Regulators of Centriole Duplication and Structure

中心粒复制和结构的调节因子

• 批准号: 8059658
• 负责人: Kevin C Slep
• 金额: $ 7.1万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8059658
• 关键词: Address; Adult; Aneuploidy; Bardet-Biedl Syndrome; Binding; Biogenesis; Biological Assay; Blindness; Boxing; Cell Culture Techniques; Cell Cycle; Cell Polarity; Cell division; Cells; Centrioles; Centrosome; Chemotaxis; Child; Chromosomes; Cilia; Cocrystallography; Complex; Coupled; Cystic Kidney Diseases; Cytoskeleton; Data; Defect; Dependence; Development; Disease; Down Syndrome; Drosophila genus; Ensure; Eukaryota; Health; Human; Hydrocephalus; Imaging Techniques; In Vitro; Integration Host Factors; Interphase; Intervention; Investigation; Lead; Left; Licensing; Licensing Factor; Light; Measures; Medical; Microtubules; Mitotic spindle; Molecular; Mothers; Mutation; Neural Tube Closure; Newborn Infant; Obesity; Organelles; Organism; Peptides; Phosphorylation; Phosphotransferases; Play; Polo-Box Domain; Polydactyly; Polymers; Primary Ciliary Dyskinesias; Process; Protein-Serine-Threonine Kinases; Proteins; Public Health; RNA Interference; Randomized; Reaction; Research; Resolution; Retinitis Pigmentosa; Role; Series; Signal Transduction; Site; Situs Inversus; Specificity; Structure; Translating; Tubulin; Work; X-Ray Crystallography; cell motility; ciliopathy; cilium biogenesis; extracellular; fluid flow; human PLK1 protein; insight; intercellular communication; mutant; research study; small molecule; therapeutic target

DESCRIPTION (Provided by Applicant): The centriole is a major organelle in eukaryotes, responsible for formation of a polarized cytoskeleton, the bipolar mitotic spindle, and cilia, both motile and primary. Defects in centriole dependent processes result in a wide spectrum of developmental diseases caused by chromosome missegregation, cell migration defects, and abnormal cilia formation including, but not limited to, Down syndrome, retinitis pigmentosa, polydactyly, situs inversus, hydrocephalus, obesity, and Bardet-Biedl syndrome. Centriole biogenesis is tightly coupled to the cell cycle, regulated by a host of factors that ensure centriole duplication is licensed once and only once per cell cycle. A master licensing factor responsible for centriole duplication is the polo-like kinase 4 (Plk4). Plk4's mechanism of action and the role a central conserved domain in Plk4 plays to modulate kinase specificity and kinase activity remains to be determined. This application develops the hypothesis that Plk4's unique central conserved domain plays a critical role in modulating kinase activity and substrate recognition. Three series of experiments examine the role of Plk4's central conserved domain in modulating kinase activity and substrate recognition in relation to centriole duplication. Studies will focus on Drosophila Plk4 given the high level of research conducted on Drosophila Plk4 and the power and ease of translating findings into Drosophila developmental studies. The high degree of identity between human and Drosophila Plk4 facilitates the transfer of Drosophila Plk4 mechanistic insight directly to human Plk4 function. Thus, Specific Aim 1 is to define, at atomic resolution, the structure of Plk4's central domain and the determinants responsible for kinase domain binding. X-ray crystallography will be employed to determine the structure of the central domain alone and in complex with the kinase domain. Specific Aim 2 is to ascertain the role Plk4's central conserved domain plays in centriole duplication. High resolution fluorescent imaging techniques will be employed to examine the localization of Plk4 constructs and the effect these constructs have on centriole duplication. This examination will use Drosophila S2 cells depleted of endogenous Plk4 using RNAi. Specific Aim 3 is to determine the role of Plk4's central conserved domain on kinase activity. This study will use standard kinase assays to measure the activity of Plk4 constructs that include or lack the conserved central domain. The long term objectives of this investigation are to ascertain the mechanism of Plk4-dependent centriole duplication, identify centriole-specific Plk4 binding and phosphorylation targets, as well as determine the downstream effectors these Plk4 targets interact with. A fundamental understanding of centriole biogenesis, identifying regulatory components and therapeutic targets, will enhance our understanding of centriole biogenesis in human health and development. Ameliorating defects in centriole-dependent processes, including bipolar mitotic spindle formation, cell migration, and ciliogenesis, will have far reaching medical implications in the development of healthy newborn children and the healthy, independent, and productive functioning of adults.

描述（由申请人提供）：中心粒是真核生物中的主要细胞器，负责形成极化细胞骨架、双极有丝分裂纺锤体和纤毛（运动性和初级性）。 中心粒依赖性过程的缺陷导致由染色体错误分离、细胞迁移缺陷和纤毛形成异常引起的多种发育疾病，包括但不限于唐氏综合症、色素性视网膜炎、多指症、内脏逆位、脑积水、肥胖症和巴代-比德尔综合症。 中心粒的生物发生与细胞周期紧密耦合，受到许多因素的调节，确保每个细胞周期中心粒复制被许可一次且仅一次。 负责中心粒复制的主要许可因子是 polo 样激酶 4 (Plk4)。 Plk4 的作用机制以及 Plk4 中央保守结构域在调节激酶特异性和激酶活性中的作用仍有待确定。 该应用提出了这样一个假设：Plk4 独特的中央保守结构域在调节激酶活性和底物识别中发挥着关键作用。 三个系列的实验检验了 Plk4 的中央保守结构域在调节与中心粒复制相关的激酶活性和底物识别中的作用。 鉴于对果蝇 Plk4 进行的高水平研究以及将研究结果转化为果蝇发育研究的能力和便利性，研究将集中在果蝇 Plk4 上。 人类和果蝇 Plk4 之间的高度同一性有助于将果蝇 Plk4 机制的见解直接转移到人类 Plk4 功能。 因此，具体目标 1 是以原子分辨率定义 Plk4 中心结构域的结构以及负责激酶结构域结合的决定因素。 X射线晶体学将用于确定单独的中心结构域以及与激酶结构域复合的中心结构域的结构。 具体目标 2 是确定 Plk4 的中央保守结构域在中心粒复制中所起的作用。 将采用高分辨率荧光成像技术来检查 Plk4 构建体的定位以及这些构建体对中心粒复制的影响。 该检查将使用通过 RNAi 耗尽内源性 Plk4 的果蝇 S2 细胞。 具体目标 3 是确定 Plk4 中央保守结构域对激酶活性的作用。 本研究将使用标准激酶测定来测量包含或缺乏保守中心结构域的 Plk4 构建体的活性。 这项研究的长期目标是确定 Plk4 依赖性中心粒复制的机制，识别中心粒特异性 Plk4 结合和磷酸化靶标，以及确定与这些 Plk4 靶标相互作用的下游效应器。 对中心粒生物发生的基本了解，确定调控成分和治疗靶点，将增强我们对人类健康和发育中中心粒生物发生的理解。 改善中心粒依赖性过程的缺陷，包括双极有丝分裂纺锤体形成、细胞迁移和纤毛发生，将对健康新生儿的发育以及成年人的健康、独立和生产性功能产生深远的医学影响。