Analysis of Centrosome Dynamics

中心体动力学分析

• 批准号: 7681102
• 负责人: Karen F 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/7681102
• 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执行两个关键功能：（1）它们指导微管成核/锚定物质的积累以形成中心体，中心体在细胞分裂期间发挥关键作用，以及（2）它们作为基体指导称为纤毛的基于微管的细胞突起的形成，纤毛执行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，这一过程在分子水平上仍然知之甚少。具体目标1和2的拟议研究使用C。线虫胚胎作为模型系统进行中心粒复制周期中的步骤的分子表征。在C.除α/β-微管蛋白外，秀丽线虫还鉴定了5种定位于中心粒并为其装配所需的蛋白质。其中，只有两个（SAS-4和SAS-6）具有稳定缔合结构组分的性质。在第一个目标中，复制循环中的步骤将通过使用基于荧光显微镜的方法来确定，以监测SAS-4和SAS-6掺入中心粒的动力学。与SAS-4和SAS-6一样，α/β-微管蛋白稳定地掺入中心粒中并且是其复制所需的，γ-微管蛋白，一种涉及微管成核的特化微管蛋白同种型也在中心粒组装中具有保守的作用。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鉴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γ-微管蛋白的主要作用是否是中心粒微管的成核。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）有关，这是一种与脑体积减小相关的疾病。我们的工作还涉及SAS-4相关蛋白在致命的胎儿脑发育障碍。最后，在C.线虫和哺乳动物细胞将确定这些人类疾病中的突变是否与中心粒的复制缺陷有关，或者与中心粒的两个关键功能之一（中心体组装或纤毛发生）有关。在人类中，中心粒模板组装至少8种不同类型的纤毛，推动粘液和液体，协调发育事件，并在生殖过程中移动卵子和精子。除了有助于我们理解脑大小的调节之外，表征中心粒在细胞分裂和纤毛发生期间如何形成和功能将可能提供与与纤毛缺陷相关的大范围疾病的诊断和治疗相关的见解，包括：进行性失明、不育、多囊肾病、Bardet-Biedel综合征、脑积水和Kartagener三联征。