Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 7921835
• 负责人: Karen F Oegema
• 金额: $ 16.56万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921835
• 关键词: Bardet Biedel syndrome; Biological Models; Blindness; Brain; C-Microtubule; Caenorhabditis elegans; Cell Cycle; Cell division; Centrioles; Centrosome; Cilia; Daughter; Defect; Development; Diagnosis; Disease; Electron Microscopy; Elements; Embryo; Eukaryota; Event; Fluorescence Microscopy; Homologous Gene; Human; Hydrocephalus; Infertility; Kinetics; Lead; Life; Link; Liquid substance; Mammalian Cell; Methods; Microcephaly; Microtubules; Modeling; Molecular; Monitor; Mucous body substance; Mutate; Mutation; Parents; Play; Polycystic Kidney Diseases; Primary Ciliary Dyskinesias; Process; Property; Protein Isoforms; Proteins; RNA Interference; Regulation; Reproduction; Research; Research Personnel; Role; Sensory; Series; Structure; Tube; Tubulin; Work; base; brain size; cilium biogenesis; developmental disease; dimer; egg; fetal; genetic analysis; insight; kinetosome; mutant; nervous system disorder; programs; research study; role model; sperm cell

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.

Centrioles在真核生物中执行两个关键功能：（1）它们指导微管的积累 成核/锚定材料以形成中心体，在细胞分裂过程中起着至关重要的作用，（2）它们 作为指导基于微管的蜂窝项目纤毛的基本机构， 执行各种母亲和感觉功能。 Centrioles在A中精确复制了一次每个细胞周期 在分子水平上仍然了解得很糟糕的过程。提议的特定目的1和2 研究使用秀丽隐杆线虫胚作为模型系统来执行步骤的分子表征 在中心复制周期中。秀丽隐杆线虫中的全面基于RNAi的筛选和遗传分析 除了CT/P-微管蛋白外，还确定了5种蛋白质，它们本地化为中心元素，并且需要其 集会。其中，只有两个（SAS-4和SAS-6）具有稳定相关结构的特性 成分。在第一个目标中，将使用荧光微观镜检查来定义重复周期中的步骤 基于监测SAS-4和SAS-6基础结构动力学的方法。像SAS-4和 SAS-6，O/P-微管蛋白稳定地掺入中心元中，其重复Y-微管蛋白是必需的 在微管成核中实施的专用小管蛋白同工型在中心中也具有保守的作用 集会。在第二个目标中，监测生物胚胎中的中心子结构的形成的能力 将用于识别需要微管组装和`Y微管蛋白的重复周期中的步骤。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元的成核 微管。最近，SAS-4的假定人同源物中的突变与常染色体有关 隐性原发性小头畸形（MCPH），这是一种与脑大小降低有关的疾病。我们的工作也有 在致命的胎儿脑发育障碍中实施了SAS-4相关蛋白。在最终目标中 在秀丽隐杆线虫和哺乳动物细胞中进行的一系列平行实验将决定是否确定 这些人类疾病中的突变与中心元重复的缺陷有关，或 他们的两个关键功能，中心体组装或纤毛发生。在人类中，中心模板 组装至少8种不同类型的纤毛，推动粘液和流体，协调发育事件， 并在繁殖过程中移动鸡蛋和精子。除了为我们的理解做出贡献 调节脑大小 可能会提供与大量疾病的诊断和治疗有关的洞察力 与睫状缺陷有关，包括：进行性失明，不育，多囊性肾脏疾病，Bardet- Biedel综合征，脑积水和Kartagener的三合会。