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，是动物细胞中主要的微管组织中心，在细胞周期的有序进行中起着关键作用。由于中心体的错误组装和复制导致细胞分裂异常，从而导致各种人类疾病，阐明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病因学的关键一步。通过结合细胞生物学、生物物理方法和x射线晶体学，我们证明了Cep152和Cep63两种中心粒周围支架具有共相分离成凝聚物的内在活性，并形成异四聚体复合物，作为在中心粒周围产生纳米级圆柱形自组装的基石。值得注意的是，两个名为自组装基序（Self-Assembly Motifs）的短而未表征的区域（分别来自Cep63和Cep152）协同赋予了它们的物理化学性质，使它们能够经历密度转变并自组装成圆柱形结构。有趣的是，Cep152-Cep63凝析物表现出快速的转换，经历了与其他组合的融合，并在凝析物中进行了很大程度的内部重排。在平面基板上自组装的Cep152-Cep63圆柱形结构显示出下降但仍可检测到的动态周转水平。有趣的是，中心粒生物发生的关键调控因子polo样激酶4 （Plk4）也在其c端隐性polo-box结构域自磷酸化后，从中心粒周围的cep152结合状态（即环状状态）动态地分相分离为点状、低纳米级球形凝结物（即点状态）。另外的体外和体内数据表明，Plk4凝析物通过聚集下游的前中心粒组装成分（如STIL和Sas6），在未来的前中心粒组装位点充当组装体，并促进Plk4介导的中心粒生物发生。因此，生物分子凝聚体的形成似乎是一个基本步骤，不仅促进了中心粒周围结构的自组装，而且还触发了中心粒复制的过程。随着这一进展，我们一直专注于研究中心周围材料（PCM）组织的机制，中心周围支架蛋白的自组装活性，构建高阶PCM结构的分子基础。为此，我们进行了尺寸排除色谱、沉降平衡超离心和干涉散射质谱分析，结果表明，异四聚体构建块以浓度依赖的方式产生八聚体和十六聚体配合物，表明圆柱形自组装是通过逐步形成的。通过使用MINFLUX纳米显微镜，在三维空间中提供低纳米尺度的定位精度，我们进一步发现，在形成Cep63-Cep152异四聚体方面存在缺陷的突变体表现出中心粒周围Cep152组织的缺陷，从而无法促进polo-like kinase 4 （Plk4）从中心粒周围到未来中心粒前组装位点的动态再定位，也无法促进Plk4介导的中心粒复制。值得注意的是，整个自组装过程可以由Cep63和Cep152中两个短的、未表征的区域（我们将其命名为“自组装模块”）驱动，这些区域能够在体外进行相分离并产生圆柱形自组装。光漂白后的荧光恢复显示，自组装的结构是高度动态的，在组装内部进行重排，同时与周围的组件交换其组件。在人类中心体中也观察到中心周围Cep63和Cep152的动态周转。有趣的是，在人类癌症组织中发现了多种与癌症相关的Cep63和Cep152突变（Catalogue of Somatic mutations in cancer; https://cancer.sanger.ac.uk/cosmic），但在gnomAD中却没有发现（https://gnomad.broadinstitute.org），后者通常代表野生型变异。其中一些突变存在于形成异四聚体Cep63-Cep152复合物的区域内。因此，研究突变的意义可以更深入地了解Cep63-Cep152复合物的结构-功能关系。它还可能有助于揭示构建Cep63-Cep152自组装的新原理，并为pcm相关人类疾病的原因提供有价值的见解。鉴于PCM的进化保守性，这项工作可以作为研究其他生物中心体支架结构和功能的范例。

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.

人类增强子丝1（HEF1）的磷酸化刺激了与类似Polo样激酶1的相互作用，从而导致HEF1定位与局灶性粘连。

• 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

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

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