Mechanism controlling centrosome duplication

控制中心体复制的机制

• 批准号: 8059287
• 负责人: Barbara E. Tanos
• 金额: $ 5.3万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-13 至 2013-08-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059287
• 关键词: Address; Anaphase; Animals; Biochemical; Biological; Biological Assay; Cancerous; Cell Cycle; Cell Cycle Regulation; Cell division; Cells; Centrioles; Centrosome; Cessation of life; Chromosome Segregation; Chromosomes; Cilia; Complex; Disease; Dwarfism; Enzymes; Genomic Instability; Growth; Health; Human; In Vitro; Laboratories; Licensing; Link; Malignant Neoplasms; Mediating; Metaphase; Methods; Microcephaly; Microscopy; Microtubule-Organizing Center; Mitosis; Molecular; Nature; Normal Cell; Pathway interactions; Phenotype; Ploidies; Process; Property; Proteins; Proteomics; RNA Interference; Reagent; Recruitment Activity; Research Proposals; Role; S Phase; Series; Stereotyping; Subcellular structure; Techniques; Testing; United States; Work; Xenopus; cancer cell; cancer therapy; ciliopathy; cohesin; egg; expression cloning; human PLK1 protein; intercellular communication; kinetosome; member; novel; reconstitution; separase

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.

描述（由申请人提供）：中心粒在动物细胞中具有两个关键功能：（1）它们募集中心粒周围物质以形成中心体或微管组织中心，以及（2）它们作为纤毛形成模板的基体。这些功能对于正确的染色体分离、细胞分裂、细胞信号传导和细胞周期控制至关重要。中心体数目失调导致基因组不稳定，这是癌症的标志。复制过程的一个进化上保守的特征是中心粒构型，其在“接合”状态和“脱离”状态之间循环，其中在S期期间两个中心粒彼此正交生长，在有丝分裂晚期或G1早期期间中心粒不再紧密相对。利用非洲爪蟾卵提取物和人类细胞，我们发现中心粒分离发生在有丝分裂晚期，并由polo样激酶（Plk 1）和分离酶介导。在即将到来的S期，只有脱离的中心粒，而不是参与的中心粒，可以被复制。因此，Plk1和separase“许可”中心粒复制。我提出以下两个目标，以进一步了解这两种酶在分离过程中的分子参与，并确定相关的底物。在目标1中，我将采用两步分离试验，使用非洲爪蟾卵提取物，以检查是否Plk 1中期活动所需的分离。此外，我将测试Plk1和分离酶是否独立发挥作用，或者在同一途径中驱动中心粒脱离。此外，我将使用体外重建中心粒脱离试验，以确定Plk 1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动。在目标2中，我计划通过候选方法和我们先前验证的无偏倚体外生化筛选来鉴定Plk1底物。分离酶和Plk1共同作用，从染色体上去除粘着蛋白复合物，以促进后期染色体分离，这与中心粒分离相一致。因此，我将通过首先通过显微镜检查hScc1和粘附素复合物的其他成员的定位来确定粘附素复合物是否可以是分离活性的底物。我还将评估hScc1和SAS 2（复合物的另一个成员）是否参与中心粒接合，通过RNAi诱导敲除这些蛋白质，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，我将测试是否一组最近通过蛋白质组学分析人类中心体确定的蛋白质，可以作为分离酶和Plk1底物。为此，我已经产生了几种试剂的生化筛选，以确定分离酶和Plk1的新底物，使用“体外表达克隆”（IVEC），已被广泛用于确定许多生物酶的底物的方法。 公共卫生相关性：癌症占美国所有死亡人数的25%，因此是一个重要的健康问题。为了了解癌细胞的基本生物学特性，首先必须了解这些特性是如何从正常状态发展起来的，以便制定策略来修复“癌”状态（即癌症治疗）。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。