Molecular Analysis of Microtubule Organization

描述(申请人提供)：我们目前的研究重点是 两个普遍的假设。首先，Y-微管蛋白负责锚定 通过精确的相互作用在特定的极化方向上的微管 具有纺锤体的微管亚基和Spc97p/Spc98p亚基 杆子。第二，y-微管蛋白参与了有丝分裂检查点通路。 这是由于它在纺锤体极成核微管组装中所起的作用。这个 第一个假设是基于别人和我们之前的工作。第二 假设是基于我们收集的人类和S.条件等位基因。 庞贝Y-微管蛋白。正常情况下，纺锤体组装的异常会阻止细胞在 前中期，进入后期和随后的细胞质分裂被抑制。 出乎意料的是，我们的具有异常纺锤体的Y-微管蛋白突变体的子集没有 停滞，但通过后期和胞质分裂进行。其中最引人注目的是 是三个冷敏感突变等位基因。其中两个条件突变体可以 充分挽救内源性Pombeγ-微管蛋白的致死性缺失 允许温度。此外，我们还分离到了一个突变的等位基因 RAD2l+，一个姐妹染色单体粘附素亚基编码基因，作为一种 伽马微管蛋白突变等位基因。我们在确定分子机制方面的进展 这一新的伽马微管蛋白功能包括一个热敏突变体sgtl的克隆 (伽马微管蛋白的抑制因子)和一个相关基因(46%相似的蛋白质 产品)、SGT2+。SGTLP和SGT2P各有两个蛋白质-蛋白质相互作用 调解WW域。重要的是，SGT1DELTA的多拷贝抑制子编码 一种新的蛋白质Ssglp以及Y-微管蛋白和Ssglp都含有推测的WW 结构域结合基序。我们的具体目标是，1)定义准确的性质 Y-微管蛋白与微管和纺锤体极的相互作用，2) 确定Y-微管蛋白突变等位基因是否消除了有丝分裂检查点，3) 研究观察到的遗传相互作用的分子机制 γ-微管蛋白和SGTLP/SGT2p，以及4)定义之间的机制联系 伽马微管蛋白和有丝分裂检查点通路。我们提出了一种组合方案 实现这些目标的遗传、生化和细胞方法。每一个 这项建议的具体目标充分体现在我们最近的进展和 我们在过去赠款期间的进展。我们最近的进步使我们陷入了 对机械理解做出独特贡献的地位 Ytubuin在有丝分裂过程中发挥作用。

DESCRIPTION (provided by applicant): Our current research focus is driven by two general hypotheses. First, y-tubulin is responsible for anchoring microtubules in a specific, polarized orientation through precise interactions with microtubule subunits and with the Spc97p/Spc98p subunits of the spindle pole. Second, y-tubulin is involved in the mitotic checkpoint pathway apart from its role in nucleating microtubule assembly from the spindle poles. The first hypothesis is based on previous work by others and us. The second hypothesis is based on our collection of conditional alleles of human and S. pombe y-tubulin. Normally, abnormalities in spindle assembly arrest cells in prometaphase, and entry into anaphase and subsequent cytokinesis is inhibited. Unexpectedly, a subset of our y-tubulin mutants with abnormal spindles does not arrest but proceeds through anaphase and cytokinesis. Among the most striking are three cold sensitive mutant alleles. Two of the conditional mutants can fully rescue the lethal deletion of the endogenous S. pombe gamma-tubulin at the permissive temperature. Furthermore, we have isolated a mutant allele of rad2l+, a sister chromatid cohesin subunit encoding gene, as a suppressor of a gamma-tubulin mutant allele. Our progress in defining the molecular mechanisms of this novel gamma-tubulin function includes cloning of a heat sensitive mutant sgtl (suppressor of gamma TUbulin) and a related gene (46 percent similar protein product), sgt2+. Sgtlp and Sgt2p each have two protein-protein interaction mediating WW domains. Importantly, a multicopy suppressor of SGT1DELTA encodes a novel protein, Ssglp, and both y-tubulin and Ssglp contain putative WW domain-binding motifs. Our specific aims are, 1) to define the precise nature of y-tubulin interactions with microtubules and the spindle pole, 2) to determine if y-tubulin mutant alleles abrogate the mitotic checkpoint, 3) to investigate the molecular mechanism of the observed genetic interaction between gamma-tubulin and Sgtlp/Sgt2p, and 4) to define the mechanistic link between gamma-tubulin and the mitotic checkpoint pathways. We propose a combination of genetic, biochemical, and cellular approaches to achieve these aims. Each of the specific aims of this proposal is well founded in our recent progress and our progress in the past grant period. Our recent progress has put us in a position to make unique contributions to the mechanistic understanding of ytubulin functions during mitosis.