Identifying molecular mechanisms that suppress centriole amplification.

识别抑制中心粒扩增的分子机制。

• 批准号: 9267488
• 负责人: Gregory Charles Rogers
• 金额: $ 28.52万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267488
• 关键词: Aneuploidy; Binding; Biochemical; Biological Assay; Biological Models; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell division; Cells; Centrioles; Centrosome; Chromosomal Instability; Chromosome Segregation; Chromosomes; Cilia; Complement; Complex; Congenital Abnormality; Coupled; DNA biosynthesis; Dangerousness; Data; Daughter; Defect; Disease; Drosophila genus; Drosophila polo protein; Elements; Ensure; Etiology; Event; Future; Genome; Goals; Growth; In Vitro; Inherited; Island; Knowledge; Laboratories; Licensing; Link; Location; Mediating; Meiosis; Microcephaly; Microtubules; Mission; Mitosis; Mitotic; Modification; Molecular; Mothers; National Institute of General Medical Sciences; Normal Cell; Oncogenic; Organelles; PLK1 gene; Pathway interactions; Pattern; Phosphorylation; Population; Process; Production; Proteins; Public Health; Regulation; Regulatory Pathway; Research; Resistance; Role; S Phase; Scaffolding Protein; Seckel syndrome; Series; Site; Spontaneous abortion; Spottings; Surface; System; Testing; Ubiquitin; Work; anaphase-promoting complex; cancer cell; ciliopathy; daughter cell; driving force; experimental study; in vivo; insight; mutant; novel; overexpression; prevent; programs; protein degradation; public health relevance; stem cell division; tumorigenesis; ubiquitin ligase

 DESCRIPTION (provided by applicant): Centrioles are the duplicating elements of centrosomes, organelles which nucleate microtubule growth to build mitotic/meiotic spindles and cilia. During each normal cell division, centrioles duplicate once and once only. Like DNA replication, centriole duplication is a semi-conservative process tightly coupled to the cell cycle and occurs only during S-phase. Centriole number must be closely regulated because a deficiency or an excess of centrioles may interfere with correct chromosome segregation and asymmetric stem cell division. The presence of too few centrioles contributes to various ciliopathies, including microcephaly, and Seckel syndrome. In contrast, too many centrioles directly promotes chromosomal instability (CIN) a driving force for aneuploidy that induces miscarriage, birth defects, and tumorigenesis. Furthermore, the presence of too many centrioles is frequently observed in cancer cells. Normally, each `mother' centriole assembles only a single `daughter', but centrioles have the capacity to assemble multiple daughters simultaneously - an aneuploidy- generating condition called centriole amplification. Normally, only a single, restricte site on the mother centriole is used to spawn a daughter. However, the mechanism limiting assembly to only this site is unknown. Experiments in this proposal test a hypothesis that explains why centriole duplication is normally restricted to a single event per mother centriole. Specifically, we link three conserved centriolar proteins, Polo-like kinase 4 (Plk4), its binding-partner and scaffolding protein Asterless (Asl), and the centriole assembly protein Anastral Spindle 2 (Ana2) into a single regulatory pathway. Overexpression of any of these proteins induces assembly of multiple daughter centrioles. How the cell cycle control program regulates these key proteins to limit daughter centriole assembly to only a single event is unknown. Here, we hypothesize that cell cycle regulators, including the Anaphase Promoting Complex (APC) ubiquitin-ligase, control the destruction of a Plk4/Asl/Ana2 complex to alter their pattern/activit on mother centrioles and generate a single site for daughter assembly. Our preliminary data indicate that 1) the Plk4 pattern on centrioles changes during mitotic progression: first coating the entire mother centriole but then being pruned to a single asymmetric spot by mitotic exit; 2) Plk4 and Ana2 are APC targets; 3) phosphorylation protects subpopulations of these proteins from degradation; and 4) Asl is a Plk4 substrate that stabilizes mitotic Plk4. In this proposal, we will (1) determine if APC regulation of Plk4 localization on centrioles defines the site of daughte centriole assembly, (2) determine the functional interaction between Plk4 and Asl which assists in defining a single site of centriole assembly, and (3) determine if Ana2 regulates Plk4 activation and whether Ana2 is controlled by APC. These studies will provide mechanistic insight into a molecular process that governs the fidelity of centriole duplication. Understanding the process at the molecular level will guide future studies to explore the etiology of centriole amplification during tumorigenesis and other centriole-related diseases.

 描述（由申请人提供）：中心粒是中心体的复制元件，中心粒是使微管生长成核以构建有丝分裂/减数分裂纺锤体和纤毛的细胞器，在每次正常细胞分裂期间，中心粒复制一次，并且仅复制一次。与细胞周期紧密耦合的半保守过程 并且仅发生在 S 期，必须严格调节中心粒数量，因为中心粒的缺乏或过多可能会干扰正确的染色体分离和不对称干细胞分裂。中心粒过少会导致各种纤毛病，包括小头畸形和塞克尔畸形。相反，过多的中心粒会直接促进染色体不稳定（CIN），这是导致流产、出生缺陷和肿瘤发生的非整倍性的驱动力。此外，在癌细胞中经常观察到过多中心粒的存在。通常，每个“母”中心粒仅组装一个“子”中心粒，但中心粒具有同时组装多个子细胞的能力——一种称为中心粒扩增的非整倍体生成条件。通常，母体中心粒上只有一个限制性位点用于产生子体，但是，仅在该位点进行组装的机制尚不清楚，该提案中的实验检验了解释其原因的假设。中心粒复制通常仅限于每个母中心粒的单个事件，具体而言，我们将三种保守的中心粒蛋白、Polo 样激酶 4 (Plk4)、其结合伴侣和支架蛋白 Asterless (Asl) 以及中心粒组装蛋白 Anastral Spindle 连接起来。 2 (Ana2) 进入单一调控途径。任何这些蛋白质的过度表达都会诱导多个子体中心粒的组装。细胞周期控制程序如何调节这些关键蛋白质以限制子体。中心粒组装到单个事件尚不清楚。在这里，我们认为细胞周期调节剂，包括后期促进复合物 (APC) 泛素连接酶，控制 Plk4/Asl/Ana2 复合物的破坏，以改变其在母体上的模式/活性。我们的初步数据表明，1）中心粒上的 Plk4 模式在有丝分裂过程中发生变化：首先覆盖整个母中心粒，然后被修剪以形成中心粒。有丝分裂退出的单个不对称点；2) Plk4 和 Ana2 是 APC 靶点；3) 磷酸化可保护这些蛋白质的亚群免于降解；4) Asl 是稳定有丝分裂的 Plk4 底物。 将 (1) 确定 APC 对中心粒上 Plk4 定位的调节是否定义了子中心粒组装的位点，(2) 确定 Plk4 和 Asl 之间的功能相互作用，这有助于定义中心粒组装的单个位点，以及 (3) 确定 Ana2 是否调节 Plk4 激活以及 Ana2 是否受 APC 控制。这些研究将为控制中心粒复制保真度的分子过程提供机制见解。指导未来的研究探索肿瘤发生和其他中心粒相关疾病期间中心粒扩增的病因。