Fig. 5: The RBD and C-terminal regions of Pa2G4 are required for multiciliation.
From: Proliferation associated 2G4 is required for the ciliation of vertebrate motile cilia
a ΔRBD and ΔC mutants did not co-localize with the acetylated tubulin signal (magenta). The images were taken by 3D-SIM. Scale bars, 5 µm. b Protein domain schematic of Xenopus Pa2G4 wild-type and deletion mutants used for rescue experiments. RBD, RNA binding domain; Lys, Lysine rich region. c ΔRBD and ΔC mutants failed to rescue ciliary phenotypes upon Pa2G4 knockdown. The indicated mRNAs and MOs were injected into one ventral blastomere at the eight-cell stage. Phalloidin (cyan) stains apical actin and Centrin-RFP marks basal bodies. Cilia was visualized by acetylated tubulin staining. Scale bars, 20 µm. d Western blot of the indicated exogenous Pa2G4 proteins in (c). Quantification of acetylated tubulin signal (e) and phalloidin intensity (f) in an MCC shown in (c). ****, P < 0.0001, one-way ANOVA; image number for acetylated tubulin staining analysis, n = 50; embryos per group (e), n = 25; MCCs for phalloidin intensity quantification, n = 50; embryos per group (f), n = 10; Data are mean ± SD. The expression of ΔRBD and ΔC failed to recover basal body docking (g) and Rac1 activity (h, i) in Pa2G4 morphant MCCs. g The cocktail of the indicated MOs and mRNAs was microinjected into one ventral blastomere at the eight-cell stage. The representative z-stack confocal images were projected in the x-z plane. MCC apical surfaces marked by arrowheads. Scale bars, 5 µm. h pGBD-GFP and Centrin-RFP mRNAs were co-injected with the indicated Pa2G4 mRNAs and MOs (4 ng) into one ventral blastomere of four-cell stage embryos. Anti-acetylated tubulin staining represents multi-cilia (gray). Scale bars, 5 µm. i Quantification of pGBD-GFP intensity in (h), MCC n = 48; embryos per group n = 12; one-way ANOVA, ****, p < 0.0001; error bars represent SD.
