Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 8301084
• 负责人: Karen F Oegema
• 金额: $ 9.92万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8301084
• 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; beta Tubulin; brain size; cilium biogenesis; developmental disease; dimer; egg; fetal; gamma Tubulin; genetic analysis; insight; kinetosome; mutant; nervous system disorder; programs; research study; role model; sperm cell

DESCRIPTION (provided by applicant): 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 alpha/beta-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, alpha/beta-tubulin is stably incorporated into centrioles and is required for their duplication, gamma-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 gamma-tubulin. A comparison of these results will indicate whether the primary role of gamma-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.

描述（由申请人提供）：中心粒在真核生物中发挥两个关键功能：(1)它们指导微管成核/锚定物质的积累，形成中心体，在细胞分裂过程中发挥关键作用；(2)它们作为基底体，指导微管细胞突起（称为纤毛）的形成，纤毛具有各种运动和感觉功能。中心粒在每个细胞周期精确复制一次，其过程在分子水平上仍然知之甚少。本研究的具体目标1和2使用秀丽隐杆线虫胚胎作为模型系统，对中心粒复制周期的步骤进行分子表征。在秀丽隐杆线虫中基于rnai的综合筛选和遗传分析发现，除了α / β -微管蛋白外，还有5种蛋白质定位于中心粒，并且是中心粒组装所必需的。其中，只有两个（SAS-4和SAS-6）具有稳定相关结构组件的特性。在第一个目标中，复制周期的步骤将通过使用基于荧光显微镜的方法来监测SAS-4和SAS-6融入中心粒的动力学来定义。与SAS-4和SAS-6一样，α / β -微管蛋白稳定地结合到中心粒中，并且是中心粒复制所必需的，γ -微管蛋白是一种与微管成核有关的特殊微管蛋白异构体，在中心粒组装中也起着保守的作用。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。这些结果的比较将表明-微管蛋白的主要作用是否为向心微管的成核。推定的SAS-4人类同系物中的一个突变最近与常染色体隐性原发性小头畸形（MCPH）有关，这是一种与脑尺寸减小相关的疾病。我们的工作也暗示了sas -4相关蛋白与致命的胎儿大脑发育障碍有关。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确定这些人类疾病中的突变是否与中心粒复制缺陷有关，或者与中心粒两个关键功能之一有关，中心体组装或纤毛发生。在人类中，中心粒为至少8种不同纤毛的组装提供模板，这些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。除了有助于我们对大脑大小调节的理解外，描述细胞分裂和纤毛发生过程中中心粒的形成和功能，可能会为与纤毛缺陷相关的一系列疾病的诊断和治疗提供见解，这些疾病包括：进行性失明、不孕症、多囊肾病、Bardet- Biedel综合征、脑积水和Kartagener's triad。