Molecular mechanisms of polarized endocytic traffic in epithelial cells

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Project Summary Epithelial tissues consist from polarized cells that are capable of selectively transporting substances across epithelial monolayer. This selective transport is achieved by the partitioning of the plasma membrane into distinct domains: apical and basolateral, with both of these plasma membrane compartments having distinct lipid and protein compositions. In addition to polarity at a single-cell level, individual polarized epithelial cells are organized around central apical lumen. Since the fidelity of trans-epithelial protein transport and epithelial tissue organization is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct cell and tissue polarization. Rab11 GTPases are the members of small monomeric GTPase super-family that have been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, deciphering the roles of these effector proteins is a key step in understanding the function of epithelial cell. During the last decade, several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Work from several laboratories, including ours, has shown that FIP5 member of FIP family regulate polarized protein transport, as well as microvilli and apical lumen formation in epithelial cells. Furthermore, we have shown that FIP5 acts as a scaffolding factor by binding to and activating sorting nexin 18, kinesin II and cingulin. Based on recently published results and on our preliminary data, we propose the following hypotheses. First, FIP5/Cingulin complex mediates apical endosome targeting to the site of apical lumen formation, and that this complex is regulated by GSK3¿ kinase. Second, FIP5-dependent endocytic transport of Rap2A mediates microvilli formation in polarized epithelial cells. Thus, the main goal of this proposal is to identify and characterize the roles of FIP5 and its interacting proteins in mediating epithelial polarization. I propose three different aims designed to test these hypotheses in vitro and in vivo. In the aim #1 we will analyze the role of FIP5 and cingulin interaction in regulating apical protein transport during apical lumen formation. In the aim #2 we will characterize the role of FIP5 and its binding proteins during microvilli formation. Finally, in aim #3 we will test the role of FIP5-dependent endocytic transport during epithelial tissue morphogenesis using zebrafish intestinal tract formation model. Completion of this project will provide a novel insight in understanding the molecular machinery and regulation of epithelial cell polarization and apical lumen formation during epithelial tissue morphogenesis and remodeling.