Rac directly regulates DPak3 recruitment to the PLS. (A) Sns, Sltr, and Rac remain localized with the F-actin foci in dpak3^zyg mutant embryos. Stage 14 embryos triple labeled with phalloidin (green), α-Sns (a) or α-Sltr (b) or α-Rac1 (c, red), and α-Duf (blue). (B) Localization of DPak3 in fusion mutants. Stage 14 embryos triple labeled with phalloidin (green), α-DPak3 (red), and α-Duf (blue). DPak3 was enriched in the F-actin foci (arrowheads) of sltr (b) and kette (c), but not sns (a) and rac1,rac2 (d) mutant embryos. (C) Colocalization of DPak3 and Rac at sites of fusion. Stage 14 wild-type (wt) embryo triple labeled with phalloidin (green), α-DPak3 (red), and α-Rac1 (blue). (D) Transgenic rescue of dpak3^zyg mutant with V5-DPak3^H29,31L driven by twi-GAL4. Stage 15 embryo double labeled with α-MHC (green) and α-V5 (red). Note that the transgene expression failed to rescue the fusion defect. (E) DPak3^H29.31L does not colocalize with F-actin foci at sites of fusion. Stage 14 embryos triple labeled with phalloidin (green), α-V5 (red), and α-Duf (blue). Unlike wt V5-DPak3, which was enriched at the F-actin foci (a, arrowhead), DPak3^H29.31L was not recruited to the F-actin foci (arrows) in wt (b) or dpak3^zyg mutant (c) embryos. Bars: (A–C and E) 5 µm; (D) 20 µm.