Actomyosin perturbation plays a key role in the nuclear instability of Wdr1-deficient neutrophils. (A and B) Whole-mount immunofluorescence confocal images of transgenic 2.5-dpf Tg(lyz:GFP) carmin mutant vand WT sibling probed with s19-pMLC antibody (red) and phalloidin (cyan). (A) In neutrophils of sibling embryos, pMLC appears both cortical (together with phalloidin-labeled F-actin) and diffusely cytoplasmic. (B) In mutants, intense caps of pMLC appear at the periphery of neutrophils, quite apart from their phalloidin-labeled cytoplasmic F-actin clumps. (C and D) Quantification of the mean intensity (C) and colocalization (D) of pMLC and F-actin in the 3D extent of individual mutant (n = 12) versus WT sibling (n = 12) neutrophils, all from the same experiment. For the Manders’ coefficients, A is F-actin and B is pMLC. Data are mean ± SEM. For statistical analysis, Welch’s t test was used; ****, P < 0.0001. (E–G) WT Tg(mpx:GFP; lyz:H2b-mCherry) embryos were incubated in 200 µM Rockout at 2 dpf for 8–16 h. (E) Immunostaining confirmed decreased amounts of p-MLC upon Rockout treatment. (F and G) Live confocal imaging revealed within the neutrophils of Rockout treated embryos a reproduction of the characteristic chromatin (red) unwinding (F, arrow) and cell fragmentation (G) seen in neutrophils of Wdr1-deficient embryos. G shows selected time points of the time-lapse sequence shown in Video 8; time is indicated in h:min. (H–J) Immunostaining for Lamin B2 (red) in control (H, n = 24) versus Rockout-treated embryos (I, n = 25) demonstrated loss of NE in some neutrophils after Rockout treatment (I and J); all points are from the same experiment; for statistical analysis, a χ2 test was used; **, P < 0.01. Scale bars, 5 µm.
