FUNCTION OF ALPHA TUBULIN IN MICROTUBULE DYNAMICS

微管结构的形成和功能取决于动态不稳定性，平衡条件下微管的异步缓慢生长和快速收缩。对动态不稳定性的完整理解将有助于确定细胞如何调节微管，并为开发用于化疗的抗有丝分裂药物提供见解。动态不稳定性源于α / β微管蛋白二聚体的固有特性。微管蛋白在组装成微管时水解GTP。这种依赖于组装的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。本研究方案验证了α -微管蛋白在组装时通过与α -微管蛋白的中间二聚体接触刺激GTP水解的假设。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标记和荧光显微镜在活细胞中可视化。改变微管动力学的突变微管蛋白将被纯化，并在组装过程中测定其GTPase活性。在即将测试的突变体中，α -微管蛋白突变体tub1-828将被研究，原因如下：(i)它含有谷氨酸-255 （E255）突变，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酶的活性，而不影响GTP的结合。一种具有加速GTPase活性的β -微管蛋白突变体，tub2- t17k，将被测试，看它是否抑制tub1-828。tub1-828中的两个突变E255A和D232A将被分离并单独检测，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α -微管蛋白在动态不稳定性中的功能，并在酵母中改变的体内微管动力学和微管蛋白突变体的整体表型之间建立迄今为止缺失的联系。

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.