MOLECULAR ANALYSIS OF THE YEAST ACTIN CYTOSKELETON

酵母肌动蛋白细胞骨架的分子分析

• 批准号: 6018786
• 负责人: DAVID G DRUBIN
• 金额: $ 20.76万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018786
• 关键词: Saccharomyces cerevisiae; actin binding protein; actins; adenosine triphosphate; biological signal transduction; cytoskeleton; gene expression; gene mutation; genetic regulatory element; histogenesis; hydrolysis; laboratory rabbit; molecular cloning; nucleic acid sequence; nucleotides; protein biosynthesis; protein structure function; second messengers; yeast two hybrid system

Biochemical and genetic approaches will be used to study actin assembly in yeast. Principles established are likely to apply to more complex eukaryotes where actin dynamics underlie diverse cellular processes and where defective cytoskeleton function contributes to conditions such as muscular dystrophy, certain hereditary anemias, and cancer. The following aims will be pursued: (1) Actin nucleotide-binding pocket mutant. To test physiological importance of actin ATP hydrolysis and Pi release, and to genetically identify regulators of filament stability, we will take advantage of a unique actin mutant. The V159N mutation uncouples the actin nucleotide cycle from filament destabilization. Specifically, we will test the role of rapid actin filament turnover in pheromone-induced cellular morphogenesis and cortical actin patch motility, and will use the V159N mutant to genetically identify regulators of actin dynamics. (2) Biochemical and structure-function analysis of the cofilin-actin interaction. Our isolation of yeast cofilin, elucidation of its three dimensional molecular structure, and demonstration that it promotes actin filament disassembly in vivo, provide a strong foundation for further studies of this important and ubiquitous protein. The molecular models of yeast cofilin and actin filaments will now be docked, providing novel insights into the mechanism of cofilin-promoted filament disassembly. To more fully elucidate steps regulating the assembly/disassembly cycle, we will identify factors which stimulate formation of ATP-actin monomers from ADP-actin:cofilin complexes formed during disassembly. (3) Genetic analysis of cofilin regulation and regulation of actin filament stability. In response to regulatory signals, changes in actin assembly typically occur on time scales that mandate regulation by second messengers and posttranslational modification. Since genetic approaches provide powerful avenues to elucidation of regulatory pathways, proteins which regulate cofilin will be identified by genetic suppression. These studies are important for determining how cells trigger rapid cytoskeletal rearrangements required for diverse processes including morphogenesis and cytokinesis.

生化和遗传学方法将用于研究肌动蛋白组装 在酵母中。 建立的原则可能适用于更复杂的情况 真核生物中，肌动蛋白动力学是多种细胞过程的基础 细胞骨架功能缺陷会导致以下情况： 肌营养不良症、某些遗传性贫血和癌症。 这 将追求以下目标：（1）肌动蛋白核苷酸结合口袋 突变体。 测试肌动蛋白 ATP 水解的生理重要性 Pi 释放，并从基因上鉴定丝的调节因子 稳定性，我们将利用独特的肌动蛋白突变体。 V159N 突变使肌动蛋白核苷酸循环与丝状体脱钩 不稳定。 具体来说，我们将测试快速肌动蛋白的作用 信息素诱导的细胞形态发生中的细丝周转和 皮质肌动蛋白补丁运动性，并将使用 V159N 突变体 从基因上识别肌动蛋白动力学的调节因子。 (2)生化及 丝切蛋白-肌动蛋白相互作用的结构-功能分析。 我们的 酵母丝切蛋白的分离及其三维结构的阐明 分子结构，并证明它促进肌动蛋白丝 体内拆卸，为进一步研究提供坚实的基础 这种重要且普遍存在的蛋白质。 酵母的分子模型 丝切蛋白和肌动蛋白丝现在将被对接，提供新的见解 进入 cofilin 促进丝分解的机制。 到更多 充分阐明调节组装/拆卸周期的步骤，我们将 确定刺激 ATP-肌动蛋白单体形成的因素 ADP-肌动蛋白：分解过程中形成丝切蛋白复合物。 (3) 遗传 丝切蛋白调控和肌动蛋白丝调控分析 稳定。响应调节信号，肌动蛋白组装发生变化 通常发生在要求以秒为单位进行监管的时间范围内 信使和翻译后修饰。 自从基因方法 为阐明调控途径、蛋白质提供强有力的途径 调节丝切蛋白的基因将通过基因抑制来鉴定。 这些 研究对于确定细胞如何快速触发非常重要 不同过程所需的细胞骨架重排，包括 形态发生和胞质分裂。