Additional file 4: of Two-step interphase microtubule disassembly aids spindle morphogenesis

Hypo-osmotic shock can be used to mimic changes in tubulin concentration induced by NEP. A) Representative time-lapse confocal images (x-y maximum projection, lower panel: pseudo-color, spectra LUT) of HeLa cells stably expressing H2B-mRFP (not imaged) and mEGFP-α-tubulin, treated with Nocodazole, to show changes in cell diameter and in mEGFP-α-tubulin intensity before and after hypo- or hyper-osmotic shock treatment relative to control treatment. Quantifications of changes in mEGFP-α-tubulin intensity (B) and in cell diameter (C) induced by osmotic shock relative to control treatment. Mean intensity of mEGFP-α-tubulin signal and cell diameter was measured in cells before and after control (blue, seven cells), hypo- (red, eight cells) or hyper- (green, eight cells) osmotic shock treatments (two independent experiments) as described in Methods. Lower panels show comparison between values at –0.5 min and 2 min relative to osmotic shock treatments. Repeated measures two-way ANOVA, Dunnett's multiple comparisons test, ****P = 0.0001. D) Representative time-lapse confocal images (x-y maximum projection, lower panel: pseudo-color, spectra LUT) of HeLa cells stably expressing H2B-mRFP (not imaged) and mEGFP-α-tubulin showing that hypo-osmotic shock affects mitotic spindle, whereas it does not have impact on interphase microtubules. White arrows indicate mitotic spindle. E) Representative time-lapse confocal images (x-y maximum projection) of HeLa cells stably expressing H2B-mRFP and mEGFP-α-tubulin and transiently overexpressing Rap1 treated with Lamin A siRNA and ESCRT-III siRNA during mitotic entry. Boxed areas are zoomed below. Control cell represents a Lamin A siRNA and ESCRT-III siRNA treated cell entering mitosis. The following cells represent accordingly a cell where nuclear envelope rupture was induced in late prophase (close to NEP) followed by immediate disassembly of microtubules and a cell where nuclear envelope rupture was induced in early prophase without triggering immediate disassembly of microtubules. F) Quantifications of timing of changes in centrosomal and non-centrosomal microtubule levels relative to NEP or to nuclear envelope (NE) ablation in cells represented in E as described in Fig. 2b. Scale bars represent 10 μm. (PDF 6682 kb)