Cofilin is constitutively active in Wdr1-deficient neutrophils. (A–H) Whole-mount immunodetection in 2.5-dpf Tg(lyz:GFP) carmin and sibling embryos of whole cofilin (A–D) and Ser3-p-cofilin (E–H). (A) In neutrophils of WT sibling embryos, cofilin is detected throughout the cytoplasm with some enrichment at the cell cortex (arrow) clearly delineated by F-actin (cyan). (B) In mutant embryos, cofilin forms aggregates that correlate with the F-actin cytoplasmic clumps (arrows). (C and D) Quantitative analysis of the mean density (C) and colocalization (D) of pMLC and F-actin in the 3D extent of individual mutant (n = 25) versus WT sibling (n = 15) neutrophils, all from the same experiment. For the Manders’ coefficients, A is F-actin and B is cofilin. (E and F) In the neutrophils of sibling embryos (E), p-cofilin was readily detected as a diffuse signal throughout the cell, whereas in the neutrophils of mutant embryos (F), p-cofilin was not visible. (G and H) Quantitative analysis of the mean density (G) and colocalization (H) of p-cofilin and F-actin in the 3D extent of individual mutant (n = 18) versus WT sibling (n = 19) neutrophils, all from the same experiment. For the Manders’ coefficients, A is F-actin and B is p-cofilin. In all graphs, data are mean ± SEM; for statistical analysis, Welch’s t test was used. ns, nonsignificant (P > 0.05); **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Scale bars, 5 µm.