Kidney and gut epithelial cells are structurally and functionally polarized to transport specific molecules while maintaining a trans-epithelial barrier This cellular asymmetry is essential for the proper functioning of epithelial tissues and depends on polarized endocytic transport routes. Additionally, epithelial cells coordinate their polarizatin with neighboring cells to form an apical lumen, a key step in the establishment of renal and gut architecture, and thereby function. Indeed, malfunctions in polarized epithelial transport and apical lumen formation are responsible for a variety of renal and intestinal disorders. This proposal aims to fill critical gaps in our understanding of epithelial cell biology, which are the molecular mechanisms mediating apical protein targeting and coordinated formation of apical lumen during kidney morphogenesis and function. Recent work, including work published from our preceding grant, identified Rab11 GTPase as a key regulator of epithelial cell polarity and epithelial tissue morphogenesis. As all Rab GTPases, Rab11 functions by binding and activation various effector proteins. 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 regulates polarized protein transport 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. Finally, we have shown that FIP5/cingulin complex is recruited to the midbody during late telophase, the event that plays a key role in establishing a single nascent apical lumen. Based on recently published results and on our preliminary data, we propose the following hypotheses. First, FIP5/cingulin complex mediates apical endosome targeting during apical lumen formation. Second, FIP5/cingulin targeting around the midbody during late telophase is required for apical lumen formation. Thus, this proposal is designed to test these hypotheses in both in vitro (MDCK cells) and in vivo (zebrafish) models. This proposal has three specific aims. 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 test the role and regulation of the midbody and cytokinesis for the initiation of the apical lumen. Finally, in aim #3 we will test the role of FIP5/cingulin-dependent endocytic transport during epithelial tissue morphogenesis using zebrafish pronephric tube and intestinal tract formation models. 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. During three 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. In this study we will identify the function and regulation of these epithelial luen organizing proteins as well as their role in apical lumen formation in vitro and in vivo.
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