Characterizing the molecular mechanisms of centriole duplication, growth and maturation

项目概要/摘要 中心体是用于构建基于微管的蛋白质机器的细胞器，包括有丝分裂纺锤体和 纤毛。在中心体的核心是一对“母女”中心粒，这是一种桶形结构， 复制细胞器的元素。正常情况下，中心粒对在每个细胞周期仅复制一次， 在有丝分裂进入的过程中，中心粒募集一层中心粒周围物质（PCM）--这一过程称为“成熟”-- 微管从中生长。它们不仅是真核细胞中最大的蛋白质复合物之一， 它是最古老的细胞器之一，自19世纪末被发现以来，一直吸引着细胞生物学家 世纪。在过去的20年里，成像、蛋白质组学和功能基因组筛选的进步导致了 到中心体领域的发现爆炸。目前，我们有一个完整的库存的蛋白质 包括中心体。在我们的模型系统果蝇中，中心体是由一个小得惊人的 组件数量（约20个）。尽管取得了这些进展， 无人回应虽然只有两个保守的主调节因子，波罗激酶和波罗样激酶4（Plk 4），启动 中心粒成熟和复制，目前尚不清楚它们是如何被激活，特别是在 中心粒还有，这些激酶的磷酸化靶点是什么，它们如何促进中心粒 复制和成熟？母中心粒是如何被限制在每个细胞只产生一个子体的 循环？如何控制中心粒长度？在分子水平上理解这些过程很重要 因为中心体功能或数量的改变会导致许多严重的病理，包括出生 缺陷、纤毛病变和癌症。plk 4一直是我们研究计划的核心，因为它既是 当过度表达时，诱导中心体过度复制（扩增）是必要的和足够的， 在癌细胞中观察到。我们已经发表了一系列研究，定义了Plk 4的调控，并确定了 它的几种基质。值得注意的是，Plk 4利用多种控制机制来抑制其活性并防止其在体内的表达。 猖獗的中心体过度复制，使用自磷酸化，泛素化和 自我抑制我们继续追求两个总体目标：1）确定分子机制， 抑制中心体扩增（由R 01 GM 110166资助）和2）表征内在机制 控制中心体功能和复制（由R 01 GM 126035资助）。在我们的进步基础上， 在过去的五年里，我们建议扩大我们的研究，以定义这五种机制的基础 组装过程中的连续步骤。具体来说，我们将确定（i）如何一个单一的网站的女儿 中心粒组装是在母中心粒上选择的，（ii）前原中心粒的组成及其如何形成， (iii)新生子中心粒如何组装，（iv）中心粒生长如何控制，以及（v） 中心体成熟

PROJECT SUMMARY/ABSTRACT Centrosomes are organelles used to build microtubule-based protein machines, including mitotic spindles and cilia. At the centrosome core lies a pair of `mother-daughter' centrioles, barrel-shaped structures that act as the duplicating elements of the organelle. Normally, the centriole pair duplicates only once each cell cycle and, during mitotic entry, centrioles recruit a shell of pericentriolar material (PCM) – a process called `maturation' – from which microtubules grow. Not only are they one of the largest protein complexes in eukaryotic cells but one of the most ancient of organelles, and have fascinated cell biologists since their discovery in the late 19th century. During the past 20 years, advances in imaging, proteomics and functional genomic screens have led to an explosion of discoveries in the centrosome field. At present, we have a complete inventory of the proteins comprising centrosomes. In our model system, Drosophila, centrosomes assemble from a surprisingly small number of components (approximately 20). Despite these advances, many important questions remain unanswered. Although only two conserved master-regulators, Polo kinase and Polo-like kinase 4 (Plk4), initiate centriole maturation and duplication, respectively, it is not known how they are activated specifically on centrioles. Also, what are the phosphorylation targets of these kinases and how do they promote centriole duplication and maturation? How are mother centrioles restrained to spawn only a single daughter once per cell cycle? How is centriole length controlled? Understanding these processes at the molecular level is important because alterations in centrosome function or number cause a number of serious pathologies, including birth defects, ciliopathies and cancer. Plk4 has been the centerpiece of our research program because it is both necessary and sufficient to induce centrosome overduplication (amplification) when overexpressed, a scenario observed in cancer cells. We have published a series of studies that have defined Plk4 regulation and identified several of its substrates. Notably, Plk4 utilizes multiple mechanisms of control to restrain its activity and prevent rampant centrosome overduplication, using an elaborate combination of autophosphorylation, ubiquitination and autoinhibition. We continue to pursue two overarching goals: 1) identifying the molecular mechanisms that suppress centrosome amplification (funded by R01 GM110166) and 2) characterizing the inherent mechanisms that govern centrosome function and duplication (funded by R01 GM126035). Building on our progress during the past five years, we propose to extend our studies that will define the mechanisms underlying the five sequential steps in the assembly process. Specifically, we will determine (i) how a single site of daughter centriole assembly is selected on mother centrioles, (ii) the composition of the pre-procentrioles and how it forms, (iii) how nascent daughter centrioles assemble, (iv) how centriole growth is controlled, and (v) the initial steps in centrosome maturation.