Characterizing the molecular mechanisms of centriole duplication, growth and maturation

表征中心粒复制、生长和成熟的分子机制

• 批准号: 10640273
• 负责人: Gregory Charles Rogers
• 金额: $ 56.24万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640273
• 关键词: Behavior Control; Biological Models; Cell Cycle; Cells; Centrioles; Centrosome; Cilia; Congenital Abnormality; Daughter; Drosophila genus; Drosophila polo protein; Elements; Equipment and supply inventories; Etiology; Eukaryotic Cell; Explosion; Funding; Goals; Growth; Image; Knowledge; Length; Malignant Neoplasms; Microtubules; Mission; Mitotic; Mitotic spindle; Molecular; Mothers; Organelles; PLK1 gene; Pathology; Phosphorylation; Phosphotransferases; Procentriole; Process; Proteins; Proteomics; Public Health; Publishing; Regulation; Research; Series; Shapes; Site; Structure; Ubiquitination; United States National Institutes of Health; cancer cell; ciliopathy; fascinate; functional genomics; human disease; overexpression; prevent; programs; protein complex; recruit; restraint; tumorigenesis

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.

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 纤毛位于中心体核心，有一对“母女”中心粒，即充当纤毛的桶形结构。 细胞器的复制元件通常，中心粒对每个细胞周期仅复制一次，并且， 在有丝分裂进入期间，中心粒募集中心粒周围物质（PCM）的外壳——这个过程称为“成熟”—— 微管不仅是真核细胞中最大的蛋白质复合物之一，而且是从中生长的。 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 在过去的 20 年中，成像、蛋白质组学和功能基因组筛选的进步引领了这一趋势。 目前，我们已经拥有完整的蛋白质清单。 在我们的果蝇模型系统中，中心体是由一个非常小的细胞组装而成的。 尽管取得了这些进步，但仍然存在许多重要问题。 尽管只有两个保守的主调节因子 Polo 激酶和 Polo 样激酶 4 (Plk4) 启动， 中心粒分别成熟和复制，目前尚不清楚它们是如何被特异性激活的 此外，这些激酶的磷酸化靶标是什么以及它们如何促进中心粒。 复制和成熟如何限制母体中心粒每个细胞只产生一个子体？ 如何控制中心粒长度？在分子水平上了解这些过程很重要 因为中心体功能或数量的改变会导致许多严重的病变，包括出生 缺陷、纤毛病和癌症一直是我们研究项目的核心，因为它两者都是。 当过度表达时，诱导中心体过度重复（扩增）是必要和充分的，一种情况 我们已经发表了一系列研究，定义了 Plk4 调控并确定了这一点。 值得注意的是，Plk4 利用多种控制机制来抑制其活性并防止其发生。 猖獗的中心体过度复制，利用自磷酸化、泛素化和 我们继续追求两个总体目标：1）确定自我抑制的分子机制。 抑制中心体扩增（由 R01 GM110166 资助）和 2) 表征固有机制 控制中心体功能和复制（由 R01 GM126035 资助）。 在过去的五年里，我们建议扩大我们的研究，以确定这五个因素背后的机制 具体来说，我们将确定 (i) 子节点的单个站点的顺序。 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形成方式， (iii) 新生子中心粒如何组装，(iv) 如何控制中心粒生长，以及 (v) 初始步骤 中心体成熟。