Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes^1,2,3,4. There are at least two NPC assembly pathways—one during the exit from mitosis and one during nuclear growth in interphase—but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.

了解核孔复合体 (NPC) 的组装方式对于掌握其基本功能背后的机制并了解其在真核生物进化过程中的作用至关重要^1,2,3,4 。至少有两种 NPC 组装途径——一种是在有丝分裂退出期间，另一种是在间期核生长期间——但我们目前缺乏这些事件的定量图谱。在这里，我们使用荧光相关光谱校准内源性荧光标记核孔蛋白的实时成像来绘制人类 NPC 在单个分裂细胞中组装过程中七个主要模块的组成和化学计量的变化。这个系统的定量图揭示了这两种组装途径具有不同的分子机制，其中两个大的结构成分（中央环复合物和核丝）的添加顺序是相反的。整合动态化学计量数据以创建 NPC 组装途径的时空模型并预测有丝分裂后 NPC 组装中间体的结构。