MOLECULAR ANALYSIS OF MICROTUBULE ORGANIZATION

细胞生物学中一个未解的问题是--哺乳动物细胞是如何 在细胞分裂间期和细胞分裂期间， 分裂周期，它们如何将间期微管（MT）阵列 进入有丝分裂器 本建议的重点是确定 负责微管组织MT的机制 哺乳动物细胞中的组织中心（MTOC）。 具体目标是 由MTOC的保守组分的一般假设驱动， γ-微管蛋白通过启动MT组装来组织细胞MT阵列 和/或将MTS连接到中心体。 我们将首先确定是否在 体外合成的γ-微管蛋白与MT末端相互作用， 生物化学和免疫细胞化学方法。 利用这种生化物质 系统改变的丙氨酸扫描的测定和体外合成 突变的γ-微管蛋白分子，我们将确定哪些表面残基 在γ-微管蛋白内的相互作用对于MT相互作用是重要的。 这些信息 了解MT附着的精确关系是必要的 到中心体，一个包含10 4个伽马分子的细胞器， 微管蛋白作为其组成部分。 接下来，使用基因替换策略 在裂殖酵母中，S. pombe，我们将测试γ-微管蛋白的能力， 突变以在功能上替代酵母γ-微管蛋白。 我们的下一个目标是 基于在哺乳动物中γ-微管蛋白过度表达的观察， 细胞在整个细胞质中产生MTS， 细胞周期的正常协调阶段，M期和S期。 通过在体内产生胞外微管，我们处于一种独特的 位置来检验有丝分裂检查点“读取” 运动舞蹈-MT相互作用。 最后，如果能资助五年，我们将 利用丙氨酸中产生的γ-微管蛋白的任何条件等位基因， 通过启动基因外抑制基因分析进行扫描诱变， 鉴定与γ-微管蛋白相互作用的细胞蛋白。 的基础 这些实验是建立在有希望的初步研究，包括 哺乳动物γ-微管蛋白cDNA分离、特异性 似乎阻断γ-微管蛋白功能的抗体， 使用体外转录/翻译来合成这种 稀有蛋白质 我们现在处于一个非常有利的位置， 假设γ-微管蛋白组织细胞MT阵列， 启动MT组装并将MT连接到中心体。 从伽玛射线开始- 微管蛋白似乎是MTOC的一种普遍存在的成分， 系统的纤毛上皮细胞，小鼠胚胎和植物细胞， 这项研究的结果将是感兴趣的一个不同的群体， 研究人员包括肿瘤学家、生殖和发育 生物学家

One of the unanswered questions in cell biology is--how do mammalian cells organize radial microtubule arrays in the interphase, and during the cell division cycle, how do they transform interphase microtubule (MT) array into the mitotic apparatus? This proposal focuses on the determination of mechanisms responsible for the organization of MTs by microtubule organizing centers (MTOC's) in mammalian cells. The specific aims are driven by the general hypothesis that a conserved component of MTOC's, gamma-tubulin, organizes cellular MT arrays by initiating MT assembly and/or attaching MTS to the centrosomes. We will first determine if in vitro synthesized gamma-tubulin interacts with the MT ends using biochemical and immunocytochemical approaches. Using this biochemical assay and the in vitro synthesis of systematically altered alanine scanning mutant gamma-tubulin molecules, we will determine which surface residues within gamma-tubulin are important for MT interactions. This information will be necessary to understand the precise relationship of MT attachment to the centrosome, an organelle that contains 10 4 molecules of gamma- tubulin as its integral component. Next, using a gene replacement strategy in the fission yeast, S. pombe, we will test the ability of gamma-tubulin mutations to functionally replace the yeast gamma-tubulin. Our next aim is based upon the observation that gamma-tubulin over -expression in mammalian cells produces MTS throughout eh cytoplasm and it uncouples the two normally coordinated phases of the cell cycle, the M-phase and the S-phase. By producing extracentrosomal microtubules in vivo, we are in a unique position to test the hypothesis that the mitotic check point "reads" the kinetochore-MT interactions. Finally, if funded for five years, we will exploit any conditional alleles of gamma-tubulin generated in the alanine- scanning mutagenesis by initiating an extragenic suppressor analysis to identify cellular proteins that interact with gamma-tubulin. The basis for these experiments is founded in promising preliminary studies including isolation of mammalian gamma-tubulin cDNA, development of specific antibodies that seem to block gamma-tubulin function, and the successful use of the in vitro transcription/translation for the synthesis of this rare protein. We are now in an excellent position to rigorously examine the hypothesis that gamma-tubulin organizes cellular MT arrays by initiating MT assembly and attaching MTs to the centrosomes. Since gamma- tubulin seems to be a ubiquitous component of MTOC's from as diverse systems as the ciliated epithelia, mouse embryo, and plant cells, the results of this study will be of interest to a diverse group of investigators including oncologists, reproductive and developmental biologists.