The objective of this research is to learn how actin interacts with plasma membrane. Three examples of actin-membrane linkages are being studied. (1) We are determining the molecular composition of the actin to membrane cross filament in intestinal microvilli. a) We propose to isolate the microvillar 110K protein, a candidate for the cross filament, and to determine whether and how it interacts with actin and the microvillar membrane in vitro. b) We will test the proposed roles of the microvillar core proteins (fimbrin, villin, 110K, actin, and calmodulin) in the structure and membrane association of the core bundle of actin filaments by immunoultrastructural localization of these proteins in situ, using 50 angstroms Fab' fragments of monoclonal antibodies complexed to 50 angstroms gold particles. c) We will screen for microvillar proteins which link actin to membrane by a reconstitution binding assay employing 3H-actin, microvillar membranes, and fractionated microvillar proteins. (2) We are analyzing the association of vinculin-like proteins with the plasma membrane. We have discovered two new proteins in smooth muscle, 150K meta-vinculin, and 300K vinculin-like polypeptide (300K-VLP) that are antigenically related to 130K vinculin, but which unlike vinculin, have solubility properties of amphiphilic membrane proteins and could be direct links between actin filaments and the cell membrane. We propose to analyze the molecular basis for the antigenic similarity and solubility difference between these proteins by peptide mapping, charge shift electrophoresis, biosynthetic labelling and pulse-chase experiments, isolation of native metavinculin and 300K-VLP and characterization of their interaction with actin, plasma membrane and liposomes in vitro. (3) We have defined a cell surface actin (CSA) on some murine lymphocytes by immunofluorescence and flow cytometry. a) We propose to determine by two-color immunofluorescence and flow cytometry if CSA is a marker for new murine and human lymphocyte subpopulations within the known subdivisions of T and B-cells. b) We will assay for biochemical differences between CSA and intracellular actin by determining if CSA is a glycoprotein and if it has a hydrophobic domain. c) We plan to test for the possible function of CSA in mediating adherence of lymphocytes to fibronectin and to cells of the lymphoreticuloendothelial system, and for the possible function of CSA as part of a cell surface growth control complex.
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