Mechanism controlling centrosome duplication

描述(申请人提供)：中心粒在动物细胞中有两个关键功能：(1)它们招募中心周物质形成中心体或微管组织中心；(2)它们作为基准体，模板纤毛的形成。这些功能对适当的染色体分离、细胞分裂、细胞信号传递和细胞周期控制至关重要。中心体数量的放松导致了基因组的不稳定，这是癌症的一个标志。复制过程的一个进化保守的特征是中心粒构型，它在S时期两个中心粒垂直生长的“接合”状态和有丝分裂后期或G1早期的“脱节”状态之间循环，在有丝分裂晚期或G1早期，中心粒不再紧密相对。利用非洲爪哇卵提取物和人类细胞，我们发现中心粒解离发生在有丝分裂后期，并由Polo-like Kinase(Plk1)和分离酶介导。在即将到来的S阶段，只能复制脱离的中心粒，而不是参与的中心粒。因此，PLK1和分离物“许可”中心粒进行复制。我提出以下两个目的，以进一步了解这两种酶在脱离过程中的分子参与，并确定相关底物。在目标1中，我将使用非洲爪哇卵提取液的两步脱附试验来检测Plk1中期酶活性是否是脱附所必需的。此外，我还将测试Plk1和Separase是独立发挥作用，还是以相同的途径驱动中心粒脱离。此外，我将使用体外重建中心粒分离实验来确定Plk1和分离酶是否足以触发中心粒分离，或者是否需要额外的活动。在目标2中，我计划通过候选方法和我们之前验证过的无偏见的体外生化筛选来鉴定Plk1底物。已知分离酶和Plk1共同作用，从染色体上移除粘连蛋白复合体，以促进后期的染色体分离，这与中心粒分离是一致的。因此，我将首先在显微镜下检查hScc1和粘附素复合体的其他成员的定位，以确定粘附素复合体是否可以作为解离活性的底物。我还将评估hScc1和SAS2(复合体的另一个成员)是否参与了中心粒的结合，方法是通过RNAi诱导这些蛋白的敲除，然后使用我们实验室开发的显微镜技术分析中心粒的表型。此外，我还将测试最近通过对人类中心体的蛋白质组学分析确定的一组蛋白质是否可以作为分离酶和Plk1底物。为此，我使用了一种广泛用于鉴定许多生物酶底物的方法--“体外表达克隆”(IVEC)，为生化筛选产生了几种试剂，用于鉴定分离酶和Plk1的新底物。 公共卫生相关性：癌症占美国所有死亡人数的25%，因此它是一个重大的健康问题。为了了解癌细胞的基本生物学特性，首先要了解这些特性是如何从正常状态发展而来的，这样才能设计出修复“癌症”状态的策略(即癌症疗法)。我们的研究计划旨在了解正常细胞如何限制称为中心体的亚细胞结构的复制，中心体在人类癌症中过度复制。这种过度复制被认为会导致DNA含量的变化和不受控制的生长。

DESCRIPTION (provided by applicant): Centrioles have two key functions in animal cells: (1) They recruit pericentriolar materials to form centrosomes or microtubule-organizing center and (2) they serve as basal bodies that template the formation of cilia. These functions are critical for proper chromosome segregation, cell division, cell signaling, and cell cycle control. Deregulation of centrosome number contributes to genome instability, a hallmark of cancer. One evolutionarily conserved feature of the duplication process is centriole configuration, which cycles between the "engaged" state, where the two centrioles grow orthogonally to one another during S phase, and the "disengaged" state during late mitosis or early G1, where centrioles are no longer tightly opposed. Using Xenopus egg extracts and human cells, we have discovered that centriole disengagement occurs at late mitosis, and is mediated by polo-like kinase (Plk1) and separase. Only the disengaged centrioles, and not engaged ones, can be duplicated in the upcoming S phase. Plk1 and separase, therefore, "license" centrioles for duplication. I propose the following two aims to further understand the molecular involvement of these two enzymes in the disengagement process, and to identify the relevant substrates. In Aim 1, I will employ a two-step disengagement assay using Xenopus egg extracts to examine whether Plk1 metaphase activity is required for disengagement. Additionally I will test whether Plk1 and separase function independently, or in the same pathway to drive centriole disengagement. Furthermore, I will use an in vitro reconstitution centriole disengagement assay to determine whether Plk1 and separase are sufficient to trigger centriole disengagement, or whether additional activities are required. In Aim 2, I plan to identify Plk1 substrates through both a candidate approach, and an unbiased in vitro biochemical screen that we have previously validated. Separase and Plk1 are known to work together to remove the cohesin complex from chromosomes to facilitate chromosome segregation during anaphase, which coincides with centriole disengagement. Thus, I will determine if the cohesin complex can be the substrate of the disengagement activity by first examining the localization of hScc1 and other members of the cohesin complex by microscopy. I will also assess whether hScc1 and SAS2 (another member of the complex) are involved in centriole engagement, by RNAi-induced knockdown of these proteins followed by analysis of centriolar phenotypes with microscopy techniques developed in our laboratory. In addition, I will test whether a group of proteins recently identified through a proteomic analysis of human centrosomes, can function as separase and Plk1 substrates. For this purpose, I have generated several reagents for a biochemical screen to identify novel substrates of separase and Plk1, using "In Vitro Expression Cloning" (IVEC), a method that has been widely used to identify substrates of many biological enzymes. PUBLIC HEALTH RELEVANCE: Cancer is responsible for 25% of all deaths in the United States, and it is therefore a significant health issue. In order to understand the basic biological properties of cancer cells, it is critical to first understand how these properties developed from a normal state so strategies can be devised to fix the "cancerous" state (i.e. cancer therapies). Our research proposal aims to understand how normal cells restrict the duplication of subcellular structures called centrosomes, which are over-duplicated in human cancers. This over-duplication is thought to result in changes in DNA content and uncontrolled growth.