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: 7333296
• 负责人: Sanja Sever
• 金额: $ 28.62万
• 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/7333296
• 关键词: Address; Affect; Auxilins; Binding; Biological Assay; Cell membrane; Cells; Chemicals; Clathrin; Clathrin-Coated Vesicles; Constriction procedure; DNA Sequence Rearrangement; Defect; Dominant-Negative Mutation; Dynamin; Electron Microscopy; Endocytosis; Enzymes; Event; Excision; Figs - dietary; Fluorescence; Fluorescence Microscopy; GTP Binding; Goals; Grant; Growth Factor; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Homologous Gene; Hormones; Human; Hydrolysis; Image; In Vitro; Laboratories; Lipid Binding; Lipids; Maps; Mediating; Membrane; Membrane Proteins; Microscopy; Modeling; Molecular; Molecular Chaperones; Neoplastic Cell Transformation; Nutrient; PH Domain; PTEN gene; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Process; Property; Proteins; Recruitment Activity; Regulation; Research Personnel; Role; Staging; Testing; Transcription Factor AP-2 Alpha; Tumor Suppressor Proteins; Vesicle; Work; base; coated pit; in vivo; mutant; programs; receptor; research study; self assembly

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（由申请方提供）：细胞质膜上网格蛋白包被囊泡的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、生长因子和营养素。内吞作用的错误调节可引起肿瘤转化。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。胞吞作用的早期事件包括网格蛋白包被的小凹的形成、小凹内陷、收缩和游离小泡的出芽。内吞作用所需的基本因素之一是发动蛋白，一种不寻常的GTdR，其作用机制仍不确定。最近的证据表明，发动蛋白是内吞作用的主要调节因子，利用其GTdR循环来招募执行内陷囊泡收缩的蛋白质机制。这个实验室现在已经确定Hsc 70和它的辅助分子伴侣生长素是两种与发动蛋白GTP特异性相互作用的蛋白质。生长素与发动蛋白相互作用的两个结构域已经被绘制出来，并且从体外内吞测定中免疫耗竭生长素抑制收缩囊泡的形成。这些结果与Hsc 70/auxilin是动力蛋白效应子一致。重要的是，已知Hsc 70/生长素催化从游离囊泡去除网格蛋白外壳。因此，生长素和Hsc 70作用于内吞作用的早期（囊泡形成）和晚期（囊泡脱壳）阶段。结果表明，在其中许多，或者也许是所有的，阶段的内吞驱动Hsc 70/生长素介导的网格蛋白外套的重排模型。据设想，Hsc 70/生长素对网格蛋白外壳的具体后果是通过与发动蛋白的相互作用来调节的。该提案解决了以下问题：具体目标1使用电子显微镜和生长素依赖性体外内吞测定的组合，以确定网格蛋白包被的囊泡形成需要生长素的所有步骤（起始，内陷，收缩，裂变）。在具体目标2中，缺乏生长素结合的发动蛋白突变体将用于证实生长素是发动蛋白效应子的模型。具体目标3通过测试发动蛋白的严重组装突变体是否支持内吞作用来检查发动蛋白自组装的功能。突变体的组装缺陷将使用荧光寿命成像显微镜（FLIM）在体内确认。具体目标4建立在生长素结合脂质的发现上，并将测试生长素是否作为脂质磷酸酶。将使用半透性细胞测定来检查生长素在胞吞作用中的脂质结合/磷酸酶活性的功能。