The goal of this proposed study is to elucidate the molecular mechanisms by which hepatocytes transport secretory and endocytosed proteins to defined cytoplasmic locations. Specifically, we will test the hypothesis that the microtubule cytoskeleton and its associated ATPases, or motor enzymes, play a major role in the organization, transport, and targeting of different vesicle populations within the hepatocyte. Numerous studies have implicated microtubules in vesicular transport and liver pathology. However, previous approaches have been largely indirect and have relied on drug perturbation either in intact animals or perfused organ systems and have yielded provocative but incomplete and conflicting results. Thus, it has not been established that microtubules and associated ATPases are required for these movements. At present, the mechanisms by which the hepatocyte discriminates between secretory granules, endosomes, lysosomes and other vesicular compartments to direct them to either sinusoidal or canalicular surfaces with precision and efficiency are totally undefined. Our goal is to conduct a definitive and novel study on the role of the microtubule-based cytoskeleton in vesicular trafficking within the hepatocyte using state of the art cell biological techniques.The proposed has three specific aims. First, we will examine how microtubules are organized and polarized within the hepatocyte in respect to the sinusoidal and canalicular surfaces by: a) confocal- immunofluorescent and electron microscopic examination in conjunction with computer-aided reconstruction morphometry; b) microtubule polarity assays of cultured hepatocytes. Second, we will determine what secretory and endocytotic components move along microtubules in cultured hepatocyte couplets by combining microinjection of fluorescent probes and unique inhibitory antibodies or drugs with computer-enhanced, fluorescent, video microscopy. Third, we will test the participation of microtubules and associated motor enzymes (kinesin, dynein, dynamin) in vesicle transport through morphological and biochemical manipulation of permeabilized and homogenized hepatocytes, and cell-free systems using purified vesicular and cytoskeletal components. The technology and experiments described in this proposal are unique to the study of vesicular transport in hepatocytes. They will expand greatly our understanding of how liver cells secrete/excrete proteins and how these critical processes are disrupted by drugs or diseased states such as cholestasis and alcohol-induced cirrhosis.
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