Functional properties of centrosomes in somatic cells

描述（由申请人提供）：本文提出的研究的广泛目标是阐明脊椎动物体细胞中心体的功能，以及中心体实现这些功能的分子机制。为了实现这些目标，我们正在使用一种独特的方法，其中中心体（标记有GFP）被激光微束消融。这种方法使我们能够创造出缺乏中心体的细胞，并将它们的行为与拥有这种细胞器的遗传相同的细胞进行比较。此外，我们将联合收割机激光显微手术与siRNA灭活单个中心体蛋白相结合。这种协同方法提供了通过其他手段无法获得的新信息。我们目前的研究重点是我们在过去几年的赠款中描述的两种现象。首先，我们发现，中心体的存在是正常细胞所必需的，而不是癌细胞在细胞周期的间期。我们还证明了没有中心体的癌细胞会重新形成这个细胞器。我们现在研究特定的分子机制，协调细胞周期的进展和激活的中心体从头形成途径。为此，我们在不同的转化阶段消融人类细胞系中的中心体（例如，p53、pRb或p21缺陷），以确定在缺乏中心体的情况下，需要这些途径中的哪一种来阻断细胞周期进程（Aim 1）。我们还消融（通过siRNA）耗尽不同中心体蛋白的细胞中的中心体，以确定单个蛋白质如何影响中心体从头形成（Aim 2）。我们的第二条研究路线是阐明中心体在有丝分裂中的作用。我们已经证明，中心体是不是必不可少的有丝分裂纺锤体的形成，然而，在无中心体细胞的有丝分裂是错误的倾向。我们现在调查的相对贡献的中心体和中心体独立的有丝分裂纺锤体形成的途径。目标3和4测试两个特定的假设：1。中心体是有效纠正人工染色体定向不良所必需的; 2.着丝粒纤维的形成是通过不依赖于中心体的机制捕获在着丝粒附近成核的短微管而发生的。由于中心体异常是恶性肿瘤的标志，因此这些研究中获得的知识将有助于设计治疗癌症的新策略。

DESCRIPTION (provided by applicant): The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we are using a unique approach in which the centrosome (labeled with GFP) is ablated by laser micro beam. This approach allows us to create cells that lack the centrosome, and compare their behavior with genetically identical cells that possess this organelle. Further, we combine laser microsurgery with inactivation of individual centrosomal proteins by siRNA. This synergistic methodology provides new information not obtainable by other means. Our current research focuses on the two phenomena that we described during past years of the grant. First, we discovered that the presence of the centrosome is required in normal but not in cancerous cells to progress through the cell cycle during interphase. We also demonstrated that cancerous cells born without centrosomes re-form this organelle de novo. We now investigate particular molecular mechanisms that coordinate cell cycle progression and activation of the centrosome de novo formation pathway. For that we ablate centrosomes in human lines at different stages of transformation (e.g., deficient in p53, pRb, or p21) to determine which of these pathways is needed to block cell cycle progression in the absence of centrosomes (Aim 1). We also ablate centrosomes in cells depleted (by siRNA) of different centrosomal proteins to determine how individual proteins affect centrosome de novo formation (Aim 2). Our second line of research is to clarify the role of the centrosome in mitosis. We have demonstrated that centrosomes are not essential for the formation of mitotic spindles, however, mitosis in acentrosomal cells is error-prone. We now investigate the relative contributions of the centrosomal and centrosome-independent pathways for mitotic spindle formation. Aims 3 and 4 test two specific hypothesis: 1. That the centrosomes are required for efficient correction of synthetic chromosome mal-orientation; and 2. That the initiation of kinetochore fiber formation occurs via capture of short microtubules nucleated in the vicinity of kinetochores by centrosome-independent mechanisms. Since centrosome abnormalities are a hallmark of malignant tumors, the knowledge obtained in these studies will be useful for designing new strategies for the treatment of cancers.