Goldberg M W, Wiese C, Allen T D, Wilson K L
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, UK.
J Cell Sci. 1997 Feb;110 ( Pt 4):409-20. doi: 10.1242/jcs.110.4.409.
We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare 'dimples' (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular 'star-ring' subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5'-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N',N'-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.
我们使用场发射内透镜扫描电子显微镜来检查非洲爪蟾卵提取物中新组装的、正在生长的核膜。在核孔复合体之间散布着罕见的“凹坑”（外膜凹陷，直径5 - 35纳米）、更丰富的具有各种边缘几何形状的孔（35 - 45纳米直径；占结构的3.3%）、含有一到八个三角形“星环”亚基的孔（占总数的2.1%）以及更复杂的结构。当将高浓度（>500微克/毫升）的麦胚凝集素（它结合O - 糖基化核孔蛋白）直接添加到组装反应中时，既不会形成成熟复合体，也不会形成这些新结构；低浓度（10微克/毫升）则没有影响。然而，在中等浓度（50 - 100微克/毫升）时，麦胚凝集素会导致“空”孔和其他结构的显著的、糖可逆性积累；这种效应与凝集素诱导的非洲爪蟾每种主要麦胚凝集素结合核孔蛋白的可变比例沉淀相关。另一种抑制剂二溴 - BAPTA（5,5'-二溴 - 1,2 - 双[邻氨基苯氧基乙烷 - N,N,N',N'-四乙酸]），根据其添加到组装反应中的时间不同而有不同效果。当在时间零时添加1毫摩尔二溴 - BAPTA时，不会形成与孔相关的结构。然而，当在组装开始40 - 45分钟后将二溴 - BAPTA添加到正在生长的细胞核中时，星环和其他结构会积累，这表明二溴 - BAPTA可以抑制核孔复合体组装的多个阶段。我们提出组装始于穿过核膜的孔（通道）的形成和稳定，并且凹坑、孔、星环和细环是核孔复合体组装中的结构中间体。
