Laboratory of Metabolism, National Cancer Institute, National Institutes of Health , Bethesda, MD, USA.
Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health , Bethesda, MD, USA.
Cell Cycle. 2020 Dec;19(24):3437-3457. doi: 10.1080/15384101.2020.1843777. Epub 2020 Nov 18.
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 and a significant level of internal rearrangemefnt within a condensate . 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. 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.
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关键作用。已经表明，两种中心体周围支架蛋白 Cep63 和 Cep152 形成异源四聚体复合物，自组装成围绕中心粒的更高阶圆柱形结构。然而，它们如何在巨大的细胞内空间达到其阈值浓度并产生自组装结构的机制仍然是个谜。在这里，我们证明像液态聚集物一样，Cep63 和 Cep152 协同生成无定形聚集体，能够经历动态周转和聚集体间融合，以及在凝聚体内发生显著的内部重排。一致地，1,6-己二醇，一种液-液相分离破坏剂，极大地降低了内源性 Cep63 和 Cep152 定位于中心体的能力。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。它还在大分子拥挤物存在的情况下形成凝聚体样固体球体。在分子水平上，Cep63 和 Cep152 各有一个疏水性基序，这两个基序对于生成相分离凝聚体和高分子量组装体是必需的。因此，我们提出 Cep63•Cep152 复合物的自组装是由复合物通过疏水性基序介导的相分离经历密度转变的内在性质触发的。PCM，中心体周围物质；LLPS，液-液相分离；MW，分子量；CLEM，共聚焦光镜和电子显微镜；WT，野生型；CMV，巨细胞病毒；FRAP，荧光恢复后光漂白；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，聚乙稀亚胺 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，Luria 肉汤；OD，光密度；IPTG，异丙基-β-D-1-硫代半乳糖吡喃糖苷；SDS-PAGE，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。
