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.
The goal of this project is to understand the molecular mechanisms of epithelial cell polarization and apical lumen formation in vitro and in vivo. During two previous funding cycles we have demonstrated that polarized endosome transport plays a key role in targeting proteins to the apical plasma membrane and identified the proteins that are required for this process. We also have shown that endocytic transport is required for microvilli formation and maintenance. In this study we will identify the function and regulation of epithelial lumen organizing proteins as well as their role in micorvilli formation in vitro and in vivo. To elucidate the mechanisms of epithelial protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis), renal (nephrogenic diabetes insipidus) and digestive (microvillus inclusion disease, microvillous atrophy syndrome) disorders. Thus, new insights into the mechanisms of polarized membrane traffic may lead to the identification of new drug targets as well as new therapeutic approaches aimed at the identification and treatment of membrane traffic disorders.
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