Nphp4 is essential for the organization of the subapical actin layer. (A) Serial confocal images for actin (phalloidin) and ciliary rootlets (GFP-Clamp) of cells with multiple motile cilia revealed that the subapical actin layer became irregular in nphp4-deficient Xenopus embryos, whereas apical actin pool remained unaffected. The depth of each optical section from the first section is shown on the left. A magnified image of the inset is shown on the right side (4× magnification). (B) Triple staining for actin (phalloidin, red in merge), ciliary rootlets (GFP-Clamp, green in merge), and basal bodies (γ-tubulin, blue in merge) revealed that actin filaments in the subapical actin layer connect a basal body with the ciliary rootlet of the neighboring basal body. In nphp4-deficient cells, the subapical actin layer was poorly nucleated and failed to provide the connection between basal bodies. Top panels show the maximum intensity projection of serial confocal images. Single optical sections at the level of the subapical actin pool (boxed area) are magnified and shown in the bottom panels (4.5× magnification). Right panels are higher magnification images of the single optical sections depicting the subapical actin organization (10.5× magnification). act, actin; bb, basal body; rtl, ciliary rootlet. Bars, 5 µm.