Analysis of Centrosome Dynamics

描述（由申请人提供）：中心粒是由一系列稳定的微管组成的小圆柱形细胞器，这些微管围绕9倍对称的中心轮毂形成，称为侧轮。在分裂的细胞中，中心粒吸收中心粒周围物质形成中心体，中心体通过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。多余中心体是癌细胞的共同特征，它们的存在导致染色体不稳定，从而导致中心粒复制的错误调节是肿瘤发生过程中的驱动力的假设。本研究将体外方法与模型系统秀丽隐杆线虫的体内工作相结合，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在癌症中控制中心体数量和形态的失败以及确定靶向中心粒复制和/或中心体组装用于治疗目的的环境至关重要。在中心粒组装的起始阶段，保守的中心粒成分SPD-2/Cep192将ZYG-1/Plk4激酶靶向到母中心粒上。ZYG-1/Plk4启动向心支架蛋白SAS-6聚合形成侧轮。在Specific Aim 1中，我们通过对SPD-2、ZYG-1和SAS-6的物理相互作用和磷酸化调节进行体内/体外平行分析，阐明了控制这一起始反应的机制。SAS- 6和SAS-4是中心粒组装所需的普遍保守模块的组成部分。纯化的SAS-6二聚体可以通过它们的n端球形结构域相互结合，形成9重对称的圆形片状结构。重要的问题是如何控制SAS-6聚合，片状SAS-6组件如何堆叠形成圆柱形侧轮，以及侧轮如何指导外中心粒壁的形成。在Specific Aim 2中，我们通过定义SAS-6与以下分子相互作用的性质和意义来解决这些问题：1)SAS-5，一种与SAS-6一起促进侧轮组装的保守蛋白；SAS-4是外中心粒壁的关键成分，控制中心粒微管的组装。我们还研究了SAS-6折叠构象的意义。中心粒外壁引导中心粒周围物质（PCM）聚集形成中心体，中心体成核并锚定微管。在有丝分裂进入中心体成熟的过程中，PCM的数量增加了5到10倍。PCM扩增受保守模块介导的磷酸化调控控制，该模块包括双功能中心体蛋白SPD-2/Cep192和有丝分裂激酶PLK-1和Aurora a .特异性Aim 3剖析了这种磷酸化调控以阐明控制中心体大小的机制。

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.