Mechanisms of Centrosome Reproduction in Animal Cells

描述(由申请人提供)：有丝分裂必须高保真地进行，以确保每个子细胞获得完整的基因组补充。两极纺锤体的组装是由中心体的复制指导的，中心体是哺乳动物细胞中主要的微管组织中心。每个中心体的核心是一对中心粒，由三个微管组成：中心粒的复制和组装反映了中心体作为一个整体的复制。中心体复制本身的错误可能会导致多极纺锤体的形成，这确实会导致基因组的不稳定。事实上，许多人类肿瘤细胞都有异常数量的中心体。 为了继续研究中心体复制的细胞周期调控，我们将使用活细胞视频显微镜和生物物理/细胞生物学技术来分析哺乳动物细胞和蛙卵提取物中的中心体复制。这些研究旨在揭示中心体功能的重要新方面，事实证明，这些方面很难使用分子遗传学或生物化学方法进行研究。 在目标I中，我们将确定CDK2-Cyclin E是否像我们先前的工作所建议的那样，允许非洲爪哇卵子提取物中的中心体复制。我们还将测试在S阶段捕获的细胞中的中心体重复复制是否需要许可。最后，我们验证了中心体复制不需要S阶段开始的假设，这也是我们之前的研究提出的。在AIM II中，我们将研究中心粒组装。中心粒微管被认为非常稳定，但我们对中心粒蛋白tektin的初步研究表明，它们在整个细胞周期中经历周转。我们将使用FRAP来测量活细胞中GFPtektin/中心粒微管的周转率。我们还将检验核心中心粒成分tektin是与中心体复制协调合成的假设，暗示复制本身的细胞周期调节机制。我们还将测试下调tektin的表达是否会导致中心粒复制/延伸受到抑制，以及这是否会导致细胞周期停滞。在目标III中，我们研究中心体繁殖的早期事件。我们已经开发出在哺乳动物细胞中组装中心体前体的方法。我们将使用活细胞显微镜研究前体形成的动力学。我们将分离这些前体结构，用多液相色谱质谱分析它们的蛋白质组成，并克隆候选基因。利用这些，我们将测试最初的复制事件(前体形成)是与中心粒组装相协调的，还是发生在细胞周期的早期。对中心体复制调控的详细了解将促进我们对癌症生物学的了解--一种以不受调控的细胞增殖为特征的疾病。我们的实验应该是对目前在各种模型系统中进行的遗传和生化工作的补充，并最终为下一代抗癌药物提供潜在靶点的肥沃来源。

DESCRIPTION (provided by applicant): Mitosis must be carried out with high fidelity to ensure that each daughter cell receives a complete complement of the genome. Bipolar spindle assembly is directed by the duplication of the centrosome, which serves as the major microtubule-organizing center in mammalian cells. At the core of each centrosome is a pair of centrioles, composed of triplet microtubules: the duplication and assembly of the centrioles mirrors the duplication of the centrosome as a whole. Mistakes in the duplication of the centrosome itself can lead to the formation of multipolar spindles that do contribute to genomic instability. Indeed, many human tumor cells have abnormal numbers of centrosomes. To continue the investigation of the cell cycle regulation of centrosome duplication we will use live-cell video microscopy and biophysical/cell biological techniques to analyze centrosome reproduction in mammalian cells and frog egg extracts. These studies are intended to reveal important novel aspects of centrosome function that have proved difficult to study using molecular genetic or biochemical approaches. In Aim I, we will determine if Cdk2-cyclin E licenses centrosome duplication in Xenopus egg extracts, as suggested by our previous work. We will also test whether centrosome re-duplication in cells arrested in S-phase requires licensing. Finally, we test the hypothesis that centrosome duplication does not require the onset of S-phase, also suggested by our previous studies. In Aim II, we will investigate centriole assembly. Centriole microtubules are thought to be very stable, but our preliminary work on the centriole protein tektin suggests they undergo turnover throughout the cell cycle. We will use FRAP to measure the rate of GFPtektin/ centriole microtubule turnover in living cells. We will also test the hypothesis that tektin, a core centriole component, is synthesized coordinate with centrosome duplication, suggesting a mechanism for the cell cycle regulation of duplication itself. We will also test whether knocking-down the expression of tektin leads to inhibition of centriole duplication/elongation, and whether this induces cell cycle arrest. In Aim III, we examine early events in centrosome reproduction. We have developed methods to assemble centrosome precursors in mammalian cells. We will examine the kinetics of precursor formation using live-cell microscopy. We will isolate these precursor structures, analyze their protein composition by multi-LC Mass Spectrometry, and clone candidate genes. Using these, we will test whether the initial duplication event (precursor formation) is coordinated with centriole assembly or occurs earlier in the cell cycle. A detailed understanding of the regulation of centrosome reproduction will advance our knowledge of cancer biology - a disease characterized by unregulated cell proliferation. Our experiments should complement the genetic and biochemical work currently being done in a variety of model systems, and ultimately provide a fertile source of potential targets for the next generation of anti-cancer drugs.