Analysis of Centrosome Dynamics

项目摘要 中心粒是由9倍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。在从间期到有丝分裂的过渡期间，PCM在称为中心体成熟的过程中被重塑，该过程准备中心体以催化微管的产生用于纺锤体组装。在目标1中，我们利用我们的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。令人惊讶的是，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不是其先前记录的基质扩张作用，而是产生专门的有丝分裂γ-微管蛋白复合物对接位点，使纺锤体组装。我们将采取生物化学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-微管蛋白复合物对接位点与其有丝分裂对接位点不同。在这个目标的后半部分，我们将确定间期PCM是如何在中心粒外壁上组织的，γ-微管蛋白复合物停靠在那里，以及间期PCM如何作为有丝分裂PCM组装的结构基础。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控制中心粒复制的Plk 4激酶的特异性，有效的抑制剂，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的泛素连接酶，该酶以双向方式控制acentrosomal纺锤体组装和癌细胞对Plk 4抑制的敏感性。TRIM 37的缺失促进了无中心体纺锤体的组装，而TRIM 37的过表达则严重影响了无中心体纺锤体的组装。在缺乏TRIM 37的情况下，PLK 4自组装以形成异位灶，其募集中心体蛋白，获得使微管成核的能力，并在催化微管生成以用于纺锤体组装中取代中心体。在目标2中，我们将通过进行体外测定来严格测试这一假设，以确定TRIM 37是否直接泛素化Plk 4，并通过评估这种修饰对其激酶活性和自组装能力的影响。我们还将评估阻断Plk 4自组装对中心粒复制的影响，并确定基于TRIM 37的Plk 4自组装调节是否也解释了为什么升高的TRIM 37水平对Plk 4抑制具有高敏感性。总的来说，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，以及定义PLK 4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.