Characterizing the molecular mechanisms of centriole duplication, growth and maturation

项目摘要/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。中心体核心有一对母女中心粒，这是一种桶形结构，起着 细胞器的复制分子。正常情况下，中心粒对每个细胞周期只复制一次， 在有丝分裂进入期间，中心粒招募一个由着丝点周围物质(PCM)组成的外壳--这一过程被称为“成熟”。 从中生长出微管。它们不仅是真核细胞中最大的蛋白质复合体之一，而且 最古老的细胞器之一，自19世纪末发现以来一直令细胞生物学家着迷 世纪。在过去的20年里，在成像、蛋白质组学和功能基因组筛选方面的进步引领了 对中心体领域的发现的爆炸性影响。目前，我们有一份完整的蛋白质清单。 由中心体组成。在我们的模型系统中，果蝇，中心体从一个令人惊讶的小 组件数量(大约20个)。尽管取得了这些进展，但许多重要的问题仍然存在。 无人接听。虽然只有两个保守的主调控因子，Polo激酶和Polo-like kinase4(Plk4)启动 分别是中心粒成熟和复制，目前尚不清楚它们是如何在 中心粒。另外，这些激酶的磷酸化靶点是什么，它们是如何促进中心粒的 复制和成熟？母中心粒如何被限制为每个细胞只能生育一次女儿 自行车？中心粒的长度是如何控制的？在分子水平上理解这些过程是很重要的 因为中心体功能或数目的改变会导致一系列严重的疾病，包括出生 缺陷、纤毛疾病和癌症。Plk4一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(放大)的必要条件和充分条件，这是一种情况 在癌细胞中观察到。我们已经发表了一系列研究，定义了Plk4的调控，并确定了 它的几种底物。值得注意的是，Plk4利用多种控制机制来抑制其活动并防止 猖獗的中心体过度复制，使用自动磷酸化、泛素化和 自我抑制。我们继续追求两个首要目标：1)确定 抑制中心体扩增(由R01 GM110166资助)和2)表征内在机制 管理中心体功能和复制(由R01 GM126035资助)。在我们的进步的基础上 在过去的五年里，我们建议扩大我们的研究，以确定五个基础的机制 装配过程中的连续步骤。具体地说，我们将确定(I)女儿的单一地点 中心粒组装选择在母中心粒上，(Ii)前着丝粒的组成及其形成方式， (Iii)初生的子粒如何聚集；(Iv)如何控制中心粒的生长；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.