Actin filament formation is required for the platelet shape change that accompanies activation and clot formation. Although many actin-binding proteins have been found, none seem able to mediate the elongation of filaments by monomer addition to membrane-bound barbed end of actin filaments. A central question remains: what are the key proteins that regulate this process? This work began with the identification of the Mab 2E4 antigen as a potential membrane-associated actin-binding protein involved in filament formation. This 43 kD protein is present in cells at sites where membrane- associated polymerization is occurring. It binds the barbed end of actin filaments in an ATP-sensitive manner and in preliminary experiments induces the formation of small nucleating oligomers of actin at concentrations below the critical. The DNA and amino acid sequence of the antigen reveal it to be a unique protein. The working hypothesis that guides this project is that there exists a membrane-associated polymerization machine which includes the Mab 2E4 antigen, and that this machine regulates polymerization of actin filaments off the cytoplasmic surface of the platelet plasma membrane during activation.
The specific aims i n this granting period is to test this hypothesis in two ways: 1) by dissecting in detail the interactions of the Mab 2E4 antigen with actin in vitro and in vivo using biochemical, molecular and cell biological approaches; and 2) by isolating the intact and functional polymerization machine from human platelet surface membranes using cationic beads, and identifying the components of the machine by functional, biochemical and molecular techniques. The tools to perform these experiments have been developed. These include: I) cloned cDNA for the Mab 2E4 its sequence; 2) polyclonal and monoclonal antibodies against both endogenous and recombinant protein; 3) bacterially expressed fusion protein that is soluble, abundant and active in in vitro assays; 4) sensitive assays of actin oligomer formation by fluorescence video microscopy; 5) ability to induce actin filaments with cationic beads in platelets; and 6) isolated bead-protein complexes that induce filament formation in vitro. Thus, all the necessary expertise, and molecular probes are in place to address this question. The significance of identifying nucleators of actin filament assembly in platelets transcends their role in platelet physiology. Membrane- associated actin polymerization is a well recognized event in the formation of a wide variety of physiologically important cellular structures, ranging from the leading edge of the migrating cell to the formation of stereocilia in the sensory organ of the inner ear.
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