Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 10582464
• 负责人: Karen F Oegema
• 金额: $ 10.57万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10582464
• 关键词: Biochemical; Caenorhabditis elegans; Cell division; Cells; Centrioles; Centrosome; Chemicals; Complex; Docking; Excision; Foundations; Generations; Human; Interphase; Malignant Neoplasms; Mechanics; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Modification; Organelles; Pathway interactions; Phosphotransferases; Process; Proliferating; Proteins; Role; Site; Testing; Therapeutic; Work; base; cancer cell; cancer therapy; gamma Tubulin; in vitro Assay; inhibitor; overexpression; recruit; self assembly; ubiquitin ligase

PROJECT SUMMARY Centrioles are small organelles composed of a 9-fold symmetric array of stabilized microtubules. Centrioles organize a proteinaceous matrix called the pericentriolar material (PCM) to form centrosomes. During the transition from interphase to mitosis, the PCM is remodeled in a process called centrosome maturation that prepares centrosomes to catalyze microtubule generation for spindle assembly. In Aim 1, we capitalize on our C. elegans expertise to elucidate the pathways that remodel the centrosome during mitotic entry. Surprisingly, our preliminary results suggest that the essential function of Plk1 during centrosome maturation is not its previously documented role in matrix expansion, but the generation of specialized mitotic g-tubulin complex docking sites that enable spindle assembly. We will take a biochemical approach to confirm this finding. Our work also suggests that the g-tubulin complex docking site in interphase centrosomes is distinct from its mitotic docking site. In the second half of this aim, we will determine how the interphase PCM is organized on the outer centriole wall, where the g-tubulin complex is docked, and how the interphase PCM serves as a structural foundation for assembly of the mitotic PCM. To examine the roles of centrioles in human cells, my lab collaboratively developed a specific, potent inhibitor of the Plk4 kinase that controls centriole duplication, called centrinone. Work using centrinone to deplete centrioles from cells led us to discover a ubiquitin ligase called TRIM37 that controls acentrosomal spindle assembly and the sensitivity of cancer cells to Plk4 inhibition in a bi- directional fashion. TRIM37 loss facilitates acentrosomal spindle assembly, whereas TRIM37 overexpression severely compromises it. Our preliminary work suggests that TRIM37 may perform these functions by ubiquitinating Plk4 to limit its self-assembly. In the absence of TRIM37, PLK4 self-assembles to form ectopic foci that recruit centrosomal proteins, acquire the ability to nucleate microtubules, and substitute for centrosomes in catalyzing microtubule generation for spindle assembly. In Aim 2, we will rigorously test this hypothesis by performing in vitro assays to determine if TRIM37 directly ubiquitinates Plk4, and by assessing the effects of this modification on its kinase activity and ability to self-assemble. We will also assess the impact of blocking Plk4 self-assembly on centriole duplication and determine whether TRIM37-based modulation of Plk4 self-assembly also explains why elevated TRIM37 levels impart high sensitivity to Plk4 inhibition. Collectively, we anticipate that the proposed work will lead to new understanding of the centrosome cycle and the role of centrosomes in spindle assembly, as well as define specific cancer contexts in which PLK4 inhibition may provide a therapeutic benefit.

项目摘要 中心粒是由9重对称排列的稳定微管组成的小细胞器。中心粒 组织称为中心粒周围物质（PCM）的蛋白质基质以形成中心体。期间 从间期到有丝分裂的过渡，PCM在一个称为中心体成熟的过程中被重塑， 制备中心体以催化微管生成用于纺锤体组装。在目标1中，我们利用 C. elegans的专业知识来阐明有丝分裂进入过程中重塑中心体的途径。令人惊奇的是， 我们的初步结果表明，Plk 1在中心体成熟过程中的基本功能并不是其自身的功能。 以前记录的作用，基质扩张，但产生专门的有丝分裂g-微管蛋白复合物 使主轴组装成为可能的对接点。我们将采取生物化学方法来证实这一发现。我们 这项工作还表明，间期中心体中的G-微管蛋白复合物对接位点与有丝分裂期中心体中的G-微管蛋白复合物停靠位点不同。 对接点在这个目标的后半部分，我们将确定相间PCM如何在外部组织， 中心粒壁，g-微管蛋白复合物停靠的地方，以及相间PCM如何充当结构 有丝分裂PCM组装的基础。为了研究中心粒在人类细胞中的作用，我的实验室 合作开发了一种控制中心粒复制的Plk 4激酶的特异性强效抑制剂，称为 Centrinone。使用centrinone从细胞中消耗中心粒的工作使我们发现了一种称为 TRIM 37控制无中心体纺锤体组装和癌细胞对Plk 4抑制的敏感性， 方向性时尚TRIM 37缺失促进无中心体纺锤体组装，而TRIM 37过表达 我们的初步工作表明，TRIM 37可以通过以下方式执行这些功能： 泛素化Plk 4以限制其自组装。在缺乏TRIM 37的情况下，PLK 4自组装形成异位病灶 这些细胞募集中心体蛋白，获得微管成核的能力，并在细胞中取代中心体。 催化用于纺锤体组装的微管产生。在目标2中，我们将通过以下方式严格检验这一假设： 进行体外测定以确定TRIM 37是否直接泛素化Plk 4，并通过评估这种作用， 修饰其激酶活性和自组装能力。我们还将评估封锁Plk 4的影响。 中心粒复制的自组装并确定是否基于TRIM 37调节Plk 4自组装 这也解释了为什么升高的TRIM 37水平赋予对Plk 4抑制的高敏感性。总体而言，我们预计 这项工作将使人们对中心体周期和中心体在以下方面的作用有新的认识： 纺锤体组装，以及确定其中PLK 4抑制可提供治疗性 效益