MICAL2-mediated actin network disassembly depends on cortactin. (A) Image stills from Video 10 showing GFP-tagged coronin 1C and MICAL2 (green) are still recruited to actin tails visualized with LifeAct-iRFP (magenta) in cells treated with cortactin siRNA. Scale bars = 5 µm (main image) and 2.5 µm (insert). The graph shows the percentage of actin tails recruiting GFP-MICAL2 following cortactin knockdown. The number of cells and actin tails analyzed is indicated. Student’s t test was used to determine statistical significance for n = 3 independent experiments. (B) Quantification of actin tail length in HeLa cells stably expressing RNAi-resistant GFP-tagged Arp3 (blue) or Arp3B (red) and treated with Arp3/Arp3B and cortactin siRNA. The error bars represent SEM from n = 3 independent experiments in which the length of 100 tails were analyzed per condition. (C) Quantification of the half-life of photoactivated GFP^PA-tagged Arp3 or Arp3B in actin tails in cells treated with cortactin siRNA. The graph shows the mean half-life and SEM from n = 3 independent experiments. Tukey’s multiple comparisons test was used to determine statistical significance; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01. (D) The schematic illustrates how MICAL2, coronin 1C, and cortactin cooperate to promote actin filament debranching by oxidizing Met293 of Arp3B. The plus and minus ends of the actin filaments are indicated. The MICAL2–coronin 1C complex is recruited to actin branches (step 1). MICAL2 oxidizes Met293 on Arp3B (step 2). Cortactin is required to facilitate ARP3B oxidation and/or promote subsequent debranching (step 3). It remains to be established if the daughter filament dissociates before the Arp2/3 complex (bottom row) or whether both dissociate at the same time, possibly with the complex attached to the end of the daughter filament (top row). NtA, amino-terminal acidic region; PRD, proline-rich domina; SH3, SRC homology 3 domain.