ABSTRACT

The centrosome is a unique membraneless organelle that plays a pivotal role in the orderly progression of the cell cycle in animal cells. It has been shown that two pericentriolar scaffold proteins, Cep63 and Cep152, generate a heterotetrameric complex to self-assemble into a higher-order cylindrical architecture around a centriole. However, the mechanisms underlying how they reach their threshold concentrations in the vast intracellular space and generate a self-assembled architecture remain mysterious. Here we demonstrate that, like liquid-like assemblies, Cep63 and Cep152 cooperatively generate amorphous aggregates capable of undergoing dynamic turnover and inter-aggregate fusion in vivo and a significant level of internal rearrangemefnt within a condensate in vitro. Consistently, 1,6-hexanediol, a liquid–liquid phase separation disruptor, greatly diminished the ability of endogenous Cep63 and Cep152 to localize to centrosomes. Interestingly, a purified Cep63•Cep152 complex generated either a cylindrical structure or a vesicle-like hollow sphere in a spatially controlled manner. It also formed condensate-like solid spheres in the presence of a macromolecular crowder. At the molecular level, two hydrophobic motifs, one each from Cep63 and Cep152, were required for generating phase-separating condensates and a high molecular–weight assembly. Thus, we propose that the self-assembly of the Cep63•Cep152 complex is triggered by an intrinsic property of the complex undergoing density transition through the hydrophobic-motif-mediated phase separation.

Abbreviations: PCM, pericentriolar material; LLPS, liquid–liquid phase separation; MW, molecular-weight; CLEM, correlative light and electron microscopy; WT, wild-type; CMV, cytomegalovirus; FRAP, fluorescence recovery after photobleaching; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; 3D-SIM, three-dimensional structured illumination microscopy; DMEM, Dulbecco’s Modified Eagle Medium; PEI Max, Polyethylenimine Max; PBS, phosphate-buffered saline; RT, room temperature; DAPI, 4', 6-diamidino-2-phenylindole; AOTF, acousto-optic tunable filter; LB, Luria broth; OD, optical density; IPTG, isopropyl β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

摘要

中心体是一种独特的无膜细胞器，在动物细胞的细胞周期有序进行中起着举足轻重的作用。已经表明，两个中心周支架蛋白 Cep63 和 Cep152 产生异四聚体复合物，以自组装成围绕中心粒的高阶圆柱形结构。然而，它们如何在广阔的细胞内空间中达到阈值浓度并产生自组装结构的机制仍然是个谜。在这里，我们证明，像液体样组装一样，Cep63 和 Cep152 协同产生无定形聚集体，能够在体内进行动态周转和聚集体间融合，并在体外冷凝物中进行显着水平的内部重排. 始终如一地，1,6-己二醇，一种液-液相分离干扰物，极大地降低了内源性 Cep63 和 Cep152 定位到中心体的能力。有趣的是，纯化的 Cep63•Cep152 复合物以空间控制的方式生成圆柱形结构或囊泡状空心球体。它还在大分子聚集体的存在下形成了类似冷凝物的固体球体。在分子水平上，需要两个疏水基序，每个来自 Cep63 和 Cep152，用于产生相分离缩合物和高分子量组装。因此，我们提出 Cep63•Cep152 复合物的自组装是由复合物通过疏水基序介导的相分离进行密度转变的内在特性触发的。

缩写： PCM，pericentriolar material；LLPS，液-液相分离；MW，分子量；CLEM，相关光电子显微镜；WT，野生型；CMV，巨细胞病毒；FRAP，光漂白后的荧光恢复；FITC，异硫氰酸荧光素；PCR，聚合酶链反应；3D-SIM，三维结构照明显微镜；DMEM，Dulbecco 改良 Eagle 培养基；PEI Max，聚乙烯亚胺 Max；PBS，磷酸盐缓冲盐水；RT，室温；DAPI, 4', 6-二脒基-2-苯基吲哚；AOTF，声光可调滤波器；LB，卢里亚肉汤；OD，光密度；IPTG，异丙基β-D-1-硫代吡喃半乳糖苷；SDS-PAGE，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳