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: 6870133
• 负责人: Sanja Sever
• 金额: $ 30.8万
• 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/6870133
• 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）：质膜上网格蛋白包被的囊泡形成是网格蛋白介导的内吞作用过程的基础，细胞通过该过程内化受体、跨膜通道、转运蛋白、激素生长因子和营养物质。内吞作用的错误调节可导致肿瘤转化。因此，网格蛋白介导的内吞作用的机制和调控对人类健康至关重要。内吞作用的早期事件涉及网格蛋白包被的小凹的形成、小凹内陷、游离囊泡的收缩和出芽。内吞作用所需的重要因素之一是动力蛋白，这是一种不寻常的 GTP 酶，其作​​用机制仍不确定。最近的证据表明，动力蛋白是内吞作用的主要调节者，利用其 GTP 酶循环来招募执行内陷囊泡收缩的蛋白质机制。该实验室现已鉴定出 Hsc70 及其辅助伴侣辅助素是两种与动力蛋白：GTP 特异性相互作用的蛋白质。辅助素内与动力相互作用的两个结构域已被绘制出来，体外内吞作用测定中辅助素的免疫耗竭可抑制收缩囊泡的形成。这些结果与 Hsc70/auxilin 作为动力效应器一致。重要的是，已知 Hsc70/auxilin 可以催化网格蛋白外壳从游离囊泡中去除。因此，辅助素和 Hsc70 在内吞作用的早期（囊泡形成）和晚期（囊泡脱壳）阶段起作用。结果表明，在一个模型中，许多或可能所有阶段的胞吞作用都是由 Hsc70/辅助素介导的网格蛋白外壳重排驱动的。据设想，Hsc70/auxilin 对网格蛋白外壳的具体影响是通过与动力相互作用来调节的。该提案解决了以下问题：具体目标 1 结合使用电子显微镜和辅助素依赖性体外内吞作用测定来识别需要辅助素的网格蛋白包被囊泡形成的所有步骤（起始、内陷、收缩、裂变）。在特定目标 2 中，辅助素结合缺陷的动力突变体将用于证实辅助素是动力效应子的模型。具体目标 3 通过测试动力蛋白的严重组装突变体是否支持内吞作用来检查动力蛋白自组装的功能。将使用荧光寿命成像显微镜（FLIM）在体内确认突变体的组装缺陷。具体目标 4 建立在辅助素与脂质结合的发现之上，并将测试辅助素是否充当脂质磷酸酶。将使用半透细胞测定来检查内吞作用中辅助素的脂质结合/磷酸酶活性的功能。