Supernumerary Centrosomes and Cell Proliferation

项目摘要 中心粒是后生动物的一种保守的细胞器，位于微管的核心 组织中心、中心体、纤毛和鞭毛的基部。中心粒功能障碍导致 广泛的疾病，包括纤毛病的发育缺陷;大脑发育缺陷 在小头畸形;和癌症中，多余的或有缺陷的中心体与 预后不良。中心粒复制由Plk 4促进，Plk 4使车轮蛋白磷酸化， 调解他们的集会。我们和其他人已经发现Plk 4介导的中心粒的诱导 扩增导致几种组织的增生并增加对肿瘤发生的易感性 在没有p53肿瘤抑制因子的小鼠中。 额外中心体的丢失和获得通常都阻碍细胞增殖。但 响应中心体丢失的途径不同于响应中心体丢失的几种途径。 额外中心体因此，为了鉴定发出信号或响应于存在的蛋白质， 额外的中心体，我们进行了基因组范围的筛选，当删除或敲除- 向下允许具有升高的Plk 4水平的细胞增殖。这表明，新 细胞对多余中心体反应的途径：一种以前未知的参与， 调节中心粒复制的特异性Rac介导的信号传导; 中心粒的伸长、脱离和分离;以及纤毛发生的负调节因子。 在这里，我们遵循三种方法来了解中心粒数量是如何调节增殖 细胞首先，我们将确定Arh 15 gap GT3激活蛋白如何作用于Pak 1/2， 蛋白激酶调节Plk 4水平或活性，并依赖Arp 2/3调节纺锤体相关肌动蛋白 以允许中心粒和细胞周期停滞的增加。其次，我们研究已知的组件 影响中心粒的执行和细胞周期在意想不到的点。我们将 确定Usp 33去泛素化酶如何调节中心粒加帽蛋白复合物，以保护 新生的原中心粒和如何蛋白质所需的中心粒周期为两个中心粒脱离 和中心体分离有助于在存在额外的细胞增殖的阻滞 中心体最后，我们将探讨纤毛发生的负调节因子如何阻断初级纤毛 在存在额外的中心粒的情况下以这样的方式形成，从而阻止细胞增殖。 我们预计，这项研究将促进我们对细胞在多种方式中的理解， 对多余中心体有反应。它将确定可以针对恢复的途径 中心粒数目或肿瘤细胞中的细胞周期调节或靶向这些细胞用于凋亡。这 将在开发多种癌症治疗药物策略中找到转化应用。

PROJECT SUMMARY The centriole is a conserved organelle of metazoans that is found at the core of microtubule organizing centers, centrosomes, and at the base of cilia and flagellae. Centriole dysfunction leads to a wide range of diseases including the developmental defects of ciliopathies; defective brain development in microcephaly; and in cancer where supernumerary or defective centrosomes are associated with poor prognosis. Centriole duplication is promoted by Plk4, which phosphorylates cartwheel proteins to mediate their assembly. We and others have found that induction of Plk4-mediated centriole amplification results in hyperplasia of several tissues and increases the susceptibility to tumorigenesis in the mouse in the absence of the p53 tumor suppressor. Both the loss and acquisition of extra centrosomes normally block cell proliferation. However, the pathway that responds to loss of centrosomes differs from several pathways that respond to supernumerary centrosomes. Therefore, to identify proteins that signal or respond to the presence of extra centrosomes, we carried out a genome-wide screen for genes that when deleted or knocked- down permit the proliferation of cells that have elevated levels of Plk4. This has identified new pathways whereby cells respond to supernumerary centrosomes: a previously unknown involvement of specific Rac-mediated signaling that regulates centriole duplication; proteins that regulate the elongation, disengagement and separation of centrioles; and negative regulators of ciliogenesis. Here we follow three approaches to understand how centriole number is regulated in proliferating cells. First, we will determine how the Arh15gap GTPase Activating Protein acts upon the Pak1/2 protein kinases to regulate Plk4 levels or activity and upon Arp2/3 to regulate spindle associated actin to permit an increase in centriole- and cell-cycle arrest. Second, we examine how known components of the centriole affect the execution of the centriole and cell cycles at unexpected points. We will determine how the Usp33 deubiquitinase regulates the centriole capping protein complex to protect the nascent procentriole and how proteins required in the centriole cycle for both centriole disengagement and centrosome separation contribute to the block to cell proliferation in the presence of supernumerary centrosomes. Finally, we will explore how negative regulators of ciliogenesis block primary cilium formation in the presence of extra centrioles in such a way as to arrest cell proliferation. We anticipate that this research will advance our understanding of the multiple ways in which cells respond to supernumerary centrosomes. It will identify pathways that can be targeted for restoring centriole numbers or cell cycle regulation in tumor cells or for targeting such cells for apoptosis. This will find translational application in developing multiple drug strategies for cancer treatment.