The long term goal of this program is an understanding at the molecular level of membrane functions regulated by clathrin coated pits and vesicles. At the plasma membrane of all eukaryotic cells, coated pits regulate endocytosis of extracellular molecules bound to receptors. In this proposal we will use fluorescent protein chimeras as tools to address aspects of clathrin-mediated endocytosis and intracellular membrane trafficking in living cells that have not previously been amenable to analysis. The dynamics of clathrin, AP-2 and other components in coated pit formation and in the pit to vesicle transformation will be determined. Individual endocytic events will be visualized and correlated with changes in coated pit components in real time, and the behavior of epsin, intersectin and endophilin, recently identified protein components of the endocytic pathway, will be studied. Factors responsible for AP-2 adaptor release from coated membranes, likely prerequisite for endocytic vesicle fusion, will be identified, purified and characterized. Elucidation of the detailed properties of early steps in the receptor-mediated endocytosis pathway in living cells is critical for understanding molecular mechanisms of vesicular transport in all eukaryotic organisms. It will also be useful in the design of agents to block cellular entry by pathogens and toxins, and for development of techniques for delivery of therapeutic agents into target cells.
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