"The Role of CAP and sNASP in Physiological Actin Depolymerization"

描述(由申请人提供)：肌动蛋白细胞骨架是许多细胞过程所必需的细胞结构的关键组成部分，如细胞分裂和细胞运动。肌动蛋白存在于细胞内，这种浓度有利于组装并对拆解构成严重的挑战；事实上，我们目前还不知道细胞在生理条件下如何能够拆解肌动蛋白细丝。这项建议旨在通过确定和描述肌动蛋白周期中最不被理解的方面的因素：分解来了解生理学肌动蛋白的动力学。这样的理解将对任何细胞问题依赖肌动蛋白的临床或临床前研究领域产生重大影响；其中一个领域是肾病学。遗传分析发现，在导致家族性肾脏疾病的蛋白中，肌动蛋白结合蛋白最为突出：局灶性节段性肾小球硬化中的1-肌动蛋白和福尔马林，多囊肾病中的丝氨酸和其他肌动蛋白结合蛋白。我们已经使用基于活性的生化重构识别了两个以前未被认识的蛋白质，该重构增强了先前描述的cofilin、cortin和AIP1介导的肌动蛋白解聚系统。环酶相关蛋白(CAP)允许这种三重蛋白混合物在细胞内聚合和单体肌动蛋白浓度存在的情况下解聚荧光肌动蛋白底物，当单独使用时，三重混合只能处理生理单体肌动蛋白浓度。我们建议使用一系列技术，包括大量肌动蛋白阵列的荧光显微镜以及单个肌动蛋白细丝的电子和荧光显微镜，以更好地描述完成这一反应的机制。体细胞核自身抗原精子蛋白(SNASP)的功能类似于AIP1，但这种相似性还需要进一步分析。虽然AIP1是一种细胞质蛋白，而sNASP是一种核蛋白，我们已经确定它是一种肌动蛋白分解因子，由于已知它与组蛋白结合并在染色质重塑中发挥作用，它代表了细胞骨架和染色质结构之间的可能联系。我们计划使用许多与我们将用于表征CAP相同的技术来表征sNASP，并通过额外的遗传操作来测试体内的AIP1和sNASP冗余。

DESCRIPTION (provided by applicant): The actin cytoskeleton is a key component of cell structure necessary for many cellular processes such as cell division and cell motility. Actin exists at concentrations inside cells that favor assembly and pose serious challenges to disassembly; in fact, we currently do not know how the cell is able to disassembly actin filaments under physiological conditions. This proposal aims to understand physiological actin dynamics by identifying and characterizing the factors responsible for the least understood side of the actin cycle: disassembly. Such an understanding would have a major impact on any area of clinical or pre-clinical research where cellular problems are actin-dependent; one such area is nephrology. Genetic analysis has found actin-binding proteins prominent among those proteins responsible for familial kidney disorders: 1-actinin and formins in focal segmental glomerulosclerosis and filamin and other actin bundling protein in polycystic kindney disease. We have identified two previously unappreciated proteins using an activity-based biochemical reconstitution that augment the previously characterized cofilin-, coronin-, and AIP1- mediated actin depolymerization system. Cyclase associated protein (CAP) allows this triple-protein mix to depolymerize a fluorescent actin substrate in the presence of cellular concentrations of polymeric and monomeric actin when alone the triple-mix can only deal with physiological monomeric actin concentrations. We propose to use a range of techniques including fluorescent microscopy of bulk actin arrays and electron and fluorescent microscopy of single actin filaments to better characterize the mechanism by which this reaction is accomplished. Somatic nuclear autoantigenic sperm protein (sNASP) functions similarly to AIP1, though further analysis of this similarity is needed. While AIP1 is a cytoplasmic protein, sNASP is a nuclear protein that we have identified as an actin disassembly factor, and since it is known to bind histones and function in chromatin remodeling it represents a possible link between the cytoskeleton and chromatin structure. We plan to use many of the same techniques to characterize sNASP as we will use to characterize CAP, with additional genetic manipulations to test AIP1 and sNASP redundancy in vivo.