FUNCTION OF ALPHA TUBULIN IN MICROTUBULE DYNAMICS

α 微管蛋白在微管动力学中的功能

• 批准号: 2777599
• 负责人: Kirk Richard Anders
• 金额: $ 3.17万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2777599
• 关键词: enzyme activity; gene mutation; guanosine triphosphate; guanosinetriphosphatases; intermolecular interaction; microtubules; protein structure function; tubulin; yeasts

The formation and function of microtubule structures depends on dynamic instability, the asynchronous slow growth and rapid shrinkage of microtubules under equilibrium conditions. A complete understanding of dynamic instability will be valuable in determining how cells regulate microtubules, and should provide insights for the development of antimitotic drugs for chemotherapeutic uses. Dynamic instability arises from the intrinsic properties of the alpha/beta tubulin dimers. Beta- tubulin hydrolyzes GTP upon assembly into microtubules. This assembly- dependent GTP hydrolysis is essential for the rapid shrinkage of microtubules. It is not known how assembly stimulates GTP hydrolysis. This research proposal tests the hypothesis that alpha-tubulin stimulates GTP hydrolysis by interdimer contact with beta tubulin upon assembly. Yeast tubulin mutants, altered at dimer-dimer interfaces, will be examined for their effects on microtubule dynamics and GTP hydrolysis. Microtubule dynamics will be visualized in living cells by GFP-labeling and epifluorescence microscopy. Mutant tubulins that alter microtubu1e dynamics will be purified and their GTPase activity assayed during assembly. Among the mutants that will be tested, the alpha- tubulin mutant tub1-828 will be studied for the following reasons: (i) it contains a mutation of glutamate-255 (E255), which likely interacts with beta-tubulin-bound GTP only in assembled protofilaments, (ii) mutation of the E255-cognate residue in the tubulin-like bacterial protein FtsZ eliminates GTPase activity without affecting GTP binding. A beta-tubulin mutant with accelerated GTPase activity, tub2-T1O7K, will be tested to see if it suppresses tub1-828. The two mutations in tub1-828, E255A and D232A, will be separated and examined individually to define the function of each residue. The results of these studies will define the currently unknown function of alpha-tubulin in dynamic instability and establish a heretofore missing link between altered in vivo microtubule dynamics and the overall phenotypes of tubulin mutants in yeast.

微管结构的形成和功能取决于动态不稳定性，即微管在平衡条件下的异步缓慢生长和快速收缩。 动态不稳定性的完整理解将是有价值的，在确定细胞如何调节微管，并应提供见解的抗有丝分裂药物的化疗用途的发展。动态不稳定性由α/β微管蛋白二聚体的内在性质引起。β-微管蛋白在组装成微管时水解GTP。这种组装依赖的GTP水解对于微管的快速收缩是必不可少的。目前尚不清楚组装如何刺激GTP水解。这项研究计划测试的假设，α-微管蛋白刺激GTP水解的二聚体接触β微管蛋白组装。酵母微管蛋白突变体，在二聚体-二聚体界面改变，将检查其对微管动力学和GTP水解的影响。微管动力学将通过GFP标记和落射荧光显微镜在活细胞中可视化。将纯化改变微管动力学的突变微管蛋白，并在组装过程中测定其GTdR活性。在将要检测的突变体中，将出于以下原因研究α-微管蛋白突变体tub 1 -828：（i）其含有谷氨酸-255（E255）突变，其可能仅在组装的原丝中与β-微管蛋白结合的GTP相互作用，（ii）微管蛋白样细菌蛋白FtsZ中E255同源残基的突变消除了GTP酶活性，而不影响GTP结合。将测试具有加速的GTT活性的β-微管蛋白突变体tub 2-T1 O 7 K，以观察其是否抑制tub 1 -828。将分离并单独检查tub 1 -828中的两个突变E255 A和D232 A，以确定每个残基的功能。这些研究的结果将确定目前未知的功能α-微管蛋白在动态不稳定性和建立一个迄今为止缺失的联系之间的改变在体内微管动力学和整体表型的微管蛋白突变体在酵母中。