在后生动物中,核包膜(NE)在前期分解,并在末期围绕分离的染色单体重新组装。已经使用活细胞成像广泛研究了NE形成的过程。在人体细胞中NE重组的早期阶段,内核膜(INM)蛋白的特异性模式样定位,连接到核孔复合体(NPC),在染色体末期的所谓“核心”区域和“非核心”区域中观察到,对应于“无孔”区域和“富孔”区域,分别,在早期G1间期核。我们将这些现象称为NE子域形成。为了生化研究这个过程,我们的目的是使用洋地黄皂苷透化半完整有丝分裂人类细胞共表达两种INM蛋白,开发一种体外NE重建系统。Emerin和层粘连蛋白B受体,用荧光蛋白标记。使用延时成像观察了半完整细胞中后期发病前后INM蛋白对染色体的靶向和积累。我们的体外NE重建系统概括了NE亚结构域的形成,就像在活细胞中一样,尽管没有观察到染色体分离和胞质分裂。这种体外NE重建需要添加补充有细胞周期蛋白依赖性激酶抑制剂和能源的有丝分裂胞质部分。INM蛋白靶向的细胞质可溶性因子依赖性在染色体的分离状态之间不同。此外,在分离的染色体上重建的NE表现出活跃的核质转运能力。这些结果表明,在后期开始招募NPC成分后,染色体状态会发生变化。关键词:核封套重组,内核膜蛋白,核孔隙复合物,半完整的细胞,体外重建。
In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called \"core\" region and \"noncore\" region on telophase chromosomes, which corresponded to the \"pore-free\" region and the \"pore-rich\" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.