Analysis of Centrosome Dynamics

描述(申请人提供)：中心粒是一种小的圆柱形细胞器，由一系列稳定的微管组成，这些微管围绕着一个被称为手轮的9重对称的中心枢纽形成。在细胞分裂中，中心粒招募中心粒周物质形成中心体，中心体通过其成核和锚定微管的能力有助于纺锤体的组装和定位。中心粒在每个细胞周期内精确复制一次，以确保每个有丝分裂细胞只包含两个中心体。多余的中心体是癌细胞的共同特征，它们的存在导致了染色体的不稳定，导致了中心粒复制的错误调节是肿瘤发生过程中的驱动力的假说。这项拟议的研究结合了模型系统线虫的体外方法和体内工作，以阐明控制中心粒复制和中心体组装的途径。了解这些过程对于理解癌症中未能控制中心体数量和形态以及确定靶向中心粒复制和/或中心体组装用于治疗目的是有用的环境是至关重要的。在中心粒组装的启动过程中，保守的中心粒组分Spd-2/Cep192将Zyg-1/Plk4激酶靶向母中心粒。Zyg-1/Plk4启动中心粒支架蛋白SAS-6的聚合，形成侧翻。在具体目标1中，我们通过对涉及Spd-2、Zyg-1和SAS-6的物理相互作用和磷调节进行平行的体内/体外分析，阐明了控制这种引发反应的机制。SAS-6和SAS-4是中心粒组装所需的普遍保守的模块的组成部分。纯化的SAS-6二聚体可通过其N-端球状结构域相互结合，形成9重对称的圆盘状结构。重要的问题是如何控制SAS-6的聚合，如何将板状的SAS-6组装堆积起来形成圆柱形侧翻，以及侧翻如何引导外心粒壁的形成。在特定的目标2中，我们通过定义SAS-6与以下方面相互作用的性质和意义来解决这些问题：i)SAS-5，一种与SAS-6一起发挥促进侧翻组装功能的保守蛋白质；以及ii)SAS-4，一种控制中心粒微管组装的外中心粒壁的关键成分。我们还研究了SAS-6折叠构象的意义。中心粒外壁指导着中心粒周围物质(PCM)的募集，形成中心体，形成核并锚定微管。在有丝分裂进入过程中，PCM的数量增加了五到十倍，这一过程被称为中心体成熟。PCM扩增受一个保守模块介导的磷调节控制，该保守模块包括双功能中心体蛋白SPD-2/Cep192和有丝分裂酶PLK-1和Aurora A。特殊目的3剖析这种磷酸调节以阐明控制中心体大小的机制。 公共卫生相关性：中心体是对细胞分裂的信号和机制至关重要的细胞器。正常分裂的细胞正好包含两个中心体，而癌细胞通常有更多的中心体，这导致了一种观点，即额外的中心体促进了肿瘤形成过程中发生的不适当的分裂。这项提案中的工作将阐明控制中心体复制的机制，目标是从治疗上针对这一过程，以防止癌细胞分裂。

DESCRIPTION (provided by applicant): Centrioles are small cylindrical organelles composed of an array of stabilized microtubules that forms around a 9-fold symmetric central hub called the cartwheel. In dividing cells, centrioles recruit pericentriolar material to form centrosomes that contribute to spindle assembly and positioning through their ability to nucleate and anchor microtubules. Centrioles duplicate precisely once per cell cycle to ensure that each mitotic cell contains only two centrosomes. Supernumerary centrosomes are a common feature of cancer cells and their presence causes chromosomal instability, leading to the hypothesis that misregulation of centriole duplication is a driving force during tumorigenesis. The proposed research combines an in vitro approach with in vivo work in the model system C. elegans to elucidate the pathways that control centriole duplication and centrosome assembly. Understanding these processes is essential to understand the failure to control centrosome number and morphology in cancer and to define the contexts in which it will be useful to target centriole duplication and/or centrosome assembly for therapeutic purposes. During the initiation of centriole assembly, the conserved centriole component SPD-2/Cep192 targets the ZYG-1/Plk4 kinase to the mother centriole. ZYG-1/Plk4 initiates polymerization of the centriolar scaffold protein SAS-6 to form the cartwheel. In Specific Aim 1, we elucidate the mechanisms that control this initiation reaction by performing a parallel in vivo/in vitro analysis of physical interactions and phosphoregulation involving SPD-2, ZYG-1 and SAS-6. SAS- 6 and SAS-4 are components of a universally conserved module required for centriole assembly. Purified SAS-6 dimers can associate with each other through their N-terminal globular domains to form a 9-fold symmetric circular plate-like structure. The important questions are how SAS-6 polymerization is controlled, how the plate-like SAS-6 assemblies stack to form the cylindrical cartwheel, and how the cartwheel directs formation of the outer centriole wall. In Specific Aim 2, we address these questions by defining the nature and significance of interactions of SAS-6 with: i) SAS-5, a conserved protein that functions with SAS-6 to promote cartwheel assembly; and ii) SAS-4, a key component of the outer centriole wall that controls assembly of the centriolar microtubules. We also investigate the significance of a folded conformation of SAS-6. The centriole outer wall directs the recruitment of pericentriolar material (PCM) to form centrosomes that nucleate and anchor microtubules. The amount of PCM increases five to ten-fold during mitotic entry in a process known as centrosome maturation. PCM amplification is controlled by phosphoregulation mediated by a conserved module that includes the bifunctional centrosome protein SPD-2/Cep192 and the mitotic kinases PLK-1 and Aurora A. Specific Aim 3 dissects this phosphoregulation to elucidate the mechanisms that control centrosome size. PUBLIC HEALTH RELEVANCE: Centrosomes are cellular organelles that are central to the signaling and mechanics of cell division. Normal dividing cells contain precisely two centrosomes, whereas cancer cells frequently have many more, leading to the view that extra centrosomes promote the inappropriate divisions that occur during tumorigenesis. The work in this proposal will elucidate the mechanisms that control centrosome duplication, with the goal of targeting this process therapeutically to prevent cancer cell division.