Molecular Biology of Microtubule Interacting Proteins

γ-微管蛋白在微管组织中心组装微管细胞骨架中的重要性现已得到充分证实。 然而，在当前资助期内获得的数据和在其他实验室获得的数据表明，γ-微管蛋白在组装的有丝分裂装置的功能和细胞质微管的组织中也起着重要作用。 γ-微管蛋白在这些过程中的作用知之甚少，考虑到这些过程的普遍重要性，值得研究。 在本研究期间，本实验室利用丙氨酸扫描定点诱变技术，在丝状真菌构巢曲霉中产生了41个γ-微管蛋白突变。 突变赋予多种表型。 一些等位基因是条件致死的，使得γ-微管蛋白在某些条件下功能不正常。在这些条件下，这些突变中的大多数不阻止有丝分裂纺锤体的组装，但抑制组装的纺锤体的功能。 至少有两个等位基因抑制有丝分裂中染色体向两极的运动，三个等位基因的生长通过抗微管剂恢复。 拟议的研究将利用这些突变作为工具，探索γ-微管蛋白在组装微管功能中的作用。该提案的目的是1）确定苯菌灵/诺考达唑抑制条件致死性γ-微管蛋白突变的机制，2）确定γ-微管蛋白突变是否改变微管动力学，3）研究γ-微管蛋白在检查点调节中的作用，4）确定γ-微管蛋白突变对有丝分裂进程的影响，以及5）鉴定与γ-微管蛋白相互作用的基因。

The importance of gamma-tubulin for the assembly of the microtubule cytoskeleton from microtubule organizing centers is now well established. Data obtained in the current grant period and data obtained in other labs suggest, however, that gamma- tubulin also plays an essential role in the functioning of the assembled mitotic apparatus and in the organization of cytoplasmic microtubules. The role of gamma-tubulin in these processes is poorly understood and, given the universal importance of these processes, worthy of study. In the present grant period our lab has created 41 gamma-tubulin mutations in the filamentous fungus Aspergillus nidulans by alanine-scanning site-directed mutagenesis. The mutations confer a variety of phenotypes. Some of the alleles are conditionally lethal such that gamma-tubulin functions improperly under some conditions. Under these conditions most of these mutations do not block the assembly of mitotic spindles but inhibit the functioning of assembled spindles. At least two alleles inhibit the movement of chromosomes to the the poles in mitosis and the growth of three alleles is restored by antimicrotubule agents. The proposed studies will employ these mutations as tools to explore the role of gamma-tubulin in the functioning of assembled microtubules. The aims of the proposal are 1) to determine the mechanism of benomyl/nocodazole suppression of conditionally lethal gamma- tubulin mutations, 2) to determine if microtubule dynamics are altered by gamma-tubulin mutations, 3) to investigate the role of gamma-tubulin in checkpoint regulation, 4) to determine the effects of gamma-tubulin mutations on mitotic progression, and 5) to identify genes that interact with gamma-tubulin.