Molecular mechanisms of polarized endocytic traffic in epithelial cells

项目摘要 上皮组织由能够选择性运输物质的极化细胞组成 跨上皮单分子层。这种选择性传输是通过对等离子体的分割来实现的 膜分为不同的区域：顶端和基侧，具有这两种质膜 具有不同的脂肪和蛋白质成分的隔室。除了单电池中的极性之外 水平上，单个极化上皮细胞围绕中央根尖管腔组织。由于保真度 跨上皮蛋白的运输和上皮组织的组织对各种上皮细胞至关重要 功能，上皮细胞已经发展出复杂的机制来确保正确的细胞和组织 极化。 Rab11 GTP酶是小单体GTP酶超家族的成员，已被 与调节内细胞膜运输有关。Rab GTP酶通过招募不同的 效应器蛋白到不同的细胞隔间。因此，破译这些效应器的角色 蛋白质是了解上皮细胞功能的关键步骤。在过去的十年里，有几个 已经鉴定出与Rab11结合的蛋白，其中包括Rab11家族相互作用蛋白 被称为FIP。包括我们在内的几个实验室的研究表明，FIP5是FIP的成员 家族调控极化蛋白运输以及微绒毛和顶腔的形成 上皮细胞。此外，我们已经证明，FIP5通过结合和 激活分选Nexin 18、Kinesin II和Cinglin。基于最近发布的结果和我们的 根据初步数据，我们提出了以下假设。首先，FIP5/Cinglin复合体介导心尖 内体定位于顶腔形成部位，该复合体受GSK3？调节 激活剂。第二，依赖于FIP5的Rap2A胞内转运介导了微绒毛的形成 极化的上皮细胞。因此，本提案的主要目标是确定和描述角色 FIP5及其相互作用蛋白在介导上皮极化中的作用。 我提出了三个不同的目标来在体外和体内检验这些假说。在目标中 #1我们将分析FIP5和Cinglin相互作用在调节心尖蛋白运输中的作用 在根尖管腔形成过程中。在目标#2中，我们将描述FIP5的作用及其结合 微绒毛形成过程中的蛋白质。最后，在目标3中，我们将测试依赖于FIP5的角色 斑马鱼肠道形成上皮组织形态发生过程中的细胞内转运 模特。这个项目的完成将为理解分子提供新的见解。 上皮细胞极化和根尖管腔形成的机制及调控 组织形态发生和重塑。

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.