Shumaker D K, Vann L R, Goldberg M W, Allen T D, Wilson K L
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Cell Calcium. 1998 Feb-Mar;23(2-3):151-64. doi: 10.1016/s0143-4160(98)90114-2.
We used Xenopus egg extracts to examine the effects of TPEN, a chelator with strong affinities for Zn2+, Fe2+, and Mn2+, on nuclear assembly in vitro. At concentrations above 1 mM, TPEN blocked the assembly of the nuclear lamina and produced nuclei that were profoundly sensitive to stress-induced balloon-like 'shedding' of nuclear membranes away from chromatin-associated membranes. TPEN-arrested nuclei were also defective for DNA replication, which could be explained as secondary to the lack of a lamina. Imaging of TPEN-arrested nuclei by field emission in-lens scanning electron microscopy (FEISEM) revealed clustered, structurally-perturbed nuclear pore complexes. TPEN-arrested nuclei were defective in the accumulation of fluorescent karyophilic proteins. All detectable effects caused by TPEN were downstream of the effects of BAPTA, a Ca2+/Zn2+ chelator that blocks pore complex assembly at two distinct early stages. Surprisingly, TPEN-arrested nuclei, but not control nuclei, remained active for replication in apoptotic extracts, as assayed by [32P]-dCTP incorporation into high molecular weight DNA, suggesting that TPEN blocks a metal-binding protein(s) required for nuclear destruction during programmed cell death.
我们使用非洲爪蟾卵提取物来研究TPEN（一种对Zn2+、Fe2+和Mn2+具有强亲和力的螯合剂）对体外细胞核组装的影响。在浓度高于1 mM时，TPEN会阻断核纤层的组装，并产生对压力诱导的核膜从染色质相关膜上呈气球样“脱落”高度敏感的细胞核。TPEN阻滞的细胞核在DNA复制方面也存在缺陷，这可以解释为是由于缺乏核纤层所致。通过场发射透镜内扫描电子显微镜（FEISEM）对TPEN阻滞的细胞核进行成像，发现核孔复合体聚集且结构紊乱。TPEN阻滞的细胞核在荧光亲核蛋白的积累方面存在缺陷。TPEN所引起的所有可检测到的效应都发生在BAPTA（一种Ca2+/Zn2+螯合剂，可在两个不同的早期阶段阻断孔复合体组装）的效应之后。令人惊讶的是，通过将[32P]-dCTP掺入高分子量DNA进行检测发现，TPEN阻滞的细胞核而非对照细胞核在凋亡提取物中仍具有复制活性，这表明TPEN阻断了程序性细胞死亡期间细胞核破坏所需的一种金属结合蛋白。
