NEKL-3 and NEKL-4 kinases prevent OSM-3’s premature motility. (A) Left panel: Schematic of the C. elegans amphid and phasmid cilia. The dashed boxes are enlarged in the middle panel. Right panel: Representative fluorescence images of the endogenous NEKL-3 labeled with mScarlet. Scale bar, 5 μm. (B) Diagram illustrating synthetic gRNAs (sgRNAs) for nekl-3 nekl-3 cko and the EMS-mutated site on nekl-4 null allele. The gene models are from WormBase. Scale bar, 500 nucleotides. (C) T7EI assay showing the cleavage efficiency of the nekl-3 gene conditionally mutated in animals expressing Phsp::Cas9 and PU6::nekl-3-sg without (left lane) or with heat-shock treatment (right lane). (D) Abnormal distribution of OSM-3 labeled with GFP within the phasmid cilia of nekl-3 and nekl-4 mutant animals. The yellow dotted lines indicate the ciliary base, and the orange dotted lines indicate the junction between the middle segment and the distal segment. Scale bar, 5 μm. (E) Statistics of the cilium length (mean ± SD) measured in the indicated animals. Comparisons were performed between WT and the indicated genotypes by unpaired Student’s t tests. N = 38–45 animals. n.s., not significant. (F) Statistics of the OSM-3 fluorescence intensity ratio (mean ± SD) between middle segment and distal segment in the indicated animals. Fluorescence ratio = 0.5*([mean gray value of middle segment area]−[background gray value])/([mean gray value of distal segment]−[background gray value]). N ≥ 32 animals. Data are mean ± SD. n.s., not significant; ***P < 0.001 by the unpaired Student’s t test with Welch’s correction. Green asterisks represent comparisons between WT animals and other strains; yellow asterisk represents comparison to nekl-3 cko strain. t.z., transition zone; m.s., middle segment; d.s., distal segment. Source data are available for this figure: SourceData F2.