This is a proposal to study the role of the microtubule (Mt) cytoskeleton and associated motor enzymes which support the formation, binding, and cyclic transport of nascent protein-containing vesicles between the Golgi apparatus and the plasma membrane during secretion and endocytosis in the hepatocyte. Although vesicle-based protein transport is essential for normal hepatocellular function, the molecular mechanisms which support this important process are undefined. During the past R29 funding period, pr ogress towards the original specific aims includes: 1) developing multiple experimental probes, tools, and cell models; 2) defining the basic organization and polarity of the Mt-based cytoskeleton in the hepatocyte; 3) identifying some specific subpopulations of secretory and endocytic hepatocellular organelles that interact with motor enzymes; and 4) defining how motor activity is altered by specific perturbations, such as exposure to various bile acids. These observations provide strong support for the central hypothesis of this proposal that vesicle-mediated protein trafficking in the hepatocyte is supported by an organized Mt framework and the associated molecular motor enzymes dynein, kinesin, and dynamin which are regulated differentially to meet the unique secretory and endocytic functions of the liver. To pursue this hypothesis further, the following specific aims are proposed: 1) to identify and define the different isoforms of kinesin and dynamin in the hepatocyte and establish the vesicle components to which they associate. By vesicle immunoisolation methods, immunofluorescence and immunoelectron microscopy, and gene cloning technologies, the specific secretory and endocytic vesicular compartments in hepatocytes which bind kinesin, dynein, and dynamin will be defined and compared. Secondly, functional in vivo studies to define how dynamin and kinesin mediate vesicle binding and transport in the hepatocyte will be conducted. Through the application of antibody microinjection and video/computer microscopy to isolated hepatocytes, studies will a) test if inhibition of kinesin and dynamin in living cells alters specific secretory and endocytic vesicles, b) test if activation of kinase signaling cascades alters vesicle transport, and c) define how these perturbations alter motor phosphorylation and vesicle interactions. Thirdly, the specific functional contributions of motor enzymes to membrane budding and transport, in vivo, using cell-free assays comprised of either permeabilized cells or isolated components such as vesicles, motors, and microtubules will be defined. The studies described will provide a novel and in-depth examination of the molecular mechanisms of vesicle-based transport in the hepatocyte.
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