The goal of this proposed study is to define the molecular mechanisms by which alcohol, and its oxidized metabolite, acetaldehyde, impairs secretory and endocytic vesicular protein transport in hepatocytes. Specifically, we will test the hypothesis that alcohol disrupts the microtubule cytoskeleton and associated ATPases, or motor enzymes, which play a major role in the organization, transport, and targeting of different vesicle populations within the hepatocyte. Numerous reports by others have suggested that alcohol and its oxidized metabolites inhibit protein trafficking indirectly through a disruption of the microtubule cytoskeleton upon which most of these transport events depend. Despite these extensive in vitro studies, it has not been determined if alcohol alters microtubule organization within the hepatocyte, how this disruption occurs, and if other microtubule associated enzymes (motors) are also perturbed. Our goal is to utilize state of the art cell biological techniques to conduct a definitive and novel study on the mechanism by which alcohol interferes with microtubule- based vesicular trafficking within the hepatocyte. The proposed study has three specific aims. First, we will examine and compare how microtubules are organized and polarized within healthy control vs. ethanol-treated hepatocytes by: a) confocal-immunofluorescent and electron microscopic examination in conjunction with computer-aided reconstruction morphometry; b) polarity assays of microtubules in cultured hepatocyte couplets. Second, through the application of cell microinjection with computer- enhanced, fluorescent, video microscopy we will determine if secretory and endocytotic vesicular transport along microtubules in cultured hepatocyte couplets is altered by alcohol treatment. Third, we will test for disruptive effects of alcohol on vesicle transport and fusion by measuring alterations in the activity of microtubule-associated motor enzymes (kinesin, dynein, dynamin) 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 novel to the study of alcohol-induced cell-damage and to vesicular transport in hepatocytes. They will not only greatly expand our understanding of how alcohol impairs hepatocyte function but lend insight into the basic mechanisms of secretion and endocytosis in these cells.
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