Sluder G, Thompson E A, Rieder C L, Miller F J
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545, USA.
J Cell Biol. 1995 Jun;129(6):1447-58. doi: 10.1083/jcb.129.6.1447.
Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.
核膜破裂（NEB）以及进入有丝分裂被认为是由p34cdc2 - 细胞周期蛋白B激酶复合物或有丝分裂促进因子（MPF）的激活所驱动。监测有丝分裂重要准备事件（如DNA复制）的检查点控制机制，被认为是通过下调MPF激活来阻止进入有丝分裂，直到这些事件完成。因此，当用秋水仙酰胺阻断海胆受精卵中的原核融合时，我们惊讶地发现雌原核始终比雄原核先破裂。这并非由于MPF激活时间上的区域差异，因为相互接触的原核会以相同程度异步破裂。为了测试NEB是在细胞核还是细胞质水平受到控制，我们通过在受精前用补骨脂素处理卵子或精子，使DNA的碱基配对链共价交联，从而分别在雌原核和雄原核中激活DNA合成完成的检查点。当仅母本DNA交联时，雄原核先破裂。当精子DNA交联时，相对于雌原核破裂，雄原核破裂会显著延迟，有时甚至不发生。在原核相互接触的此类受精卵中，雌原核发生NEB后使秋水仙酰胺失活，会产生一个由母本染色体和父本中心体组成的功能性纺锤体。完整的雄原核在整个有丝分裂过程中始终位于一个星体处。在其他实验中，用补骨脂素处理精子核后，超过90%的受精卵核在对照之后至少2小时内不会破裂，尽管H1组蛋白激酶活性逐渐升高至接近或高于对照有丝分裂水平。用阿非迪霉素处理正常受精卵以阻断DNA合成时也是如此。从这些结果中，我们得出结论，海胆受精卵中的NEB是在细胞核水平而非细胞质水平受到控制，并且细胞质MPF活性的有丝分裂水平不足以驱动处于检查点控制下的细胞核发生NEB。我们的结果还表明，DNA合成完成的检查点主要通过在细胞核内起作用来抑制NEB，而不是通过下调细胞质MPF的活性。
