Analysis of Centrosome Dynamics

中心粒在真核生物中有两个重要的功能：（1）指导微管的积累 成核/锚定材料以形成在细胞分裂期间起关键作用的中心体，以及（2）它们 作为基底体，指导形成基于微管的细胞突起，称为纤毛， 执行各种运动和感觉功能。中心粒在细胞周期中精确复制一次， 这一过程在分子水平上仍然知之甚少。具体目标1和2 研究使用C.线虫胚胎作为模型系统进行分子表征的步骤 在中心粒复制周期中。在C. elegans 除了CT/β-微管蛋白外，我还鉴定了5种定位于中心粒的蛋白质，它们是细胞增殖所必需的。 组装件.其中，只有两个（SAS-4和SAS-6）具有稳定缔合结构的性质， 件.在第一个目标中，复制循环中的步骤将通过使用荧光显微镜来定义 的方法来监测SAS-4和SAS-6掺入中心粒的动力学。比如SAS-4， SAS-6，o/p-微管蛋白稳定地整合到中心粒中，并且是中心粒复制所必需的，y-微管蛋白，a 一种与微管成核有关的特化微管蛋白同种型在中心粒中也具有保守的作用 组装件.在第二个目标中，能够监测活胚胎中中心粒亚结构的形成 将被用来确定复制周期中需要微管组装和γ-微管蛋白的步骤。一 这些结果的比较将表明是否y-微管蛋白的主要作用是中心粒的成核 微管在SAS-4的假定人类同源物中的突变最近与常染色体 隐性原发性小头畸形（MCPH），一种与大脑体积缩小相关的疾病。我们的工作也 表明SAS-4相关蛋白与致命的胎儿脑发育障碍有关。在最后的目标中，A 在C.线虫和哺乳动物细胞将决定 这些人类疾病中的突变与中心粒的复制缺陷有关，或者与以下之一有关： 它们的两个关键功能，中心体组装或纤毛发生。在人类中，中心粒模板 至少有8种不同类型的纤毛，推动粘液和液体，协调发育事件， 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，表征中心粒在细胞分裂和纤毛发生过程中的形成和功能 将可能提供与大范围疾病的诊断和治疗相关的见解， 与睫状体缺陷相关，包括：进行性失明、不孕症、多囊肾病、Bardet- 比德尔综合征，脑积水和卡塔格纳三联征。

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.