Functional properties of centrosomes in somatic cells

描述(申请人提供)：本研究的主要目标是阐明中心体在脊椎动物体细胞中的功能，以及它完成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，即用激光微束消融中心体(标有GFP)。这种方法允许我们创造缺乏中心体的细胞，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其他方式无法获得的新信息。我们目前的研究重点是我们在过去几年的拨款中描述的两个现象。首先，我们发现，中心体的存在在正常细胞中是必需的，但在癌细胞中并不需要，以便在间期细胞周期中进行。我们还证明，没有中心体的癌细胞可以重新形成这种细胞器。我们现在研究协调细胞周期进程和中心体新生形成途径激活的特定分子机制。为此，我们在不同的转化阶段(例如，缺乏p53、pRB或p21)消融人类细胞系中的中心体，以确定在没有中心体的情况下，需要这些途径中的哪一条来阻止细胞周期进展(目标1)。我们还消融了(通过siRNA)不同中心体蛋白耗尽的细胞中的中心体，以确定单个蛋白质如何影响中心体的新生形成(目标2)。我们的第二个研究方向是阐明中心体在有丝分裂中的作用。我们已经证明，中心体对于有丝分裂纺锤体的形成并不是必不可少的，然而，无着丝体细胞的有丝分裂很容易出错。我们现在研究中心体和中心体非依赖途径对有丝分裂纺锤体形成的相对贡献。AIMS 3和4验证了两个特定的假设：1.中心体是有效纠正合成的染色体定向错误所必需的；2.动粒纤维的形成是通过中心体无关的机制捕获在动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，在这些研究中获得的知识将有助于设计癌症治疗的新策略。

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.