific antibodies against the major integral membrane components of an ameboid membrane cytoskeleton, to explore the structure and regulation of the transmembrane linkages in this system, and to determine whether structurally similar linkages are found in other species. Of ultimate interest is the molecular basis for motility and cell-cell and cell-substrate interactions.