Centrosome Structure, Function and Duplication

描述(申请人提供)：拟议的研究中解决的中心问题是中心体是如何组装的，它如何在每个细胞周期复制一次，以及中心体数量异常的细胞中会发生什么。中心体使微管成核，并将这些微管组织起来，形成一个有用的阵列。中心体是动物细胞中主要的微管组织中心，在细胞周期开始时以单拷贝形式存在。S期中心体重复，形成的两个中心体有助于组织有丝分裂纺锤体的两极。在过去的十年里，我的实验室和其他实验室的工作已经确定了参与微管成核的分子，中心体复制的中央调节因子，参与复制的重要结构蛋白，以及控制中心体数量和连接中心体和细胞周期的控制机制。最近，人们对中心体的兴趣与日俱增，因为中心体异常与癌症的发展之间已经建立了联系。癌细胞通常有额外的中心体，这可能是这种疾病的基因组不稳定和快速进化的特征之一。拟议的实验利用了我们在研究中心体结构、功能和复制方面开发的试剂和分析方法的优势。我在这项建议中谈到了四个具体目标： 1)将区块表征为中心体重复。我们将测试阻断机制的模型，确定癌细胞是否在阻断中存在缺陷，并测试是否可以通过操纵潜在的调节器来克服阻断。 2)研究中心体数、倍体与基因组不稳定性的关系。我们将使用细胞融合方法来创建具有特定中心体数量和倍体的细胞，检测这些细胞中有丝分裂的进展和结果，并比较正常细胞和癌细胞对中心体和倍体异常的反应。 3)确定γ-微管蛋白环复合体与微管和中心体的分子相互作用。我们将使用纯化的成分来确定伽马微管蛋白环复合体、微管和中心体之间的分子相互作用。 4)探讨β-微管蛋白和β-微管蛋白在中心体功能和复制中的作用。我们将描述微管蛋白、微管蛋白和其他已知微管蛋白之间的相互作用，并通过青蛙卵提取液和人体细胞中的实验来确定微管蛋白在体外的功能。

DESCRIPTION (provided by applicant): The central problems addressed in the proposed research are how the centrosome is assembled, how it duplicates once per cell cycle, and what happens in cells in which centrosome number is aberrant. The centrosome nucleates microtubules and organizes those microtubules to create a useful array. The centrosome is the major microtubule organizing center in animal cells, and is present in a single copy at the beginning of the cell cycle. The centrosome duplicates in S phase, and the two resulting centrosomes help to organize the two poles of the mitotic spindle. Work from my lab and others over the last ten years has identified molecules involved in microtubule nucleation, the central regulators of centrosome duplication, important structural proteins involved in duplication, and control mechanisms that control centrosome number and link the centrosome and the cell cycle. Interest in the centrosome has grown recently because a correlation has been established between centrosome abnormalities and the development of cancer. Cancer cells often have extra centrosomes, which is likely to contribute to the genomic instability and rapid evolution characteristic of this disease. The proposed experiments make use of the strengths that we have developed in reagents and assays for studying centrosome structure, function and duplication. I address four specific aims in this proposal: 1) Characterize the block to centrosome reduplication. We will test models for the mechanism of the block, determine whether cancer cells have defects in the block, and test whether the block can be overcome by manipulation of potential regulators. 2) Investigate the relationship between centrosome number, ploidv, and genome instability. We will use cell fusion methods to create cells of defined centrosome number and ploidy, examine the progression and outcome of mitosis in these cells, and compare normal and cancer cells in their response to centrosome and ploidy abnormalities. 3) Define the molecular interactions of the gamma-tubulin ring complex with microtubules and with the centrosome. We will use purified components to determine the molecular interactions between the gamma-tubulin ring complex, the microtubule, and the centrosome. 4) Investigate the role of delta-tubulin and epsilon-tubulin in centrosome function and duplication. We will characterize the interactions between delta-tubulin, epsilon-tubulin and the other known tubulins, and define the function of delta-tubulin in vitro using assays in frog egg extracts, and in vivo in human cells.