Analysis of Centrosome Dynamics

项目概要 中心粒是由稳定微管的 9 重对称阵列组成的小细胞器。中心粒 组织称为中心粒周围材料（PCM）的蛋白质基质以形成中心体。期间 从间期到有丝分裂的转变，PCM 在称为中心体成熟的过程中被重塑， 准备中心体以催化纺锤体组装的微管生成。在目标 1 中，我们利用我们的 线虫的专业知识阐明了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 Plk1 在中心体成熟过程中的基本功能不是它的 先前记录的在基质扩张中的作用，但专门的有丝分裂 g-微管蛋白复合物的生成 实现主轴组装的对接站点。我们将采用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点与其有丝分裂不同 对接站点。在这个目标的后半部分，我们将确定相间相变材料如何在外部组织 中心粒壁，g-微管蛋白复合物对接的地方，以及间期 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 Plk4 激酶的特异性、有效抑制剂，称为 森特里酮。使用 centrinone 消耗细胞中的中心粒的工作使我们发现了一种称为泛素连接酶 TRIM37 控制着中心体纺锤体组装和癌细胞对 Plk4 抑制的敏感性 定向时尚。 TRIM37 缺失有利于中心体纺锤体的组装，而 TRIM37 过度表达 严重损害了它。我们的初步工作表明 TRIM37 可能通过以下方式执行这些功能 泛素化 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，获得微管成核的能力，并替代中心体 催化纺锤体组装的微管生成。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 TRIM37 是否直接泛素化 Plk4，并通过评估其影响 对其激酶活性和自组装能力进行修饰。我们还将评估阻止 Plk4 的影响 自组装对中心粒复制的影响，并确定是否基于 TRIM37 的 Plk4 自组装调制 还解释了为什么 TRIM37 水平升高会对 Plk4 抑制产生高度敏感性。总的来说，我们预计 拟议的工作将导致对中心体循环和中心体在 纺锤体组装，以及定义特定的癌症背景，其中 PLK4 抑制可能提供治疗 好处。

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.