Fig. 2: Cofilin-1 contributes to abscission by regulating F-actin depolymerization.

a Lysates of HeLa cells treated with either Control or Cofilin-1 siRNAs were blotted for endogenous Cofilin-1 and GAPDH (loading control). This experiment was repeated at least three times independently with similar results. b Left: Cumulative distribution of the fraction of cells that completed abscission (membrane cut measured by phase-contrast) as function of time after complete furrow ingression in cells treated with either Control or Cofilin-1 siRNAs (n ≥ 245 cells per condition from 4 independent experiments). Abscission times (from complete furrow ingression to abscission) were determined using phase contrast time-lapse microscopy. Right: Mean abscission time (min) ± SD in indicated cells, n ≥ 59 cells per condition, N = 4 independent experiments. Two-tailed unpaired Student’s t-test. c Left: same analysis as in (b) for cells treated with either Control or Cofilin-1 siRNAs and transiently transfected with indicated constructs (n ≥ 183 cells per condition from 3 independent experiments). Right: Mean abscission times (min) ± SD in indicated cells, n ≥ 60 cells per condition, N = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. p-values after comparison to siControl + GFP alone are indicated. n.s. = non-significant (p > 0.05). d Top: Schemes illustrating how the removal of TMP induces the sequestration of Cofilin-1-GFP at the surface of the mitochondria upon activation of anti-GFP nanobodies. Adapted from ref. ⁴⁹. Bottom: Snapshots of a spinning disk confocal microscopy movie of cells stably co-expressing Cofilin-1-GFP and the Tom20-anti-GFP nanobody-SNAP-DHFR fusion protein (labelled with SiR-SNAP) either when TMP is present or 5 and 120 min upon TMP removal, as indicated. Scale bars = 20 μm. e Left: same analysis as in (b) for cells stably co-expressing Cofilin-1-GFP and the Tom20-anti-GFP nanobody-SNAP-DHFR fusion protein, treated with either Control or Cofilin-1 siRNAs, upon removal (-) of TMP or not (+) after furrow ingression (n ≥ 164 cells per condition from 3 independent experiments). Right: Mean abscission time (min) ± SD in indicated cells, n ≥ 38 cells per condition, N = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. f Left: same analysis as in (b) for cells treated with either Control or Cofilin-1 siRNAs and incubated with either DMSO or 7.5 nM Cytochalasin D (CytoD) (n ≥ 173 cells per condition from N = 3 independent experiments). Right: Mean abscission time (min) ± SD in indicated cells, n ≥ 51 cells per condition, N = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. = non-significant (p > 0.05).