Extended Data Fig. 7: NPC degradation and nuclear envelope reshaping upon starvation.
From: In-cell architecture of the nuclear pore and snapshots of its turnover
a, western blot and quantification of the degradation of Nup133, Nup188 upon Nitrogen starvation at 25 °C and 30 °C in WT and nup116Δ strain. eGFP was measured using anti-eGPF immunoblotting. eGFP′ denotes vacuolar eGFP remnant. Dpm1 was used as a loading control. Centre values represent the mean and error bars the s.d. of n = 3 biologically independent replicates. At 30 °C, Nup133, Nup188 are degraded fourfold less in comparison to WT. b, Typical tomographic plastic section of WT, atg8Δ S. cerevisiae nuclei after 0h of starvation (negative control of Fig. 4c) or of vps4Δ S. cerevisiae nuclei after 0 or 24 h of starvation. Quantification of number of NPC-exposing herniae at 0, 5 and 24 h of starvation is shown for vps4Δ cells. We show an example of a round nucleus and of an NPC-exposing hernia from those cells. n = 85 tomograms of nuclei for WT; n = 84 for vps4Δ; n = 83 for atg8Δ; n = 62 for vps4Δ 24h starved; n = 64 for vps4Δ 5h starved; see Supplementary Table 2. N marks the nucleus. Scale bar, 200 nm. c, Quantification of of NPC-exposing herniae from plastic section tomograms as in Fig. 4c at 5h and 24h of starvation. After 24h, 82% of nuclei in the atg8Δ cells contain NPC-exposing herniae (n = 78 tomograms in average for each condition, see Supplementary Table 2). d, Quantification of deconvoluted wide-field maximum intensity projection images as in Fig. 4d from three biologically independent replicates and n = 300 per replica. e, Quantification of NPC density (NPCs/μm2) from plastic sections tomograms as in c shows clustering of NPCs in NPCs-exposing hernia. The statistical significance was evaluated with one-way ANOVA with Dunn’s multiple comparisons test (n = 20 tomograms per strain except vps4Δ NPC-exposing herniae where n = 5 tomograms; the average nuclear envelope surface measured per strain is 20 μm2, except atg8Δ NPC-exposing herniae where is 11 μm2 and vps4Δ NPC-exposing herniae where is 3.5 μm2; ****P < 0.0001; centre values represent the mean and error bars the SD). f, Phase contrast and deconvoluted wide-field max intensity projection of WT, vps4Δ and atg8Δ cells imaged live before and after 24h of starvation with eGFP-tagged Nup192 as marker. The quantification is derived from three independent biological replicates where five images with at least n = 250 nuclei per replicate; central bars represent the mean and error bars the s.d. The trend of nucleus deformation is the same seen in b. g, Same as c for WT and atg8Δ cells, but with the inner nuclear membrane marker Nsg1. Data are derived from three independent biological replicates with at least n = 250 nuclei per replicate.
