Molecular mechanisms of polarized endocytic traffic in epithelial cells

描述（由申请方提供）：上皮由极化细胞组成，极化细胞能够选择性地将物质转运穿过上皮单层。这种选择性运输是通过将质膜划分为不同的区域来实现的：顶端和基底外侧，这两个质膜区室具有不同的脂质和蛋白质组成。由于跨上皮蛋白质转运的保真度对各种上皮功能至关重要，因此上皮细胞已经开发出复杂的机制来确保蛋白质的正确转运。Rab 11 GTPases是小单体GTPases超家族的成员，参与调节内吞膜转运。Rab GT3通过将各种效应蛋白募集到不同的细胞区室来起作用。因此，了解这些效应蛋白的作用是了解上皮细胞功能的关键步骤。在过去的六年中，已经鉴定了几种Rab 11结合蛋白，其中包括Rab 11家族相互作用蛋白，也称为FIPs。包括我们在内的几个实验室的工作表明，FIP家族的Rip 11/FIP 5成员调节上皮细胞中的极化蛋白质转运。此外，它表明，FIPs作为支架因子，允许组装上皮蛋白运输所需的特定分选/转运复合物。根据最近发表的结果和初步数据，我们提出以下假设。首先，Rip 11/FIP 5介导蛋白质转运到顶端质膜。第二，Rip 11/FIP 5通过将分选蛋白连接蛋白18（SNX 18）和驱动蛋白II依次募集到内吞膜来调节顶端蛋白转运。因此，本提案的主要目标是进一步表征Rip 11/FIP 5-SNX 18和Rip 11/FIP 5-Kinesin II复合物的形成，并确定其对上皮转运的作用。我提出了三个不同的目标，旨在测试这些假设。在目标#1中，我们将分析Rip 11/FIP 5在调节顶端蛋白质转运中的作用。我们将使用tet诱导的蛋白敲低，荧光成像和体内转运分析的组合。在目标#2和目标#3中，我们将表征Rip 11/FIP 5与SNX 18和驱动蛋白II的结合。我们将使用免疫沉淀，谷胱甘肽珠下拉，酵母双杂交和等温量热法测定的组合。此外，在目标#2和目标#3中，我们将确定Rip 11/FIP 5结合蛋白在调节顶端和基底外侧膜运输中的作用。为此，我们将使用泰特诱导蛋白敲低、荧光成像、定点诱变和体内转运测定的组合。公共卫生相关性：本项目的目标是了解Rab 11依赖性极化膜交通的分子机制，并确定每个Rab 11-Rip 11/FIP 5蛋白复合物调节的膜交通步骤。此外，该项目还将确定介导Rab 11依赖的内吞蛋白分选的分子机制。这些数据将推进我们的理解，无论是概念上和机制，机制管理蛋白质靶向上皮细胞。阐明蛋白质靶向的机制是非常重要的，因为在这个过程中的缺陷会导致各种肺（囊性纤维化）和肾（肾源性尿崩症）疾病。因此，对极化膜交通机制的新见解可能导致识别新的药物靶点以及旨在识别和治疗膜交通障碍的新治疗方法。

DESCRIPTION (provided by applicant): Epithelia 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. Since the fidelity of trans-epithelial protein transport is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct transport of proteins. Rab11 GTPases are the members of small monomeric GTPase super-family that has been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, understanding the role of these effector proteins is a key step in understanding the function of epithelial cell. In the last six years several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Works from several laboratories, including ours, have shown that Rip11/FIP5 member of FIP family regulate polarized protein transport in epithelial cells. Furthermore, it was shown that FIPs act as scaffolding factors allowing the assembly of specific sorting/transport complexes required for the epithelial protein traffic. Based on recently published results and on preliminary data, we propose the following hypotheses. First, that Rip11/FIP5 mediates protein transport to apical plasma membrane. Second, that Rip11/FIP5 regulates apical protein transport by sequential recruiting of sortin nexin 18 (SNX18) and kinesin II to the endocytic membranes. Thus, the main goal of this proposal is to further characterize Rip11/FIP5-SNX18 and Rip11/FIP5-Kinesin II complex formation and determine their role on epithelial transport. I propose three different aims designed to test these hypotheses. In the aim #1 we will analyze the role of Rip11/FIP5 in regulating apical protein transport. We will use combination of tet-inducible protein knock-down, fluorescence imaging and in vivo transport assays. In the aim #2 and aim #3 we will characterize the binding of Rip11/FIP5 to SNX18 and Kinesin II. We will use the combination of immunoprecipitations, glutathione bead pull-down, yeast two-hybrid and isothermal calorimetry assays. In addition, in aim #2 and aim #3 we will determine the roles of Rip11/FIP5-binding proteins in regulating apical and basolateral membrane traffic. To that end we will use the combination of tet- inducible protein knock-down, fluorescence imaging, site-directed mutagenesis and in vivo transport assays. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the molecular mechanisms of Rab11-dependent polarized membrane traffic and define the membrane traffic steps that are regulated by each Rab11-Rip11/FIP5 protein complex. Furthermore, this project will also identify the molecular machinery mediating Rab11-dependent endocytic protein sorting. These data will advance our understanding, both conceptually and mechanistically, of the machinery governing protein targeting in epithelial cells. To elucidate the mechanisms of protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis) and renal (nephrogenic diabetes insipidus) 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.