Regulators of Centriole Duplication and Structure

描述(申请人提供)：中心粒是真核生物中的主要细胞器，负责形成极化的细胞骨架、两极有丝分裂纺锤体和纤毛，包括运动和初级纤毛。中心粒依赖过程中的缺陷导致一系列发育疾病，包括染色体错误分离、细胞迁移缺陷和纤毛形成异常，包括但不限于唐氏综合征、视网膜色素变性、多指、内翻、脑积水、肥胖和Bardet-Biedl综合征。中心粒生物发生与细胞周期紧密相连，受一系列因素调节，确保中心粒复制一次，且每个细胞周期只有一次。负责中心粒复制的主要许可因子是类Polo-kinase4(Plk4)。Plk4的S作用机制以及Plk4中的一个中心保守结构域在调节蛋白激酶的特异性和活性中所起的作用仍有待确定。这一应用发展了一种假设，即Plk4‘S独特的中央保守结构域在调节激酶活性和底物识别方面发挥着关键作用。 三个系列的实验研究了Plk4的S中心保守结构域在调节中心粒复制中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力量和简便性，研究将集中在果蝇Plk4上。人与果蝇Plk4的高度同源性有助于将果蝇Plk4的机械洞察力直接转化为人类Plk4的功能。因此，特定的目标1是在原子分辨率下定义Plk4的S中心结构域的结构和负责结合激酶结构域的决定因素。X射线结晶学将被用来确定中央结构域单独和与激活域的复合体的结构。特异目的2是确定Plk4的S中心保守区在中心粒复制中的作用。高分辨率荧光成像技术将被用来检查Plk4结构的定位以及这些结构对中心粒复制的影响。这项检查将使用RNAi技术去除内源性Plk4的果蝇S2细胞。特异目的3是确定Plk4的S中心保守结构域对蛋白激酶活性的影响。这项研究将使用标准的激酶分析来测量包含或不包含保守的中心结构域的Plk4结构的活性。 这项研究的长期目标是确定依赖Plk4的中心粒复制的机制，识别中心粒特异性的Plk4结合和磷酸化靶标，以及确定这些Plk4靶标与下游效应分子相互作用。对中心粒生物发生的基本了解，确定调节成分和治疗靶点，将增强我们对中心粒生物发生在人类健康和发育中的理解。改善中心粒依赖过程中的缺陷，包括双极有丝分裂纺锤体的形成、细胞迁移和纤毛发生，将对健康新生儿的发育和成人的健康、独立和生产功能具有深远的医学意义。

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.