Mechanism controlling centrosome duplication

描述（由申请人提供）：中心粒在动物细胞中有两个关键功能：(1)它们招募中心周围物质形成中心体或微管组织中心；(2)它们作为基底模板形成纤毛。这些功能对正确的染色体分离、细胞分裂、细胞信号传导和细胞周期控制至关重要。中心体数量的减少会导致基因组不稳定，这是癌症的一个标志。复制过程的一个进化保守特征是中心粒结构，它在“接合”状态（两个中心粒在S期彼此垂直生长）和有丝分裂后期或G1早期的“脱离”状态（中心粒不再紧密对立）之间循环。使用爪蟾卵提取物和人类细胞，我们发现中心粒脱离发生在有丝分裂后期，并由polo样激酶（Plk1）和分离酶介导。只有未接合的中心粒和未接合的中心粒可以在即将到来的S相中复制。因此，Plk1和分离酶“许可”中心粒复制。我提出了以下两个目标，以进一步了解这两种酶在脱离过程中的分子参与，并确定相关的底物。在目标1中，我将使用爪蟾卵提取物采用两步脱离试验来检查Plk1中期活性是否需要脱离。此外，我将测试Plk1和分离酶是否独立起作用，还是在同一途径中驱动中心粒脱离。此外，我将使用体外重构中心粒脱离试验来确定Plk1和分离酶是否足以触发中心粒脱离，或者是否需要额外的活性。在目标2中，我计划通过候选方法和我们之前验证的无偏体外生化筛选来鉴定Plk1底物。分离酶和Plk1共同作用，从染色体上移除黏结蛋白复合物，促进染色体在后期分离，这与中心粒脱离相一致。因此，我将首先通过显微镜检查hScc1和黏结蛋白复合物其他成员的定位来确定黏结蛋白复合物是否可以作为脱离活性的底物。我还将评估hScc1和SAS2（复合体的另一个成员）是否参与中心粒接合，通过rnai诱导这些蛋白的敲低，然后用我们实验室开发的显微镜技术分析中心粒表型。此外，我将测试最近通过人类中心体蛋白质组学分析鉴定的一组蛋白质是否可以作为分离酶和Plk1底物。为此，我制作了几种生化筛选试剂，使用“体外表达克隆”（IVEC）来鉴定分离酶和Plk1的新底物，这种方法已被广泛用于鉴定许多生物酶的底物。

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.