Fig. 5: The MT severing delay observed in Cofilin-1-depleted cells is associated with a defective recruitment of the ESCRT-III subunit CHMP1B and spastin.
From: Cytokinetic abscission requires actin-dependent microtubule severing
a Top: Staining of endogenous Cofilin-1, CHMP4B and Tubulin in a late ICB with secondary ingression (arrowhead). Scale bar = 2 μm. Bottom: Intensity profile of CHMP4B (green) and Cofilin-1 (magenta) between * and ** along the ICB from the top merge picture. This experiment was repeated at least three times independently with similar results. b Top: Snapshots of a spinning disk confocal microscopy movie of cells stably expressing CHMP4B-GFP and incubated with fluorescent SiR-Tubulin. T1: time from CHMP4B-GFP recruitment at the midbody (t = 0 min) to appearance at the secondary ingression (arrowhead); T2: time from CHMP4B-GFP appearance at the secondary ingression to the MT cut. Scale bar = 5 μm. Bottom: Mean T1 and T2 (min) ± SD in indicated cells, n = 10–35 cells per condition, N = 4 independent experiments (violin plots). Two-tailed unpaired Student’s t-test with Welch’s correction. n.s. = non-significant (p > 0.05). c Quantification of CHMP4B-GFP cone size at the timepoint just before the cut of the MTs from the movies described in (d). Mean ± SD, n = 8–32 cells per condition, N = 3 independent experiments (violin plots). Two-tailed unpaired Student’s t-test. n.s. = non-significant (p > 0.05). d Left: Snapshots of a spinning disk confocal microscopy movie (8 min intervals) of cells stably expressing CHMP4B-GFP and incubated with fluorescent SiR-Tubulin and treated with either Control or Cofilin-1 siRNAs. Arrowhead: tip of the ESCRT-III cone. Scale bar = 2 μm. Right: Quantification of the growth rate of CHMP4B cone in indicated cells. Mean ± SD, n = 8–32 cells per condition, N = 3 independent experiments (violin plots). Two-tailed unpaired Student’s t-test. e Top: Representative images of endogenous CHMP1B and Tubulin in cells treated with either Control or Cofilin-1 siRNAs. Arrowhead: secondary ingression. Scale bars = 5 μm. Bottom: mean fluorescence intensity of CHMP1B (arbitrary units) at the midbody (late ICBs without secondary ingression) or at the midbody + secondary ingression (late ICBs with secondary ingression) in indicated cells. Mean ± SD, n = 7–21 cells per condition, N = 3 independent experiments (violin plots). Intensities are normalized in each experiment to the mean intensity of siControl cells in late ICB without secondary ingression. One-way ANOVA with Tukey’s multiple comparisons test. n.s. = non-significant (p > 0.05). f Same as in (e) for endogenous spastin. Mean ± SD in indicated cells, n = 8–20 cells per condition, N = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. = non-significant (p > 0.05).
