Analysis of Centrosome Dynamics

项目总结 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 组织一种称为中心周围物质(PCM)的蛋白质基质，形成中心体。在.期间 从间期到有丝分裂，PCM在一个称为中心体成熟的过程中重塑 准备中心体以催化纺锤体组装所需的微管生成。在目标1中，我们利用我们的 线虫的专业知识，以阐明在有丝分裂进入期间重塑中心体的途径。令人惊讶的是， 我们的初步结果表明，Plk1在中心体成熟过程中的基本功能不是它的 先前已证实的在基质扩张中的作用，但特化有丝分裂G-微管蛋白复合体的产生 支持主轴组装的对接位置。我们将采取生化方法来证实这一发现。我们的 研究还表明，间期中心体中的g-微管蛋白复合体对接位置不同于它的有丝分裂。 对接地点。在这个目标的后半部分，我们将确定外部如何组织相间PCM 中心粒壁，g-微管蛋白复合体的对接位置，以及相间PCM如何作为结构 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 合作开发了一种特殊的、有效的Plk4激酶抑制剂，它可以控制中心粒复制，称为 中枢酮。使用中心素来耗尽细胞中的中心粒的工作使我们发现了一种名为 TRIM37控制无着丝体纺锤体组装和癌细胞对Plk4抑制的敏感性。 定向时尚。TRIM37缺失促进无着丝体纺锤体组装，而TRIM37过表达 严重损害了这一点。我们的初步工作表明，TRIM37可能通过 泛化Plk4以限制其自组装。在没有TRIM37的情况下，Plk4自组装形成异位灶 招募中心体蛋白，获得成核微管的能力，并取代中心体在 催化纺锤体组装所需的微管生成。在目标2中，我们将通过以下方式严格测试这一假设 进行体外实验以确定TRIM37是否直接泛素化Plk4，并评估其影响 修饰其激酶活性和自组装能力。我们还将评估屏蔽Plk4的影响 自组装对中心粒复制的影响及决定是否基于TRIM37调控Plk4的自组装 这也解释了为什么TRIM37水平升高对Plk4抑制具有高度的敏感性。总的来说，我们期待着 这项拟议的工作将导致对中心体周期和中心体在 纺锤体组装，以及定义Plk4抑制可能提供治疗作用的特定癌症背景 利益。

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.