Fig. 4

Loss of the nuclear-cytoplasmic compartment barrier is associated with the loss of tubulin polymer. a Representative time-lapse confocal images of a HeLa cell during mitotic entry stably expressing H2B-mRFP (not imaged) and mEGFP-α-tubulin and transiently overexpressing Rap1* treated with S-trityl-l-cysteine (STLC). Upper panel: upper single section (inverted greyscale) of mEGFP-α-tubulin to visualise NEP, middle panel: x-y maximum projection images of mEGFP-α-tubulin (inverted greyscale) to visualise microtubules, lower panel: sum projection of mEGFP-α-tubulin sections around the centrosome (pseudo-color, spectra LUT) to visualise centrosomal microtubules. b Changes in centrosomal microtubule levels (circles) relative to NEP (nuclear tubulin, triangles) for one cell (shown in a, upper panel) and for equivalent four cells from two independent experiments (lower panel). c Representative time-lapse confocal images of a HeLa cell expressing MS2-mCherry-NLS (nuclear marker) and Lifeact-GFP during mitotic entry. Lower panel shows lateral projections. d Quantification of volume occupied by MS2-mCherry-NLS signal in HeLa cells similar to that in c (eight cells, two independent experiments). e Quantification of MS2-mCherry-NLS intensity. Median of MS2-mCherry-NLS signal for cells in d at indicated time points. Repeated measures ANOVA with the Greenhouse-Geisser correction, Tukey's multiple comparisons test with individual variances computed for each comparison, ****P < 0.0001. f Representative time-lapse confocal images of a HeLa cell stably expressing H2B-mRFP (shown in the upper panel at –30 min and 6 min) and mEGFP-α-tubulin during mitotic entry, following treatment with Nocodazole in non-adherent chambers (upper panel: x-y maximum projection, lower panel: single section, pseudo-color, spectra LUT). g Quantification of median mEGFP-α-tubulin signal in cells similar to that in f (eight cells filmed at high resolution using spinning disc confocal microscope, one independent experiment). h Quantification of median mEGFP-α-tubulin signal in cells similar to that in f at indicated time points (20 cells filmed at low resolution with wide-field microscope, two independent experiments). Repeated measures ANOVA with the Greenhouse-Geisser correction, Tukey's multiple comparisons test, with individual variances computed for each comparison, ****P < 0.0001. For measurement details see Methods. Graphs show mean and SD. Scale bars represent 10 μm