Role of dynamin for podocytes structure and function

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。

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.