Role of dynamin for podocytes structure and function

动力对足细胞结构和功能的作用

• 批准号: 8221205
• 负责人: Sanja Sever
• 金额: $ 39.01万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8221205
• 关键词: Actins; Adaptor Signaling Protein; Affect; Affinity; Antibodies; Applications Grants; Arginine; Binding; C-terminal; Clathrin; Clathrin-Coated Vesicles; Complex; Cytoskeleton; Data; Down-Regulation; Dynamin; Endocytosis; Epithelial Cells; Fluorescence Microscopy; Foot Process; Functional disorder; Gelsolin; Glomerular Capillary; Goals; Guanosine Triphosphate Phosphohydrolases; Higher Order Chromatin Structure; In Vitro; Integral Membrane Protein; Intercellular Junctions; Kidney Diseases; Kidney Glomerulus; Laboratories; Link; Lipids; Mediating; Membrane; Microfilaments; Molecular; NCK adaptor protein 1; NPHS2 protein; Phenotype; Phosphorylation; Play; Proline; Proteins; Recruitment Activity; Regulation; Role; SH3 Domains; Signal Transduction; Staging; Structure; Testing; United States; Work; base; blood filtration; coated pit; in vitro Assay; mutant; nephrin; neuronal cell body; novel; podocyte; polymerization; slit diaphragm; small molecule

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the molecular mechanism(s) that underlie pathophysiology of proteinuric kidney diseases. Work from this laboratory recently demonstrated direct regulation of actin assembly in vitro and in podocytes by dynamin, and suggested an intricate interplay between dynamin's oligomerization cycle and actin cytoskeleton dynamics. Our findings motivate the hypothesis that signaling at the slit diaphragm alters dynamin oligomerization cycle through direct dynamin-Nck interactions, which in turn initiates actin polymerization in the membrane vicinity. In Specific Aim 1 we test whether clustering of Nck on the membrane promotes dynamin oligomerization into rings, which in turn drives actin polymerization in cultured podocytes. Using small molecules that specifically promote or inhibit dynamin oligomerization, as well as our novel actin binding domain mutants of dynamin, we test whether dynamin oligomerization cycle and actin-binding interactions are essential and/or sufficient for Nck-dependent polymerization of cortical actin cytoskeleton. In Specific Aim 2 we test whether direct interactions between dynamin and Nck alter the dynamin oligomerization cycle and dynamin-dependent actin polymerization using in-vitro assays. In Specific Aim 3, using TIR-FM we test whether direct dynamin-actin interactions are essential for clathrin mediated endocytosis. PUBLIC HEALTH RELEVANCE: In the United States alone, glomerular type of kidney diseases affects some 20 million people, and this number has roughly doubled within the last two decades. Here we propose to test the hypothesis that signaling at the slit diaphragm alters dynamin oligomerization cycle through direct dynamin-Nck interactions, which in turn initiates actin polymerization in the membrane vicinity. Positive data will identify the missing link between signaling at the slit diaphragm and organization of the actin cytoskeleton in podocytes.

描述（由申请人提供）：本提案的目的是阐明蛋白尿性肾病病理生理学基础的分子机制。该实验室最近的工作表明，在体外和足细胞中，发动蛋白直接调节肌动蛋白的组装，并提出了发动蛋白的寡聚化周期和肌动蛋白细胞骨架动力学之间复杂的相互作用。我们的研究结果激发了这样一种假设，即在狭缝隔膜处的信号通过直接的动力蛋白-Nck相互作用改变动力蛋白寡聚化周期，这反过来又在膜附近启动肌动蛋白聚合。在特定目标1中，我们测试了Nck在膜上的聚集是否促进发动蛋白寡聚成环，这反过来又驱动了培养的足细胞中的肌动蛋白聚合。使用小分子，特别是促进或抑制发动蛋白寡聚化，以及我们的新的肌动蛋白结合结构域突变体的发动蛋白，我们测试发动蛋白寡聚化周期和肌动蛋白结合的相互作用是否是必不可少的和/或足够的NCK依赖的聚合皮质肌动蛋白细胞骨架。在具体目标2中，我们测试发动蛋白和NCK之间的直接相互作用是否改变发动蛋白寡聚化周期和发动蛋白依赖性肌动蛋白聚合使用体外测定。在具体目标3中，使用TIR-FM，我们测试直接动力蛋白-肌动蛋白相互作用是否是网格蛋白介导的内吞作用所必需的。 公共卫生相关性：仅在美国，肾小球型肾病就影响了约2000万人，并且这个数字在过去二十年中大约翻了一番。在这里，我们建议测试的假设，即信号在狭缝隔膜改变发动蛋白寡聚化周期通过直接发动蛋白NCK相互作用，这反过来又引发肌动蛋白聚合在膜附近。积极的数据将确定在狭缝隔膜和组织的肌动蛋白细胞骨架在足细胞的信号之间的缺失环节。