Marshall I C, Gant T M, Wilson K L
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Cell Calcium. 1997 Feb;21(2):151-61. doi: 10.1016/s0143-4160(97)90039-7.
The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca(2+)-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.
高等真核生物的核膜在有丝分裂早期解体，随后在子代染色体周围重新组装。先前的体外研究支持这样一种假说，即通过肌醇1,4,5-三磷酸门控的Ca2+通道释放内质网腔Ca2+库对于非洲爪蟾卵提取物中的核组装是必需的。其他研究表明，内质网腔Ca2+库对于哺乳动物细胞体内的核蛋白导入是必需的，但在体外则不然。在这里，我们使用非洲爪蟾卵提取物严格测试了内质网腔Ca2+库在核组装和核蛋白导入中的作用。通过用Ca2+离子载体（离子霉素、A23187）或Ca2+螯合泵抑制剂（毒胡萝卜素、环匹阿尼酸）预处理提取物来耗尽内质网腔Ca2+库。耗尽内质网腔Ca2+库的提取物围绕去膜精子染色质组装成核。当通过光学或电子显微镜观察时，这些核在形态上与对照核没有区别。缺乏内质网腔Ca2+库的核排除了膜不透性的荧光葡聚糖，表明形成了密封的核膜，并且它们积累了一种荧光亲核蛋白核质素，表明核孔复合体是有功能的。DNA复制在缺乏内质网腔Ca2+的核中发生，尽管效率低于对照核。我们证明体外核输入不依赖于内质网腔Ca2+库，这证实了Greber和Gerace先前未发表的观察结果，并表明在离子载体处理的活细胞中看到的输入缺陷并非直接由于内质网腔Ca2+的丧失。最后，我们得出结论，与我们的预期相反，非洲爪蟾卵提取物中的核膜组装不需要内质网腔Ca2+库。
