Molecular mechanisms of polarized endocytic traffic in epithelial cells

描述(申请人提供)：上皮细胞由极化的细胞组成，这些细胞能够选择性地跨上皮单层运输物质。这种选择性转运是通过将质膜划分为不同的区域来实现的：顶端和基侧，这两个质膜区段都具有不同的脂和蛋白质成分。由于跨上皮蛋白运输的保真度对多种上皮功能至关重要，上皮细胞已经发展出复杂的机制来确保蛋白质的正确运输。Rab11 GTP酶是小单体GTP酶超家族的成员，参与调节胞膜转运。Rab GTPase通过将各种效应蛋白招募到不同的细胞隔间来发挥作用。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一步。在过去的六年中，已经发现了几种Rab11结合蛋白，其中包括Rab11家族相互作用蛋白，也称为FIP。包括我们在内的几个实验室的研究表明，FIP家族的Rip11/FIP5成员调控上皮细胞中极化的蛋白质运输。此外，研究表明，FIP作为支架因子，允许组装上皮蛋白运输所需的特定分类/运输复合体。基于最近发表的结果和初步数据，我们提出了以下假设。首先，Rip11/FIP5介导了蛋白质向顶端质膜的转运。第二，Rip11/FIP5通过将索尔丁Nexin 18(SNX18)和激动素II依次招募到细胞内膜来调节顶端蛋白的运输。因此，这项建议的主要目的是进一步表征Rip11/FIP5-SNX18和Rip11/FIP5-Kinesin II复合体的形成，并确定它们在上皮运输中的作用。我提出了三个不同的目标来检验这些假说。在目标1中，我们将分析Rip11/FIP5在调节心尖蛋白运输中的作用。我们将结合使用tet诱导的蛋白敲除、荧光成像和体内转运分析。在目标2和目标3中，我们将表征Rip11/FIP5与SNX18和Kinesin II的结合。我们将使用免疫沉淀、谷胱甘肽颗粒下拉、酵母双杂交和等温量热法相结合的方法。此外，在目标2和目标3中，我们将确定Rip11/FIP5结合蛋白在调节心尖膜和基底侧膜运输中的作用。为此，我们将使用tet诱导蛋白敲除、荧光成像、定点突变和体内转运分析的组合。公共卫生相关性：该项目的目标是了解依赖Rab11的极化膜交通的分子机制，并定义由每个Rab11-Rip11/FIP5蛋白复合体调控的膜交通步骤。此外，该项目还将确定介导Rab11依赖的内吞蛋白分选的分子机制。这些数据将在概念和机制上促进我们对管理上皮细胞中蛋白质靶向的机制的理解。阐明蛋白质靶向的机制非常重要，因为这一过程中的缺陷会导致各种肺(囊性纤维化)和肾脏(肾源性尿崩症)疾病。因此，对极化膜运输机制的新认识可能导致识别新的药物靶点以及旨在识别和治疗膜运输障碍的新的治疗方法。

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.