Function of the Amphiphysin Protein Family project I will address the role of amphiphysin and interacting proteins, dynamin, synaptojanin and AP2 in endocytosis, cell polarity and cell proliferation. Changes in the expression and function of amphiphysin isoforms and amphiphysin- interacting proteins during normal growth and following the loss of growth control it will be determined. Ypt51 Interacting Components in Endocytosis: Project II focuses on the role of Ypt51 and interacting proteins in yeast endocytosis. Functional interactions with fibrin and amphiphysin homologs indicate a role of the cortical cytoskeleton. Additional interacting genes have been identified through a synthetic lethal screen. The relationship between these gene products and Ypt51 function on the endocytic pathway will be defined. Regulated Exocytosis in Developing and Neoplastic Pancreatic Acinar Cells: Project IV concerns the role of rab proteins in the developing rat pancreas. During development, secretogenesis correlates with the movements of rab3D from the cytosol to the granule membrane and the transient appearance of rab4 on the actin terminal web. The changes in rab localization, function and protein-protein interactions will be determined. Control of Cell Polarity and Plasma Membrane Function by rho Family GTPases: Project V will explore the development and maintenance of cell polarity in epithelial cells, neurons and lymphocytes. The role of the rho family of GTPases and of mammalian homologs of yeast genes involved in polarized growth will be addressed. The changes in localization and function of these proteins in metastatic cells will be defined. Sec4 and Interacting Proteins in Cell Polarity: Project VI focuses on the contribution of Sec4 and interacting proteins to polarized cell growth in yeast. Polarized concentration of secretory vesicles depends upon Sec2, actin and a myosin. Sec 2 localizes to sites of exocytosis and directly interacts with Sec4 in a nucleotide dependent fashion. This project will define the role of Sec2, Sec4 and the cytoskeleton in polarized growth. GROWTH=P01CA461280008 The long term goal of this proposal is to improve our understanding of the properties of the cortical cell cytomatrix and of its pleiotropic effects on a variety of cell processes including endocytosis and cell proliferation. More specifically, this grant application proposes to elucidate the role of the amphiphysin/RVS family which is compromised of at least two members in man, amphiphysin I and II, and with two members in yeast, Rvs161 and Rvs167. The RVS yeast genes have been implicated in endocytosis, cell polarity, entry into stationary phase and actin dynamics. Amphiphysin I is expressed primarily by neurons, is concentrated in the peripheral cytomatrix of nerve terminals, and has a putative role in endocytosis via its interaction with the GTPase dynamin I, the inositol-5-phosphatase synaptojanin and the clathrin adaptor AP2. Amphiphysin II undergoes extensive regions of the subplasmalemmal cytoskeleton of muscle and nerve cells. Both amphiphysin I and II, like other proteins of the cortical cytoskeleton, have been linked to cancer. Amphiphysin I is ectopically expressed in some cancer cells and is a dominant autoantigen in Stiff-Man syndrome associated with breast cancer. Amphiphysin II was found to interact with the oncogene Myc and to be a tumor suppressor gene. The working hypothesis of this proposal is that proteins of the amphiphysin family are general components of the subplasmalemmal cytoskeleton, and that the pleiotropic actions of the amphiphysins reflect the multiple functions of the cortical cell cytoplasm. This application proposes 1) to perform a thorough characterization of the amphiphysin protein family in neurons, muscle and epithelial cells, 2) to identify interacting partners of amphiphysin isoforms and to study the functional role of these interactions (with emphasis on endocytosis, actin dynamics and cell proliferation) by using biochemical experiments, cell free assays, microinjection experiments, cell transfections, 3) to determine the phenotypic changes in mice caused by manipulations of the amphiphysin genes, 4) to investigate changes in amphiphysin expression in human cancer tissues and to establish whether these changes have a pathogenic role in some form of cancer. These studies will provide new important information on functional aspects of the cell periphery which are highly conserved among all eukaryotic cells, and may offer new insights into the pathogenesis of some forms of cancer, providing clues toward their early diagnosis and therapy.
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