Figure 3: MICAL1 depletion leads to F-actin accumulation in cytokinetic bridges associated with abnormal CHMP4B recruitment and abscission defects.

(a) Left: Staining of F-actin by phalloidin (green), AuroraB (red) and DAPI (blue) in control- or MICAL1-depleted cells. Right: quantification of the intensity of the phalloidin staining in bridges after control and MICAL1 depletion (N=4). *P<0.05; **P<0.01; ***P<0.001 (two-way ANOVA). n=321–365 cells per condition. Scale bars, 10 μm. (b) Correlative light-scanning electron microscopy (SEM) of dividing cells expressing GFP-actin^endogenous after control (left) or MICAL1 (right) depletion. Phase contrast (TRANS), fluorescent and SEM pictures with corresponding zooms of cytokinetic bridges are presented. Scale bars, 10 μm (except for SEM zooms: 1 μm). (c) Quantification of the phalloidin staining in bridges in control- or MICAL1-depleted cells treated with DMSO or 20 nM Latrunculin-A (LatA) (N=3). NS, not significant; *P<0.05; ***P<0.001 (two-way ANOVA). n=152–194 cells per condition. (d) Distribution of the abscission times and mean abscission times in control- and MICAL1-depleted cells treated with either DMSO or Latrunculin A, as indicated (N=3). No statistical differences between black, green and blue curves; P<0.002 between red and other curves (KS test). NS, not significant; *P<0.05; ***P<0.001 (two-way ANOVA). n=218–321 cells per condition. (e) Percentage of bridges with no CHMP4B at all, with CHMP4B only at the midbody and with CHMP4B at the midbody+ at the abscission site in control− or MICAL1-depleted cells (N=4). **P<0.01; ***P<0.001 (two-way ANOVA). n=288–320 cells per condition. The abscission site is defined as an interrupted tubulin staining with a spot of CHMP4B, on one side of the midbody. (f) Same as in e after treatment with either DMSO or Latrunculin-A, as indicated (N=3). NS, not significant; *P<0.05; **P<0.01 (two-way ANOVA). n=152–212 cells per condition. Error bars represent s.d.