The goal of this project is to identify and characterize plasma membrane anchorage sites for the cytoskeleton in cultured fibroblasts. The approach will use HAHS, a new crosslinking reagent, which transfers a radioactive moiety from an initially labelled protein to other molecules within reach of a reactive group. Neighboring molecules can then be identified simply and unambiguously by their acquired radioactivity. Two complementary methods will be used. In one, inside-out plasma membranes will be purified by letting cells phagocytose specific ligand-coated beads, then reisolating the beads; then HAHS-labelled actin, fodrin and vinculin will be reconstituted onto the membranes, photolyzed, and analyzed by electrophoresis to identify their sites of anchorage. In the other, labelled proteins will be microinjected into living cells, allowed to incorporate into cytoskeletal structures, then photolyzed and analyzed. Experiments will be done with normal vs. transformed cells, well spread vs. suspended, and rapidly dividing vs. growth arrested cells to identify proteins involved in oncogenic transformation, anchorage to the substratum and growth control. The interaction of cells with fibronectin will also be studied by labelling the fibronectin 11.5 k cell-binding fragment with HAHS, and crosslinking it to the cell surface. Once identified, the location, distribution, regulation, function and structure of these proteins will be investigated. Particularly interesting proteins will be isolated and their structure and function studied further.
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