Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 10543543
• 负责人: Karen Oegema
• 金额: $ 19.32万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543543
• 关键词: Address; Affect; Biochemical; Caenorhabditis elegans; Cancer cell line; Cell division; Cells; Centrioles; Centrosome; Chemicals; Complex; Data; Docking; Embryo; Excision; Foundations; Generations; Human; Impairment; Interphase; Malignant Neoplasms; Mechanics; Microtubule Stabilization; Microtubules; Mitosis; Mitotic; Modification; Organelles; Pathway interactions; Phosphotransferases; Physical condensation; Predisposition; Process; Proliferating; Proteins; Role; Site; Structure; Testing; Therapeutic; Ubiquitination; Work; cancer cell; cancer therapy; cancer type; gamma Tubulin; in vitro Assay; in vivo; inhibitor; overexpression; recruit; self assembly; ubiquitin isopeptidase; 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 γ-tubulin complex docking sites that enable spindle assembly. We will take a biochemical approach to confirm this finding. Our work also suggests that the γ-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 γ-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 中心元素是由稳定微管的9倍对称阵列组成的小细胞器。 Centrioles组织了一种称为丁香三元素材料（PCM）的蛋白质基质，以形成中心体。在从相间到有丝分裂的过渡过程中，PCM在称为中心体的过程中重塑以催化微管产生用于纺锤体组件。在AIM 1中，我们利用了秀丽隐杆线虫的专业知识，以阐明在有丝分裂进入过程中重塑中心体的途径。令人惊讶的是，我们的初步结果表明，中心体成熟过程中PLK1的基本功能不是其先前记录在基质扩展中的作用，而是其产生的专用有丝分裂γ-微素络合物络合物对接位点，从而启用纺锤体组件。我们将采用生化方法来确认这一发现。我们的工作还表明，间相中心体中的γ-微管蛋白复合物对接位点与其有丝分裂对接部位不同。在此目标的后半部分，我们将确定如何在γ-微管蛋白复合物被停靠的外侧中心壁上组织中的PCM，以及相间PCM如何作为有丝分裂PCM组装的结构基础。为了检查中心元素在人类细胞中的作用，我的实验室共同开发了一种控制中心重复的PLK4激酶的特定潜在抑制剂，称为中心。使用中心酮从细胞中巧妙的中心元中进行的工作使我们发现了一种称为TRIM37的泛素连接酶，该连接酶控制着艾当胶质体纺锤体组装和癌细胞对PLK4抑制的敏感性以双向方式抑制。 TRIM37损失的热门载体纺锤体组件，而TRIM37过表达严重损害了它。我们的初步工作表明，TRIM37可以通过泛素化PLK4来限制其自组装来执行这些功能。在没有TRIM37的情况下，PLK4自组件形成生态灶，以募集中心体蛋白，获得核微管的能力，并代替催化微管产生纺锤体组装的中心体。在AIM 2中，我们将通过进行体外评估来严格检验该假设，以确定TRIM37是否直接泛素PLK4，并评估这种修饰对其激酶活性和自组装能力的影响。我们还将评估阻止PLK4自组装对中心复制的影响，并确定基于TRIM37的PLK4自组装的调制是否也解释了为什么提高TRIM37水平升高对PLK4抑制的高灵敏度。总体而言，我们预计拟议的工作将导致对中心体周期和中心体在主轴组装中的作用的新理解，并定义PLK4抑制作用可以提供治疗益处的特定癌症环境。