Molecular Biology of Microtubule Interacting Proteins

γ-微管蛋白在从微管组织中心组装微管细胞骨架中的重要性现在已经得到了充分的证实。然而，在当前授权期获得的数据和在其他实验室获得的数据表明，伽马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发挥着重要作用。伽马微管蛋白在这些过程中的作用人们知之甚少，鉴于这些过程的普遍重要性，值得研究。在目前的资助期间，我们实验室已经通过丙氨酸扫描定点突变在丝状真菌nidulans中产生了41个伽马微管蛋白突变。这些突变产生了多种表型。其中一些等位基因是条件性致死的，以至于在某些条件下，伽马微管蛋白的功能不正常。在这些条件下，这些突变中的大多数不会阻止有丝分裂纺锤体的组装，但会抑制组装的纺锤体的功能。在有丝分裂过程中，至少有两个等位基因抑制了染色体向两极的移动，而三个等位基因的生长可以通过抗微管药物恢复。拟议的研究将使用这些突变作为工具来探索伽马微管蛋白在组装的微管功能中的作用。该提案的目的是1)确定苯菌灵/诺可达唑抑制条件性致死性伽马微管蛋白突变的机制，2)确定伽马微管蛋白突变是否改变了微管动力学，3)研究伽马微管蛋白在检查点调节中的作用，4)确定伽马微管蛋白突变对有丝分裂进程的影响，以及5)确定与伽马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.