The Rac-WRC-Arp2/3 pathway is required for wound closure. (A) Wild type 18 h APF old pupa specifically expressing a Lifeact-EGFP transgene in the abdominal epidermis under the control of the A58-Gal4 driver. The imaged area of the monolayered epithelium is boxed in yellow. (A′) Scale bar represents 250 µm (A′) Schematic of the in vivo wounding model. Laser-induced single-cell ablation starts at t = 0 min. In the first two min (t = 2 min), F-actin assembles into broad lamellipodial protrusions (green) within cells at the wound edge; lamellipodial protrusions reach a maximum size between 5 and 10 min after wounding. Later on (t > 5 min), an acto-myosin ring (red) is formed at the leading edge of the wound (according to Lehne and Bogdan [2023]). (B–E) Frames of spinning disc microscopy videos of 18 h APF old epidermis expressing a Lifeact-EGFP transgene under the control of the A58-Gal4 driver. The genotypes are indicated. Images were taken at indicated time points. Ablation of a single cell (yellow asterisk) starts at t = 0 min. Scale bar represents 50 µm. (F and F′) Quantification of wound closure in wild type (WT; n = 13) and after knockdown of arp2 (n = 15), arp3 (n = 16) and wave (n = 16) by RNAi. (F) Lamellipodia size was measured every 5 min and normalized to the initial size of the unwounded cell. Scale bar represents 50 µm. (F′) After 60 min wound closure was assessed by comparison of remaining wound size normalized to unwounded cell size. To evaluate statistical significance in F and F′, the two-way ANOVA analysis with Dunnett correction was used and P values was obtained: P value: 0.12 (ns), 0.033 (*), 0.002 (**), <0.001 (***). Error bars represent SD. At least three independent experiments for each genotype were performed.