Regulation of Actin Dynamics

肌动蛋白动力学的调节

• 批准号: 6899294
• 负责人: DAVID C AMBERG
• 金额: $ 29.24万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6899294
• 关键词: Saccharomyces cerevisiae; actins; cellular polarity; fungal genetics; fungal proteins; molecular assembly /self assembly; protein localization; protein structure function

DESCRIPTION (provided by applicant): The actin cytoskeleton participates in diverse cellular functions including cellular organization, polarized cell growth, membrane traffic, cell shape changes, cytokinesis and cell motility. These functions require precise control of the assembly, dynamics and organization of the actin filaments that constitute the core of the actin cytoskeleton. Defects in proper actin regulation can contribute to human disease including cancer cell metastasis, Wiskott-Aldrich Syndrome, and sickle cell crisis to name a few. Our lab seeks to understand how actin associated proteins of S. cerevisiae regulate the assembly and dynamics of actin filaments and how this regulation contributes to cell function. The focus is on three novel regulators of actin dynamics: Actin Interacting Protein 3 (Alp3), Actin Interacting Protein 1 (Alp1), and Old Yellow Enzyme (Oye2). Aim#1: An in vitro biochemical analysis of Aip3 activity will be undertaken to understand how Aip3p cooperates with other cellular proteins to coordinate cell-cycle controlled, polarized actin assembly. This will be complemented by genetic analyses designed to uncover the networks of genes that cooperate with AlP3 to regulate polarization of the actin cytoskeleton. Aim #2: Aip1 is a novel actin filament severing protein whose activity is constrained to filaments decorated with cofilin, another actin binding protein. Through mutagenesis we have begun to uncover the structure/function relationships between domains of Aip1 and cofilin. This has led to hypotheses concerning how Aiplp negatively auto-regulates its severing activity and possible mechanisms for the severing reaction. We will test these hypotheses by identifying protein interaction defects of mutant alleles and correlating those defects with biochemical defects in Alp1 regulation and filament severing. Genetic analysis will be used to uncover the genes that interact with an aip1delta allele, these will be tested against our battery of aip1mutant alleles to define a genome level, genetic and biochemical structure/function map of ALP1.Aim#3: Preliminary analysis of oyedelta strains suggests that Oye2p regulates reduction of an intramolecular disulfide bond in actin. This form of actin oxidation in red blood cells contributes to sickle cell crisis by reducing the dynamic behavior of actin filaments. We will test this comparison by quantifying the amount of oxidized actin that accumulates in oyedelta yeast strains and by directly measuring reduction of red blood cell actin by Oye2p. Genetic analysis will be used to define those genes and pathways that require proper redox regulation of the actin cytoskeleton. These experiments will help to establish the yeast oyedelta strain as a model system for human disease states (such as sickle cell crisis) that are affected by actin oxidation.

描述（由申请人提供）：肌动蛋白细胞骨架参与各种细胞功能，包括细胞组织，极化细胞生长，膜流量，细胞形状变化，细胞因子和细胞运动。这些功能需要精确控制构成肌动蛋白细胞骨架核心的肌动蛋白丝的组装，动力学和组织。适当的肌动蛋白调节缺陷会导致人类疾病，包括癌细胞转移，Wiskott-Aldrich综合征和镰状细胞危机。我们的实验室试图了解静脉葡萄球菌的蛋白质如何调节肌动蛋白丝的组装和动力学以及该调节如何促进细胞功能。重点是肌动蛋白动力学的三个新型调节剂：肌动蛋白相互作用蛋白3（ALP3），肌动蛋白相互作用蛋白1（ALP1）和旧的黄色酶（OYE2）。 AIM＃1：将进行AIP3活性的体外生化分析，以了解AIP3P如何与其他细胞蛋白合作以协调细胞周期控制的偏振肌动蛋白组装。这将通过旨在揭示与Alp3合作以调节肌动蛋白细胞骨架极化的基因网络的遗传分析进行补充。 AIM＃2：AIP1是一种新型的肌动蛋白丝切断的蛋白质，其活性限制为用Cofilin（另一种肌动蛋白结合蛋白）装饰的细丝。通过诱变，我们已经开始发现AIP1和Cofilin域之间的结构/功能关系。这导致了关于AIPLP如何自动调节其切断活性和可能对切断反应的可能机制的假设。我们将通过鉴定突变等位基因的蛋白质相互作用缺陷来检验这些假设，并将这些缺陷与ALP1调节和细丝切断中的生化缺陷相关联。 Genetic analysis will be used to uncover the genes that interact with an aip1delta allele, these will be tested against our battery of aip1mutant alleles to define a genome level, genetic and biochemical structure/function map of ALP1.Aim#3: Preliminary analysis of oyedelta strains suggests that Oye2p regulates reduction of an intramolecular disulfide bond in actin.红细胞中这种肌动蛋白氧化形式通过减少肌动蛋白丝的动态行为来促进镰状细胞危机。我们将通过量化积聚在Oyedelta酵母菌菌株中的氧化肌动蛋白的量以及直接测量OYE2P对红细胞肌动蛋白的还原来测试这种比较。遗传分析将用于定义那些需要适当的肌动蛋白细胞骨架调节的基因和途径。这些实验将有助于确定受肌动蛋白氧化影响的人类疾病状态（例如镰状细胞危机）的模型系统（例如镰状细胞危机）的模型系统。