Role of dynamin-auxilin interactions in endocytosis

动力-辅助素相互作用在内吞作用中的作用

• 批准号: 6998869
• 负责人: Sanja Sever
• 金额: $ 30.08万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6998869
• 关键词: cell component structure /function; clathrin; dynamin; electron microscopy; endocytosis; enzyme activity; fluorescence microscopy; guanosinetriphosphatases; membrane activity; membrane transport proteins; molecular assembly /self assembly; protein binding; protein biosynthesis; protein protein interaction; protein structure function; vesicle /vacuole

DESCRIPTION (provided by applicant): Clathrin-coated vesicle formation at the plasma membrane underlies the process of clathrin-mediated endocytosis by which the cell internalizes receptors, transmembrane channels, transporters, hormones growth factors and nutrients. Mis-regulation of endocytosis can cause neoplastic transformation. Therefore, the mechanism and regulation of clathrin-mediated endocytosis are of critical importance for human health. The early events of endocytosis involve formation of clathrin-coated pits, pit invagination, constriction and budding of the free vesicle. One of the essential factors required for endocytosis is dynamin, an unusual GTPase whose mechanism of action remains uncertain. Recent evidence suggests that dynamin is a master regulator of endocytosis, using its GTPase cycle to recruit the protein machinery that executes the constriction of invaginated vesicles. This laboratory has now identified Hsc70 and its co-chaperone auxilin as two proteins that specifically interact with dynamin:GTP. Two domains within auxilin that interact with dynamin have been mapped, and immunodepletion of auxilin from an in vitro endocytosis assay inhibits formation of constricted vesicles. These results are consistent with Hsc70/auxilin being dynamin effector. Importantly, Hsc70/auxilin was already known to catalyze removal of the clathrin coat from the free vesicle. Therefore, auxilin and Hsc70 act at early (vesicle formation) and late (vesicle uncoating) stages of endocytosis. The results suggest a model in which many, or perhaps all, stages of enocytosis are driven by Hsc70/auxilin-mediated rearrangements of the clathrin coat. It is envisioned that the specific consequences of Hsc70/auxilin on the clathrin coat are regulated by interactions with dynamin. This proposal addresses the following questions: Specific Aim 1 uses a combination of electron microscopy and an auxilin-dependent in vitro endocytosis assay to identify all the steps (initiation, invagination, constriction, fission) in clathrin-coated vesicle formation that require auxilin. In Specific Aim 2, mutants of dynamin that are deficient in auxilin binding will be used to corroborate the model that auxilin is a dynamin effector. Specific Aim 3 examines the function of dynamin self-assembly by testing whether severe assembly mutants of dynamin support endocytosis. The assembly defects of the mutants will be confirmed in vivo using fluorescence lifetime imaging microscopy (FLIM). Specific Aim 4 builds on the finding that auxilin binds lipids and will test whether auxilin acts as a lipid phosphatase. The function of lipid binding/phosphatase activity by auxilin in endocytosis will be examined using a semi-permeable cell assay.

描述（由申请人提供）：在质膜上形成的网格蛋白包被囊泡是网格蛋白介导的内吞作用的基础，通过该过程，细胞内化受体、跨膜通道、转运体、激素、生长因子和营养物质。内吞作用调节不当可引起肿瘤转化。因此，网格蛋白介导的胞吞作用机制及其调控对人体健康至关重要。胞吞作用的早期事件包括网格蛋白包被的凹坑的形成、凹坑内陷、收缩和自由囊泡的出芽。胞吞作用的关键因素之一是动力蛋白，这是一种不寻常的gtp酶，其作用机制尚不确定。最近的证据表明，动力蛋白是内吞作用的主要调节剂，利用其GTPase循环来招募执行内陷囊泡收缩的蛋白质机制。该实验室现在已经确定Hsc70及其合作伙伴auxilin是两种与动力蛋白GTP特异性相互作用的蛋白质。辅助蛋白中与动力蛋白相互作用的两个结构域已经被绘制出来，并且从体外内吞试验中免疫消耗辅助蛋白可以抑制收缩囊泡的形成。这些结果与Hsc70/auxilin为动力效应相一致。重要的是，已经知道Hsc70/auxilin可以催化网格蛋白外壳从自由囊泡中去除。因此，auxilin和Hsc70在早期（囊泡形成）和晚期（囊泡脱落）阶段起作用。结果表明，在这个模型中，胞吞作用的许多阶段，或者可能是所有阶段，都是由Hsc70/辅助素介导的网格蛋白外壳重排驱动的。可以设想，Hsc70/auxilin对网格蛋白外壳的特定影响是通过与动力蛋白的相互作用来调节的。具体目标1使用电子显微镜和辅助蛋白依赖的体外内吞试验相结合，以确定在网格蛋白包被的囊泡形成中需要辅助蛋白的所有步骤（起始，内陷，收缩，裂变）。在Specific Aim 2中，将使用缺乏辅助素结合的动力蛋白突变体来证实辅助素是动力蛋白效应器的模型。特异性目的3通过检测dynamin的严重装配突变体是否支持内吞作用来检验dynamin自组装的功能。利用荧光寿命成像显微镜（FLIM）在体内证实突变体的组装缺陷。特异性目的4建立在辅助素结合脂质的发现上，并将测试辅助素是否作为脂质磷酸酶起作用。脂质结合/磷酸酶活性的功能辅助素在内吞作用将检查使用半透性细胞试验。