Molecular mechanisms of polarized endocytic traffic in epithelial cells

项目摘要 上皮组织由极化细胞组成，极化细胞能够选择性地转运物质 穿过上皮单层。这种选择性运输是通过等离子体的分配来实现的 膜分为不同的领域：顶端和基底外侧，这两个质膜 具有不同的脂质和蛋白质组成的隔室。除了单细胞的极性外 在水平上，单个极化上皮细胞围绕中央顶腔组织。因为忠诚 跨上皮蛋白运输和上皮组织组织的形成对各种上皮细胞的生长至关重要。 功能，上皮细胞已经开发出复杂的机制，以确保正确的细胞和组织 极化 Rab 11 GTP酶是小单体GTP酶超家族的成员， 参与调节胞吞膜转运。Rab GTestival通过招募各种 不同的细胞区室的效应蛋白。因此，破译这些效应器的作用， 蛋白质是了解上皮细胞功能的关键步骤。在过去的十年里，一些 已经鉴定了Rab 11结合蛋白，其包括Rab 11家族相互作用蛋白，还 称为FIP。包括我们在内的几个实验室的工作表明，FIP的FIP 5成员 家族调节极化蛋白运输，以及微绒毛和顶腔的形成， 上皮细胞此外，我们已经表明FIP 5作为支架因子，通过结合并 激活分选连接蛋白18、驱动蛋白II和扣带蛋白。根据最近发表的结果和我们的 根据初步数据，我们提出以下假设。首先，FIP 5/Cingulin复合物介导顶端 内体靶向顶端腔形成的位点，并且该复合物受GSK 3调节。 激酶。第二，Rap 2A的FIP 5依赖性内吞转运介导微绒毛的形成。 极化上皮细胞因此，本提案的主要目标是确定和描述 FIP 5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的，旨在测试这些假设在体外和体内。在目标 #1我们将分析FIP 5和扣带蛋白相互作用在调节顶端蛋白转运中的作用 在顶腔形成期间。在目标#2中，我们将描述FIP 5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FIP 5依赖的作用。 利用斑马鱼肠道形成在上皮组织形态发生期间的内吞转运 模型该项目的完成将为理解分子生物学提供新的见解。 上皮细胞极化和顶端腔形成的机制和调节 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.