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: 3304698
• 负责人: ALISON E ADAMS
• 金额: $ 13万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304698
• 关键词: Saccharomyces cerevisiae; actin binding protein; actins; autoradiography; cell motility; chickens; cytoskeleton; fluorescence microscopy; fungal genetics; gene complementation; gene expression; gene interaction; gene mutation; genetic manipulation; human tissue; laboratory rabbit; molecular cloning; molecular genetics; oligonucleotides; protein biosynthesis; protein sequence; protein structure function; radionuclides; site directed mutagenesis; temperature sensitive mutant; ultraviolet spectrometry

The actin cytoskeleton has been implicated in a large variety of motile functions, as well as in the structural and mechanical properties of the cytoplasmic matrix. Except in muscle cells, however, very little is known about its composition, roles, or mechanism of action. The proposed research is designed to determine at the molecular level how the actin cytoskeleton functions in the yeast Saccharomyces cerevisiae. Yeast can be readily manipulated genetically, and should allow rapid and detailed analysis of these complex processes. Because actin is highly conserved from yeast to man, what is learned from these studies is likely to apply to higher cells, where defective actin cytoskeleton function is implicated in pathological conditions such as muscular dystrophy and neoplastic transformation. The following specific questions will be addressed: 1. How does SAC6p, an actin-binding protein, function in the cytoskeleton? Conditional-lethal mutations in the SAC6 gene will be produced by in vitro mutagenesis. Biochemical and physiological analyses of a full spectrum of mutants will then allow activities observed in vitro to be correlated with functions seen in vivo. 2. What residues or domains are important for function of SAC6p and its higher-cell homologs? Fine-structure mapping of mutations will be used to identify regions of the gene associated with various C6p activities. Specific hypotheses concerning sequences such as the putative actin-binding domain (a highly conserved 25 amino acid sequence found in a number of actin-binding proteins) will be tested by site-specific mutagenesis. Information from these studies will be used in an analysis of human plastin and chicken fimbrin, which are 40% identical to SAC6p. 3. What other proteins are involved in the function of the actin cytoskeleton? The diversity of actin cytoskeletal function must be mediated by non-actin proteins that regulate actin assembly, establish interactions between the cytoskeleton and other cell structures, and generate the force for intracellular motility. A variety of genetic methods will be used to identify additional components of the actin cytoskeleton, and proteins that interact with it.

肌动蛋白细胞骨架与多种运动性疾病有关。 功能，以及在结构和机械性能方面 细胞质基质。然而，除了肌肉细胞，人们对此知之甚少。 关于它的组成、作用或作用机制。拟议的研究 是为了在分子水平上确定肌动蛋白细胞骨架 酿酒酵母的功能。酵母可以很容易地 在基因上被操纵，并应允许快速和详细地分析 这些复杂的过程。因为肌动蛋白从酵母到酵母高度保守 伙计，从这些研究中学到的东西很可能适用于更高级的细胞， 肌动蛋白细胞骨架功能缺陷与病理 肌肉营养不良和肿瘤转化等情况。这个 将讨论以下具体问题： 1.肌动蛋白结合蛋白SAC6p在细胞骨架中是如何发挥作用的？ SAC6基因的条件致死突变将在体外产生 诱变。全谱生化和生理分析 然后，突变体将允许在体外观察到的活动与 在活体中看到的功能。 2.哪些残基或结构域对SAC6p及其功能起重要作用 更高级的细胞同源物？突变的精细结构图谱将用于 确定与各种C6p活动相关的基因区域。 关于序列的特定假设，如假定的肌动蛋白结合 结构域(高度保守的25个氨基酸序列，存在于许多 肌动蛋白结合蛋白)将通过定点突变进行测试。 来自这些研究的信息将被用于分析人类纤溶酶 和鸡毛蛋白，它们与SAC6P有40%的同源性。 3.肌动蛋白的功能还涉及哪些其他蛋白质 细胞骨架？肌动蛋白细胞骨架功能的多样性必须被调节 通过非肌动蛋白调节肌动蛋白组装，建立相互作用 在细胞骨架和其他细胞结构之间，并产生力 用于细胞内的运动。将使用各种遗传方法来 确定肌动蛋白细胞骨架的其他成分，以及 与之互动。