Fig 1: NPC assembly relies on and consumes almost half of the material inherited from the mother cell within one hour after mitosis.FCS-calibrated 3D confocal microscopy was performed as in Fig. 2, and the Nup copy number was quantified in whole dividing cells (for details see Methods). The number of Nups in cytoplasm (dark blue), nucleoplasm (medium dark blue) and nuclear envelope (light blue) are plotted against time after anaphase onset. The plot is the mean of 15, 20, 13, 14, 22, 22, 19, and 24 cells for Nup107, Seh1, Nup205, Nup93, Nup62, Nup214, Tpr and Nup358, respectively. Source data
Fig 2: Integrative model of the postmitotic NPC assembly pathway.a, Protein density (grey) overlaid with the nuclear envelope surface (wireframe model, grey) at each time point12 used for integrative modelling. C, cytoplasm; N, nucleoplasm. The dome-like density in the nucleoplasmic side at 5 and 6 min is the noise from electron tomography. b, The best-scoring model of the postmitotic assembly pathway (top and side views). The uncertainty of each Nup localization is indicated by the density of the corresponding colour. The Y-complex is shown in green; an isolated fraction of Nup155 that is not forming a complex with Nup93, Nup188 and Nup205 is shown in orange; Nup93–Nup188–Nup155 and Nup205–Nup93–Nup155 complexes are in blue; and the Nup62–Nup58–Nup54 complex is in purple. c, The enlargement of one of the spokes. The model has a much higher score than all the other lower-scoring pathways (Extended Data Fig. 8). The pseudoatomic model of the native NPC structure27,28 we imposed as the endpoint of the assembly pathway does not include the Y-complex-bound fraction of Nup205 or the Nup214-bound fraction of Nup62, and thus contains only 16 of the 40 copies of Nup20533,34 and 32 of the 48 copies of Nup6235 in the fully mature NPC. How the remaining 24 copies of Nup205 and 16 copies of Nup62 are assembled remains elusive.
Fig 3: The variations of the integrative models for postmitotic assembly pathway.Detailed views of the best and lower-scoring pathways. The side views of the 3 spokes are shown. The NE surface is indicated by wireframe model (grey). The uncertainty of each Nup localization is indicated by the density of the corresponding color: The Y-complex (green), isolated fraction of Nup155 (orange), Nup93-Nup188-Nup155 and Nup205-Nup93-Nup155 complexes (blue), and Nup62-Nup58-Nup54 complex (purple). The posterior model likelihood for each pathway is indicated (see Methods for details). The variations in the structural models for the intermediates at 5 and 6 min after anaphase onset would be due to their lower protein density (lower signal-to-noise ratio) in the electron tomography images.
Fig 4: Quantitative imaging of GFP-knock-in Nup cell lines.a, Confocal microscopy of genome-edited HeLa cells with homozygous expression of mEGFP-tagged Nups. Fluorescence intensity was converted to protein concentration (colour bar) using FCS-calibrated imaging22. Images were filtered with a median filter (kernel size: 0.25 × 0.25 µm) for presentation purposes. Scale bar, 10 µm. b,c, Stimulated emission depletion (STED) microscopy of genome-edited cells stained with Nup62 antibody. Cells were imaged (b) and the density of nuclear pores was quantified (c). n = 6 (Nup107), 6 (Seh1), 6 (Nup205), 5 (Nup93), 6 (Nup62), 6 (Nup214), 6 (Tpr) and 4 (Nup358) cells. Scale bar, 1 µm. d, Calculated copy number of Nups per nuclear pore. n = 241 (Nup107), 37 (Seh1), 41 (Nup205), 20 (Nup93), 41 (Nup62), 26 (Nup214), 55 (Tpr) and 28 (Nup358) cells. The horizontal line represents the median.Source data
Fig 5: Kinetic decomposition of the two assembly processes for each nucleoporin.The fluorescence intensities at non-core (brown) and inner-core (yellow) regions were plotted and fitted with a sequential model of NPC assembly (bold lines) as in Extended Data Fig. 5. Dots represent the average and s.d. of measurements from 15, 20, 13, 14, 22, 22, 19, 24, 14 and 13 cells for Nup107, Seh1, Nup205, Nup93, Nup62, Nup214, Tpr, Nup358, Nup153, and Pom121, respectively. Single confocal slices of cells at 20 min, 40 min, and 60 min after AO are shown. Images were filtered with a median filter (kernel size: 0.25 × 0.25 µm) for presentation purposes. Scale bars, 10 µm. Source data
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