Rac1 stimulates FHOD1 recruitment to actin tails. (A) Representative images of HeLa cells transfected with dsRed-FHOD1 and infected with WR strain of vaccinia. Bars, 10 µm. Localization of FHOD1 (red channel) to actin tails (green channel) was assessed in mock-treated (mock) or Rac1-depleted (Rac1si) cells. The inset in the top panels (mock) shows a representative actin tail decorated with FHOD1. Bars, 1 µm. Closed arrowheads indicate examples of FHOD1-positive actin tails. The inset in the bottom panels (Rac1si) shows a representative FHOD1-negative actin tail. (B) Quantitative analysis of FHOD1 localization to actin tails in mock-treated and Rac1-depleted cells. Data are presented as the mean ± SEM. ***, P < 0.0001. (C) Representative images of HeLa cells infected with the WR strain of vaccinia virus. Control cells were stained with phalloidin (red channel) to visualize actin tails. Dominant-negative Rac1 (DN Rac1) cells were transfected with GFP-Rac1 N17 (green channel) the day before infection and stained with phalloidin (red channel) to visualize actin tails. DN-Rac1 + FHOD1 cells were cotransfected with GFP-Rac1 N17 (blue channel) and dsRed-FHOD1-ΔDAD (red channel) the day before infection and stained with phalloidin (yellow channel) to visualize actin tails. Bars, 10 µm. (D) Quantitative analysis of the number of actin tails per cell in control cells (control), cells expressing dominant-negative versions of the small GTPases Rho N19 (DN-Rho), Cdc42 N17 (DN-Cdc42), and Rac1 N17 (DN-Rac1), or cells coexpressing DN-Rac1 + NWASP, DN-Rac1 + FHOD1wt, DN-Rac1 + FHOD1-ΔDAD, or DN-Rac1 + FHOD1-ΔFH2. ***, P < 0.0001, compared with control; **, P = 0.0022, for cells expressing DN-Cdc42.