The major focus of this research program continues to be the study of a major physiological process performed by the hepatocyte, mainly, the organized cyclic transport of proteins and lipids between the sinusoidal plasma membrane and the trans-Golgi network (TON). Because plasma protein secretion and bile formation are essential liver functions, we have focused our efforts on defining and understanding the molecular machinery that supports these processes. Specifically, we are actively investigating the role of three prominent interacting proteins that comprise the central vesicle forming machinery in the hepatocyte. These proteins include: a large mechanochemical GTPase called dynamin (Dyn2) that is expressed in a tissue-specific manner and believed to constrict membrane during vesicle formation, the actin cross-linking protein cortactin that binds Dyn2 and is phosphorylated by src kinase, and the EH-domain containing protein Epsl5 that is required for clathrin-coated vesicle formation. Based on our previously published work and extensive new preliminary data, the central hypothesis of this proposal predicts that Dyn2, cortactin, the actin cytoskeleton, and Epsl5 comprise a conserved molecular machinery that functions differentially at both the plasma membrane and TGN to mediate vesicle formation in the hepatocyte during endocytosis and secretion. Using our unique array of molecular and immunological reagents to Dyn2, cortactin and Epsl5, we will perform mechanistic and morphological studies combining molecular, cell biological, and state of the art imaging technologies to address three related but distinct specific aims. First, we will test the hypothesis that Dyn2 and cortactin participate in multiple endocytic events in the hepatocyte that include: (a) distinct forms of fluid phase endocytosis and, (b) a novel organization of the clathrin-based endocytic machinery. Second, we will test the hypothesis that specific subdomains distinguish Dyn2 from other dynamin family members in the hepatocyte and are alternatively spliced to associate with: (a) clathrin coated pits vs. other organelles, and (b) specific endocytic and secretory compartments such as caveolae, micropinosomes and the Golgi apparatus. Third, we will test the hypothesis that Dyn2 interacts with cortactin and Epsl5 in the hepatocyte secretory pathway to: (a) physically associate with a specific compartment of the Golgi apparatus (TGN), and (b) support the formation of TGN-derived vesicles containing nascent secretory proteins. Information gathered from this proposal will provide important insights into the complex hepatocyte trafficking machinery that is often disrupted during a variety of liver diseases.
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