Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 7491099
• 负责人: Karen Oegema
• 金额: $ 30.42万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7491099
• 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; Eukaryotic Cell; Event; Fluorescence Microscopy; Homologous Gene; Human; Hydrocephalus; Infertility; Kinetics; Lead; Life; Link; Liquid substance; Localized; 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; SAS; 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.

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.在分子水平保持不足的过程中，Centrioles在每个细胞周期中精确地复制了一次。拟议的研究的具体目的1和2使用秀丽隐杆线虫胚作为模型系统，以对中心重复周期中的步骤进行分子表征。除α/β-微管蛋白外，秀丽隐杆线虫中的全面基于RNAi的筛选和遗传分析还鉴定了5种蛋白质，这些蛋白质本地化为中心元素，并且是其组装所必需的。其中，只有两个（SAS-4和SAS-6）具有稳定相关的结构成分的特性。在第一个目标中，将使用基于荧光微观显微镜的方法来定义重复周期中的步骤，以监测SAS-4和SAS-6掺入中心元中的动力学。像SAS-4和SAS-6一样，α/β-微管蛋白稳定地掺入中心元素中，并且是重复的，γ-微管蛋白是一种与微管成核有关的专门小管蛋白同工型，在中心组装中也具有保守的作用。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确定需要微管组装和γ-微管蛋白的重复周期中的步骤。这些结果的比较将表明γ-微管蛋白的主要作用是否是中心微管的成核。最近，SAS-4的假定人同源物的突变与常染色体隐性原发性小头畸形（MCPH）有关，这是一种与脑大小降低有关的疾病。我们的工作还暗示了与致命的胎儿脑发育障碍中的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确定这些人类疾病中的突变是否与中心元重复的缺陷有关，或者在其两个关键功能之一，即中心体组装或纤毛生成中。在人类中，Centrioles模板至少需要8种不同类型的纤毛，这些纤毛可以推动粘液和液体，协调发育事件，并在繁殖过程中移动卵和精子。 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.