Molecular basis of centriole duplication

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会导致细胞分裂异常，从而导致各种人类疾病，因此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疾病的病因学的关键一步。通过结合细胞生物学、生物物理方法和X射线结晶学，我们证明了两种中心周围支架Cep152和Cep63具有内在的共相分离成凝聚体的活性，并形成了一个异四聚体复合体，作为围绕中心粒生成纳米级柱状自组装的构建块。值得注意的是，两个名为自组装基序的短的未表征区域(分别来自Cep63和Cep152)协同赋予物理化学性质，使它们经历密度转换并自组装成圆柱形结构。有趣的是，Cep152-Cep63凝析油表现出快速周转，与其他组件融合，并在凝析油中进行了显著程度的内部重排。在平面基板上自组装的Cep152-Cep63圆柱形架构显示出降低但仍可检测到的动态周转水平。有趣的是，作为中心粒生物发生的关键调节因子，Polo-like kinase4(Plk4)也动态地从中心粒周围的Cep152结合状态(即环状状态)分离为点状、低纳米尺度的球形凝聚体(即点状状态)，当其C末端隐蔽的Polo-box结构域自动磷酸化时。更多的体外和体内数据表明，Plk4缩合物通过聚集下游的前着丝点组装成分如STIL和Sas6，作为未来前着丝点组装部位的集合体，促进Plk4介导的中心粒的生物发生。因此，生物分子凝聚体的形成似乎是一个基本步骤，它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过程。随着这一进展，我们目前正在研究中心周围物质(PCM)组织的机制，中心周围支架蛋白的自组装活性，以及构建高阶PCM结构的分子基础。

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 are currently examining the mechanism underlying pericentriolar material (PCM) organization, self-assembling activity of pericentriolar scaffold proteins, molecular basis of building higher-order PCM architectures.