Analysis of Centrosome Dynamics

在真核生物中，中心粒起着两个重要的作用：(1)指导微管的积累 成核/锚定物质以形成在细胞分裂中起关键作用的中心体，以及(2)它们 作为基础体，指导基于微管的细胞投影的形成，称为纤毛，它 执行各种运动和感官功能。在一个细胞周期中，中心粒精确复制一次 这一过程在分子水平上仍然知之甚少。建议的具体目标1和2 研究使用线虫胚胎作为模型系统来执行步骤的分子表征 在中心粒复制循环中。基于RNAi的线虫综合筛选和遗传分析 除了ct/p-微管蛋白，我还鉴定了5种定位于中心粒的蛋白质，它们是中心粒所必需的。 集合。其中，只有两个(SAS-4和SAS-6)具有稳定缔合结构的特性 组件。在第一个目标中，将使用荧光显微镜来定义复制周期中的步骤 基础方法监测SAS-4和SAS-6掺入中心粒的动力学。如SAS-4和 Sas-6，o/p-微管蛋白稳定地结合到中心粒中，是中心粒复制所必需的。 与微管成核有关的特殊微管蛋白亚型在中心粒中也具有保守的作用 集合。在第二个目标中，监测活胚胎中中心粒亚结构形成的能力 将用于确定复制周期中需要微管组装和γ-微管蛋白的步骤(S)。一个 这些结果的比较将表明y-微管蛋白的主要作用是否是中心粒成核。 微管。最近，人们推测的SAS-4同源物中的一个突变与常染色体有关 隐性原发小头畸形(MCPH)，一种与脑体积缩小相关的疾病。我们的工作还包括 与致命的胎儿大脑发育障碍有关。在最终目标中，一个 在线虫和哺乳动物细胞中进行的一系列平行实验将确定 这些人类疾病中的突变与中心粒复制的缺陷有关，或者在 它们的两个关键功能，中心体组装或纤毛发生。在人类中，中心粒以 由至少8种不同类型的纤毛组成，它们推动粘液和液体，协调发育活动， 并在繁殖过程中移动卵子和精子。除了有助于我们理解 大脑大小的调节，描述中心粒在细胞分裂和纤毛发生过程中的形成和功能 可能会为诊断和治疗大范围疾病提供相关的见解 与纤毛缺陷相关的疾病包括：进行性失明、不孕不育、多囊肾病、Bardet- 比德尔综合征、脑积水和卡氏三联症。

Centrioles perform two critical functions in eukaryotes: (1) they direct the accumulation of microtubule nucleating/anchoring material to form centrosomes that play a critical role during cell division, and (2) they serve as basal bodies that direct the formation of microtubule-based cellular projections called cilia, which perform a variety of motile and sensory functions. Centrioles duplicate precisely once per cell cycle in a process that remains poorly understood at a molecular level. Specific Aims 1 and 2 of the proposed research use the C. elegans embryo as a model system to perform a molecular characterization of the steps in the centriole duplication cycle. Comprehensive RNAi-based screens and genetic analysis in C. elegans have identified 5 proteins, in addition to ct/p-tubulin, that localize to centrioles and are required for their assembly. Of these, only two (SAS-4 and SAS-6) have the properties of stably-associated structural components. In the first aim, steps in the duplication cycle will be defined by using fluorescence-microscopy based methods to monitor the kinetics of incorporation of SAS-4 and SAS-6 into centrioles. Like SAS-4 and SAS-6, o/p-tubulin is stably incorporated into centrioles and is required for their duplication, y-tubulin, a specialized tubulin isoform implicated in microtubule nucleation also has a conserved role in centriole assembly. In the second aim, the ability to monitor the formation of centriolar substructures in living embryos will be used to identify the step(s) in the duplication cycle that require microtubule assembly and ¿y-tubulin. A comparison of these results will indicate whether the primary role of y-tubulin is nucleation of centriolar microtubules. A mutation in the putative human homolog of SAS-4 was recently linked to autosomal recessive primary microcephaly (MCPH), a disorder associated with reduced brain size. Our work has also implicated a SAS-4-associated protein in a lethal fetal brain developmental disorder. In the final aim, a series of parallel experiments performed in C. elegans and mammalian cells will determine whether the mutations in these human disorders are associated with defects in the duplication of centrioles, or in one of their two critical functions, centrosome assembly or ciliogenesis. In humans, centrioles template the assembly of least 8 different types of cilia that propel mucus and fluids, coordinate developmental events, and move the egg and sperm during reproduction. In addition to contributing to our understanding of the regulation of brain size, characterizing how centrioles form and function during cell division and ciliogenesis will likely provide insight relevant to the diagnosis and treatment of the large spectrum of diseases associated with ciliary defects including: progressive blindness, infertility, polycystic kidney disease, Bardet- Biedel syndrome, hydrocephalus, and Kartagener's triad.