半贴壁细胞有丝分裂染色体上核膜亚域形成的重建

IF 2 4区 生物学 Q4 CELL BIOLOGY
Tomoko Funakoshi, Naoko Imamoto
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引用次数: 0

摘要

在后生动物中,核包膜(NE)在前期分解,在端期围绕分离的染色体重新组合。人们利用活细胞成像技术对核包膜的形成过程进行了广泛研究。在人类细胞NE重新组合的早期阶段,我们在端期染色体上所谓的 "核心 "区域和 "非核心 "区域观察到了与核孔复合体(NPC)相连的核内膜(INM)蛋白的特异性模式定位,这两个区域分别对应于G1间期早期细胞核中的 "无孔 "区域和 "富孔 "区域。我们将这些现象称为NE亚域的形成。为了对这一过程进行生化研究,我们开发了一种体外 NE 重构系统,该系统使用地高辛渗透稳定的半有丝分裂人体细胞,共表达两种 INM 蛋白(emerin 和层粘连蛋白 B 受体),并用荧光蛋白标记。利用延时成像技术观察了INM蛋白在半有丝分裂期开始前后在染色体上的靶向和聚集。我们的体外 NE 重组系统再现了 NE 亚域的形成,就像在活细胞中一样,尽管没有观察到染色体分离和细胞分裂。这种体外 NE 重组需要加入有丝分裂的细胞质部分,并辅以细胞周期蛋白依赖性激酶抑制剂和能量源。染色体分离状态不同,INM 蛋白靶向的细胞质可溶性因子依赖性也不同。此外,在分离染色体上重组的 NE 表现出活跃的核胞质转运能力。这些结果表明,无丝分裂期开始后,染色体的状态会发生变化,从而招募NPC成分。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells

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.

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来源期刊
Cell structure and function
Cell structure and function 生物-细胞生物学
CiteScore
2.50
自引率
0.00%
发文量
6
审稿时长
>12 weeks
期刊介绍: Cell Structure and Function is a fully peer-reviewed, fully Open Access journal. As the official English-language journal of the Japan Society for Cell Biology, it is published continuously online and biannually in print. Cell Structure and Function publishes important, original contributions in all areas of molecular and cell biology. The journal welcomes the submission of manuscripts on research areas such as the cell nucleus, chromosomes, and gene expression; the cytoskeleton and cell motility; cell adhesion and the extracellular matrix; cell growth, differentiation and death; signal transduction; the protein life cycle; membrane traffic; and organelles.
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