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、平衡条件下微管的非同步缓慢生长和快速收缩。对动态不稳定性的全面了解将有助于确定细胞如何调节微管，并应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性质。β-微管蛋白在组装成微管后将GTP水解成微管。这种依赖装配的GTP水解对于微管的快速收缩是必不可少的。目前尚不清楚组装是如何刺激GTP水解的。这项研究提案测试了这一假设，即α-微管蛋白通过组装时通过二聚体与β-微管蛋白接触来刺激GTP水解酶的假设。在二聚体-二聚体界面改变的酵母微管蛋白突变体，将被检测它们对微管动力学和GTP水解的影响。通过GFP标记和荧光显微镜观察活细胞中微管的动态变化。改变微管动力学的突变微管蛋白将被提纯，并在组装过程中检测其GTP酶活性。在将被测试的突变体中，将研究α-微管蛋白突变体tub1-828，原因如下：(I)它包含谷氨酸-255(E255)突变，它可能只在组装的原丝中与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。一种具有加速GTP酶活性的β-微管蛋白突变体tub2-T107K将进行测试，以确定它是否抑制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，并在体内微管动力学改变和酵母中微管蛋白突变体的整体表型之间建立迄今缺失的联系。

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.