Molecular Analysis of Microtubule Function in Yeast

酵母微管功能的分子分析

• 批准号: 6917992
• 负责人: TIM C HUFFAKER
• 金额: $ 33.95万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-11-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917992
• 关键词: Saccharomyces cerevisiae; binding proteins; cell growth regulation; centromere; chromosome movement; electron microscopy; fluorescence microscopy; fungal genetics; gene expression; gene mutation; green fluorescent proteins; immunoprecipitation; microtubule associated protein; microtubules; mitotic spindle apparatus; molecular cloning; phenotype; phosphorylation; polymerase chain reaction; protein biosynthesis; protein protein interaction; protein structure function; transcription factor; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Microtubules are dynamic polymers that play essential roles in a variety of cellular processes such as organelle transport and chromosome segregation. The long-term objective of this proposal is to gain a better understanding of the biological role of microtubule dynamics and the ways in which these dynamic properties are regulated in the cell. Microtubule dynamics in cells is likely to be determined by the complex interplay of proteins that bind to microtubules and regulate their assembly. Three microtubule-binding proteins in the yeast S. cerevisiae, Stu2p, Bik1p and Bim1p, interact with each other in all pairwise combinations. The combinatorial effects of these proteins and the functional significance of their protein-protein interactions in regulating microtubule dynamics in vivo will be determined by making and analyzing specific mutants. In addition, the effects of these proteins, alone and in combination, on the dynamic properties of microtubules in vitro will be assayed. A fourth microtubule-binding protein, Stu1p, is found on spindle microtubules and recent findings indicate a role for Stu1p at the kinetochore. Experiments are proposed to determine if Stu1p localizes to kinetochores, the dynamics of kinetochore microtubules in cells lacking Stu1p, and the intrinsic effects of Stu1p on the dynamic properties of microtubules in vitro. Although the dynamic properties of microtubules are believed to be central to their function, the extent to which cells rely on microtubule dynamics has not been tested experimentally. Mutations in beta-tubulin that alter the dynamic properties of microtubules in S. cerevisiae will be constructed. The effect of these mutations on microtubule dynamics and function in vivo and on microtubule dynamics in vitro will be measured. The aim of this work is to understand how intrinsic changes in the dynamic properties of microtubules affect microtubule dynamics and function in the cell.

描述（由申请人提供）：微管是动态聚合物 在各种细胞过程中扮演重要角色，例如Organelle 运输和染色体分离。该建议的长期目标是更好地了解微管的生物学作用 动力学以及这些动态属性的调节方式 细胞。 细胞中的微管动力学可能由复合物确定 与微管结合并调节其组装的蛋白质相互作用。 酵母菌S.酿酒酵母，Stu2p，Bik1p和 BIM1P，以所有成对组合相互作用。这 这些蛋白质的组合作用以及功能意义 它们在调节体内微管动力学方面的蛋白质蛋白质相互作用 将通过制造和分析特定突变体来确定。另外， 这些蛋白质（单独和组合）对动态特性的影响 将分析微管的体外。第四个微管结合蛋白， Stu1p在纺锤微管上发现，最近的发现表明 stu1p在动力学上。提出了实验以确定STU1P是否 定位于动力学，动力学微管在细胞中的动力学 缺乏Stu1p，以及Stu1p对动态特性的内在影响 体外微管。 尽管微管的动态特性被认为是 它们的功能，细胞依赖微管动力学的程度尚未 经过实验测试。改变动态的β-微管蛋白突变 将构建酿酒酵母中微管的特性。效果 这些关于微管动力学和功能在体内和微管上功能的突变 将测量体外动力学。这项工作的目的是了解如何 微管动态特性的内在变化会影响微管 细胞中的动力学和功能。