Regulation of Clathrin and Adaptor Function

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DESCRIPTION (provided by applicant): Membranes define distinct structural and functional boundaries between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is largely mediated by vesicular transport, and protein coats, of which the clathrin coated membrane is a paradigm, are invariably associated with these processes. Phosphoinositides are recognized to play important roles as regulators of membrane trafficking, and identifying mechanisms by which inositides are locally accumulated or generated at sites of action, and how they interact with effector proteins are unresolved questions. During the last award period we found that a Class II PI 3-kinase, PI3K-C2?, binds specifically to clathrin. Upon overexpression, PI3K-C2? can induce the proliferation of numerous intracellular clathrin coated buds containing both proteins and which move extremely rapidly. Our more recent work has demonstrated that similar rapidly moving structures can be detected in live cells in the absence of exogenous PI3K-C2? expression, that they contain clathrin, PI3K- C2?, and endocytosed transferrin, as well as associated proteins suggesting involvement in membrane recycling. This proposal tests the hypothesis that these `Fast-clathrin' (F-clathrin) structures are involved in membrane recycling and other steps in the endocytic pathway by (1) testing the hypothesis that F- clathrin structures are coated membrane tubules with unique adaptors, cargos and motor proteins, and probing their relationship to peripheral sorting endosomes at the ultrastructural level; (2) using rapid imaging and simultaneous acquisition of multiple signals as well as photoactivatable GFP probes to determine how cargo passes through the F-clathrin compartment in live cells; (3) dissecting the regulation of F-clathrin formation and cargo loading in intact cells by individual F-clathrin components and by small G proteins and their activators and effectors; and (4) elucidating the interactions between PI3K-C2? and the clathrin heavy chain terminal domain, linker and ankle by crystallography and solution studies, facilitating the production of dominant-negative forms of both proteins. Although the pathways of membrane trafficking in mammalian cells are broadly understood, exactly how they function at some steps mechanistically, and even morphologically, remain obscure and will be addressed by this work. Public Health Relevance: This project focuses upon the trafficking pathways by which individual cells maintain their complex intracellular structure and carry out functions in specialized compartments. These pathways are ubiquitous in all eukaryotic cells, and thus these are central issues in cell biology with relevance for many normal biological processes such as signaling, development and intercellular communication in tissues. Correspondingly, they also are sites of derangement in many disease processes including cancer, cardiovascular and neurological diseases.