Molecular basis of centriole duplication

中心粒复制的分子基础

• 批准号: 10926238
• 负责人: Kyung Lee
• 金额: $ 110.5万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926238
• 关键词: 3-Dimensional; Abnormal Cell; Aneuploidy; Animals; Architecture; Binding; Biogenesis; Biological Process; C-terminal; Catalogs; Cell Cycle; Cell Cycle Progression; Cell division; Cell physiology; Cells; Cellular biology; Centrioles; Centrosome; Chromosome Segregation; Client; Complex; Data; Defect; Development; Disease; Ensure; Etiology; Eukaryotic Cell; Event; Exhibits; Fluorescence Recovery After Photobleaching; Future; Goals; Human; In Vitro; Life; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Microtubule-Organizing Center; Mitotic spindle; Molecular; Molecular Sieve Chromatography; Mutation; Names; Nanoscopy; Organelles; Organism; PLK1 gene; Phase; Physical condensation; Play; Polo-Box Domain; Procentriole; Process; Property; Protein Dynamics; Proteins; Research; Role; Scaffolding Protein; Shapes; Site; Somatic Mutation; Structure; Subcellular structure; Tissues; Ultracentrifugation; Variant; Work; X-Ray Crystallography; biophysical techniques; density; genome integrity; human disease; in vivo; insight; macromolecular assembly; mutant; nanoscale; novel; scaffold; sedimentation equilibrium; self assembly; self organization; single molecule

The centrosome, a unique membrane-less multiprotein organelle that serves as the main microtubule-organizing center in animal cells, plays a pivotal role in the orderly progression of the cell cycle. Since faulty assembly and duplication of the centrosome results in abnormal cell division, which then leads to various human disorders, elucidating the molecular mechanisms underlying centrosome assembly and function is likely a key step to understanding the etiology of centrosome-associated human diseases. By combining cell biology with biophysical methods and X-ray crystallography, we demonstrated that two pericentriolar scaffolds, Cep152 and Cep63, possess intrinsic activity of co-phase-separating into condensates and form a heterotetrameric complex that serves as a building block for generating a nanoscale cylindrical self-assembly around a centriole. Remarkably, two short uncharacterized regions named Self-Assembly Motifs (one each from Cep63 and Cep152) cooperatively conferred physicochemical properties that allowed them to undergo density transition and self-assemble into a cylindrical architecture. Interestingly, the Cep152-Cep63 condensates exhibited a rapid turnover, underwent fusion with other assemblies, and carried out a significant degree of internal rearrangement within a condensate. A Cep152-Cep63 cylindrical architecture that self-assembled on a flat substrate displayed a decreased but still detectable level of dynamic turnover. Interestingly, Polo-like kinase 4 (Plk4), a key regulator of centriole biogenesis, also dynamically phase-separated from a Cep152-bound state around a centriole (i.e., ring state) into a dot-like, low-nanoscale spherical condensate (i.e., dot state) upon autophosphorylating its C-terminal cryptic polo-box domain. Additional in vitro and in vivo data suggest that the Plk4 condensate serves as an assembling body at the future procentriole assembly site by amassing downstream procentriole assembly components such as STIL and Sas6 and facilitating Plk4-mediated centriole biogenesis. Thus, the formation of biomolecular condensates appears to be a fundamental step that not only promotes the self-assembly of a pericentriolar architecture but also triggers the process of centriole duplication. Along with this progress, we have been focusing on examining the mechanism underlying pericentriolar material (PCM) organization, self-assembling activity of pericentriolar scaffold proteins, molecular basis of building higher-order PCM architectures. To this end, we performed size-exclusion chromatography, sedimentation equilibrium ultracentrifugation, and interferometric scattering mass spectrometry and showed that the heterotetrameric building block generates octameric and hexadecameric complexes in a concentration-dependent manner, suggesting that the cylindrical self-assembly is formed through stepwise processes. By using MINFLUX nanoscopy, which offers low-nanometer-scale localization precision in a three-dimensional space, we further showed that mutants defective in forming the Cep63-Cep152 heterotetramer exhibited crippled pericentriolar Cep152 organization, consequently failing to promote polo-like kinase 4 (Plk4)'s dynamic relocalization from around the centriole to the future procentriole assembly site as well as Plk4-mediated centriole duplication. Remarkably, the entire self-assembly process could be driven by two short, uncharacterized regions (which we named "self-assembly modules") in Cep63 and Cep152 capable of cophase-separating and generating cylindrical self-assemblies in vitro. Fluorescence recovery after photobleaching revealed that the self-assembled architecture is highly dynamic, undergoing internal rearrangement within the assembly while exchanging its components with those in the surroundings. Dynamic turnover of pericentriolar Cep63 and Cep152 has also been observed in human centrosomes. Intriguingly, multiple cancer-associated Cep63 and Cep152 mutations are found in human cancer tissues (Catalogue of Somatic Mutations in Cancer; https://cancer.sanger.ac.uk/cosmic) but not in the gnomAD (https://gnomad.broadinstitute.org), which generally represents wildtype variants. Several of these mutations are present within the regions forming the heterotetrameric Cep63-Cep152 complex. Thus, investigating the mutations' significance could offer a deeper understanding about the architecture-function relationship of the Cep63-Cep152 complex. It may also help uncover new principles of building the Cep63-Cep152 self-assembly and provide valuable insights into the causes of PCM-associated human disorders. Given the evolutionarily conserved organization of PCM, this work could serve as a paradigm for investigating the structure and function of centrosomal scaffolds in other organisms.

中心体是一种独特的无膜多蛋白细胞器，充当动物细胞中的主要微管组织中心，在细胞周期的有序进展中起关键作用。由于中心体的组装故障和重复会导致异常细胞分裂，然后导致各种人类疾病，从而阐明了中心体组装和功能的分子机制，可能是理解中心体相关人类疾病的病因的关键步骤。 By combining cell biology with biophysical methods and X-ray crystallography, we demonstrated that two pericentriolar scaffolds, Cep152 and Cep63, possess intrinsic activity of co-phase-separating into condensates and form a heterotetrameric complex that serves as a building block for generating a nanoscale cylindrical self-assembly around a centriole.值得注意的是，两个简短的未表征区域称为自组装基序（一个来自CEP63和CEP152）合作赋予的物理化学特性，使他们能够进行密度过渡并自组装成圆柱形结构。有趣的是，CEP152-CEP63冷凝物表现出快速的营业额，与其他组件进行了融合，并在冷凝物内进行了很大程度的内部重排。自组装在平坦基板上的CEP152-CEP63圆柱体构造显示出降低但仍可检测到的动态营业额水平。有趣的是，类似polo样激酶4（PLK4）是中心生物发生的关键调节剂，也从Cep152结合的状态（即环状状态）中动态分离为单点，低纳米级球形冷凝物（即，在自动层上）的prolaptrapry trimal trime crian trime trimecrains triment trime。额外的体外和体内数据表明，PLK4冷凝物通过积聚下游丙烯酸酯组件（例如stil and sas6），并促进PLK4介导的中心生物发生，从而在未来的procentriole组装部位充当组装体。因此，生物分子冷凝物的形成似乎是一个基本步骤，不仅促进了丁香三醇结构的自组装，而且还触发了中心重复的过程。随着这一进展，我们一直专注于检查周围三元素材料（PCM）组织的机制，周围三醇支架蛋白的自组装活性，建立高阶PCM体系结构的分子基础。为此，我们进行了尺寸排斥色谱，沉积平衡超速离心和干涉散射质谱法，并表明异位型构建块以浓度依赖性的方式产生八聚体和十六成分复合物，表明cylindrical cylindrical cylindrical cylindrical cysembluse逐步形成了逐步的过程。 By using MINFLUX nanoscopy, which offers low-nanometer-scale localization precision in a three-dimensional space, we further showed that mutants defective in forming the Cep63-Cep152 heterotetramer exhibited crippled pericentriolar Cep152 organization, consequently failing to promote polo-like kinase 4 (Plk4)'s dynamic relocalization from around the centriole to the future Procentriole组装位点以及PLK4介导的中心复制。值得注意的是，整个自组装过程可以由CEP63和CEP152中的两个简短的，未表征的区域（我们命名为“自组装模块”）和能够在体外能够分离和产生cophase分离和产生圆柱体的自组件。光漂白后的荧光恢复表明，自组装的结构是高度动态的，在组件内进行了内部重排，同时将其组件与周围环境中的组件交换。在人的中心体中，也观察到了丁香三元素CEP63和CEP152的动态周转率。引人入胜的是，在人类癌组织（癌症中的体细胞突变目录； https://cancer.sanger.ac.ac.uk/cosmic）中发现了多个与癌症相关的CEP63和CEP152突变，但在GNOMAD（https://gnomad.broadinstitute.orgg）中，通常代表了野生。这些突变中有几个存在在形成异量式CEP63-CEP152复合物的区域内。因此，研究突变的意义可能可以更深入地了解CEP63-CEP152复合物的结构功能关系。它也可能有助于发现建立CEP63-CEP152自组装的新原则，并为PCM相关的人类疾病的原因提供宝贵的见解。鉴于PCM的进化保守组织，这项工作可以作为研究其他生物体中心体支架的结构和功能的范式。

项目成果

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

• DOI: 10.1074/jbc.m117.802587
• 发表时间: 2018-01-19
• 期刊: The Journal of biological chemistry
• 作者: Lee KH;Hwang JA;Kim SO;Kim JH;Shin SC;Kim EE;Lee KS;Rhee K;Jeon BH;Bang JK;Cha-Molstad H;Soung NK;Jang JH;Ko SK;Lee HG;Ahn JS;Kwon YT;Kim BY
• 通讯作者: Kim BY

Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis.

• DOI: 10.1038/nsmb.2846
• 发表时间: 2014-08
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8
• 作者: Park SY;Park JE;Kim TS;Kim JH;Kwak MJ;Ku B;Tian L;Murugan RN;Ahn M;Komiya S;Hojo H;Kim NH;Kim BY;Bang JK;Erikson RL;Lee KW;Kim SJ;Oh BH;Yang W;Lee KS
• 通讯作者: Lee KS

Recruitment of PP1 to the centrosomal scaffold protein CEP192.

将 PP1 募集至中心体支架蛋白 CEP192。

• DOI: 10.1016/j.bbrc.2017.02.004
• 发表时间: 2017
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Nasa,Isha;Trinkle-Mulcahy,Laura;Douglas,P;Chaudhuri,Sibapriya;Lees-Miller,SP;Lee,KyungS;Moorhead,GregB
• 通讯作者: Moorhead,GregB

Autophosphorylation-induced self-assembly and STIL-dependent reinforcement underlie Plk4's ring-to-dot localization conversion around a human centriole.

自磷酸化诱导的自组装和 STIL 依赖性强化是 Plk4 在人类中心粒周围从环到点定位转换的基础。

• DOI: 10.1080/15384101.2020.1843772
• 发表时间: 2020
• 期刊: Cell cycle (Georgetown, Tex.)
• 作者: Park,Jung-Eun;Meng,Lingjun;Ryu,EunKyoung;Nagashima,Kunio;Baxa,Ulrich;Bang,JeongKyu;Lee,KyungS
• 通讯作者: Lee,KyungS

Centrosomes in the spotlight: from organization to function to role in disease.

中心体成为焦点：从组织到功能再到在疾病中的作用。

• DOI: 10.1016/j.sbi.2021.01.001
• 发表时间: 2021
• 期刊: Current opinion in structural biology
• 影响因子: 6.8
• 作者: Lee,KyungS;Steinmetz,MichelO
• 通讯作者: Steinmetz,MichelO