The ability of Arp3B to induce short actin tails depends on MICAL2. (A) The immunoblot shows the expression level of GFP-tagged Arp3 (blue) and Arp3B (red) together with their respective glutamine mutants (yellow). The immunofluorescence images show representative actin tails (magenta) in HeLa cells stably expressing the indicated GFP-tagged proteins (green), while the graph shows the quantification of their length. Scale bar = 2.5 µm. Ab, antibody. (B) Image stills from Video 6 and Video 7 showing that GFP-tagged (green) MICAL2, but not MICAL1, is recruited to vaccinia-induced actin tails visualized with LifeAct-iRFP (magenta). Scale bars = 5 µm (main image) and 2.5 µm (insert). (C) Quantification of the half-life of photoactivated Cherry-GFP^PA-β-actin in actin tails in cells treated with MICAL1 and MICAL2 siRNA. The main graphs represent the best-fitting curve for each condition (continuous line) together with the average normalized intensity of the GFP signal at every time point (error bars represent the SEM for the indicated number of tails). The insert graphs show the mean half-life and SEM from n = 3 independent experiments. Student’s t test was used to determine statistical significance. (D) The graph shows the length of actin tails in HeLa cells treated with the indicated siRNA. (E) Quantification of the effect of the loss of MICAL2 on actin tail length in HeLa cells stably expressing GFP-tagged Arp3 and Arp3B. In all actin tail length graphs, the error bars represent SEM from n = 3 independent experiments in which the length of 100 tails was analyzed per condition. Tukey’s multiple comparisons test was used to determine statistical significance; ***, P < 0.001; **, P < 0.01; *, P < 0.05.