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。中心粒形成一种称为中心粒周围物质(PCM)的蛋白质基质，形成中心体。在从间期到有丝分裂的转变过程中，PCM在一个称为中心体成熟的过程中重塑，该过程准备中心体来催化微管的产生，以用于纺锤体的组装。在目标1中，我们利用线虫的专业知识来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，Plk1在中心体成熟过程中的基本功能不是它在基质扩张中的作用，而是产生专门的有丝分裂γ-微管蛋白复合体对接位点，使纺锤体组装成为可能。我们将采取生化方法来证实这一发现。我们的工作还表明，间期中心体中的γ-微管蛋白复合体对接位置与其有丝分裂对接位置不同。在这个目标的后半部分，我们将确定间期PCM是如何在外中心粒壁上组织的，γ-微管蛋白复合体的对接位置，以及间期PCM如何作为组装有丝分裂PCM的结构基础。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，称为中心酮。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的泛素连接酶，它以双向方式控制无着丝体纺锤体组装和癌细胞对Plk4抑制的敏感性。TRIM37的缺失促进了无着丝体纺锤体的组装，而TRIM37的过表达则严重影响了它的组装。我们的初步工作表明，TRIM37可能通过泛素化Plk4来限制其自组装来执行这些功能。在没有TRIM37的情况下，Plk4自组装形成异位焦点，招募中心体蛋白，获得成核微管的能力，并取代中心体催化微管生成以形成纺锤体。在目标2中，我们将严格检验这一假设，方法是进行体外测试，以确定TRIM37是否直接泛素化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自组装对中心粒复制的影响，并确定基于TRIM37的Plk4自组装调制是否也解释了为什么TRIM37水平升高对Plk4抑制具有高敏感性。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纺锤体组装中的作用的新理解，并定义Plk4抑制可能提供治疗益处的特定癌症背景。

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.