Regulation of Actin Dynamics

描述(申请人提供)：肌动蛋白细胞骨架参与多种细胞功能，包括细胞组织、极化细胞生长、膜运输、细胞形状变化、胞质分裂和细胞运动。这些功能需要对构成肌动蛋白细胞骨架核心的肌动蛋白细丝的组装、动力学和组织进行精确控制。适当的肌动蛋白调控缺陷可能导致人类疾病，包括癌细胞转移、Wiskott-Aldrich综合征和镰状细胞危机等。我们的实验室试图了解酿酒酵母的肌动蛋白相关蛋白如何调控肌动蛋白细丝的组装和动力学，以及这种调控如何有助于细胞功能。重点是肌动蛋白动力学的三个新的调节因子：肌动蛋白相互作用蛋白3(Alp3)、肌动蛋白相互作用蛋白1(Alp1)和老黄酶(Oye2)。目的#1：对Aip3活性进行体外生化分析，以了解Aip3p如何与其他细胞蛋白合作来协调细胞周期控制的极化肌动蛋白组装。这将得到基因分析的补充，该分析旨在揭示与AlP3合作的基因网络，以调节肌动蛋白细胞骨架的极化。目的#2：Aip1是一种新的肌动蛋白细丝切断蛋白，其活性受另一种肌动蛋白结合蛋白cofilin修饰的微丝的限制。通过突变，我们已经开始揭示Aip1结构域和cofilin之间的结构/功能关系。这导致了关于Aiplp如何负向自动调节其切断活性和切断反应的可能机制的假说。我们将通过确定突变等位基因的蛋白质相互作用缺陷并将这些缺陷与Alp1调节和细丝切断中的生化缺陷相关联来检验这些假设。遗传分析将被用来发现与aip1Delta等位基因相互作用的基因，这些基因将与我们的一组aip1突变等位基因进行测试，以确定ALP1的基因组水平、遗传和生化结构/功能图。目的#3：对OyeDelta菌株的初步分析表明，Oye2p调节肌动蛋白中分子内二硫键的减少。这种形式的肌动蛋白氧化在红细胞中通过减少肌动蛋白细丝的动态行为而导致镰状细胞危机。我们将通过量化OyeDelta酵母菌株中积累的氧化肌动蛋白的数量和直接测量Oye2p对红细胞肌动蛋白的减少来测试这种比较。基因分析将被用于定义那些需要对肌动蛋白细胞骨架进行适当的氧化还原调节的基因和途径。这些实验将有助于建立酵母OyeDelta菌株作为受肌动蛋白氧化影响的人类疾病状态(如镰状细胞危机)的模型系统。

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.