RhoGEF2, Pbl, RhoGEF3, and Tum exhibit discrete localization patterns and are required for cell wound repair. (A) Schematic diagram summarizing the localization patterns of actin, Rho1, Rac1, and Cdc42 at cell wounds. (B–H) Confocal xy projection images from Drosophila NC4–6 staged embryos coexpressing an actin marker (sGMCA or sChMCA) and fluorescently tagged Rho family GTPases: ChFP-Rho1 (B–B″), ChFP-Cdc42 (C–C″), and GFP-Rac1 (D–D″). The actin ring and halo regions are indicated in (B′). (E–H) Confocal xy projection images from Drosophila NC4–6 staged embryos coexpressing sChMCA and GFP-tagged RhoGEFs or Tum: sfGFP-Tum (E–E″), sfGFP-RhoGEF2 (F–F″), Pbl-eGFP (G–G″), and sfGFP-RhoGEF3 (H–H″). (I–K) Confocal xy projection images from Drosophila NC4–6 staged embryos coexpressing two fluorescently tagged RhoGEFs: Pbl-eGFP and RFP-RhoGEF2 (I–I’’), sfGFP-RhoGEF3 and RFP-RhoGEF2 (J–J’’), and Pbl-eGFP and sfGFP-RhoGEF3 (K–K’’). (L–P) Actin dynamics (sChMCA or sGMCA) during cell wound repair in control (GAL4 driver 7063 alone) (L), RhoGEF2^RNAi(1) (M), pbl^RNAi(1) (N), RhoGEF3^RNAi(1) (O), and Tum-i+antibodies (Abs) (P). (L’–P’) xy Kymograph across the wound area depicted in (L–P), respectively (yellow dashed lines, wound edges; yellow arrowheads, actin ring; red arrowheads, actin accumulation inside wounds). (Q–T) Quantification of the wound area over time for control, RhoGEF2^RNAi(1), and RhoGEF2^RNAi(2) (n = 10 for each; Q); control, pbl^RNAi(1), and pbl^RNAi(2) (n = 10 for each; R); control, RhoGEF3^RNAi(1), and RhoGEF3^RNAi(2) (n = 10 for each; S); and GAL4 control, 9E10 control, tum^RNAi, Tum Abs, and tum^RNAi+Abs (n = 10 for controls and n = 15 for tum^RNAi and Tum Abs; T). Time after wounding is indicated. Bars, 20 µm. Error bars represent ± SEM.