Con A containing ghosts and caps have been prepared from axenically grown amoebae of Dictyostelium discoideum and coated vesicles have been isolated from the human lymphoblastoid cell line Wil 2. Both of these structures contain regions of membrane that remain attached to actin filaments. In this study I propose to isolate the actin filament-membrane attachment sites from these structures and determine the mechanism by which this site regulates the association of actin filaments with the membrane. Actin filaments that are associated with ligan-receptor complexes through a protein-protein interaction will be isolated by ligand affinity chromatography or gradient centrifugation. The components responsible for ligand binding, actin binding and regulation of actin binding will be identified and their location in situ will be determined with immunoelectron microscopy, chemical cross-linking and vectorial probes. Similar studies will be performed on coated vesicles to identify components responsible for actin binding and regulation of actin binding. The role of cortical actomyosin in ligand induced receptor redistribution on the cell surface will be studied using an in vitro model. The mode of actin recruitment to the cell membrane in response to ligand challenge at the cell surface will be studied using isolated coated vesicles. These results will be related to the properties of the actin-membrane attachment site to construct a coherent picture of the role of actin in membrane function.
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