The phosphomimic mutation S96D inhibits OSM-3 motility in vivo. (A) Amphid and phasmid cilia in the WT, osm-3^S96D, and osm-3^S96A mutant animals. Cilia are visualized with GFP-tagged endogenous WT OSM-3 (the same as Fig. 2 D) or the corresponding mutant OSM-3. Dyf, dye-filling defective; N ≥ 100. Arrows indicate the ciliary base. Arrowheads indicate the junctions between the middle and distal segments. Scale bar, 5 μm. (B) Quantification of cilium length in WT and osm-3 mutant animals. N = 37–39 animals. Statistical significance, compared with the control with a matching color code, is based on Student’s t test. **P < 0.01; ***P < 0.001. Data are mean ± SD. (C) Histogram of anterograde IFT speeds of WT and mutant OSM-3 kinesins at the middle (top) and distal (bottom) ciliary segments. Velocities and numbers of IFT particles (N) are indicated. The plots were fit by a Gaussian distribution. Statistical significance, compared with the control with a matching color code, is based on Student’s t test. Data are mean ± SD. (D) Amphid and phasmid cilia of osm-3^S96D;nekl-3 cko double-mutant animals. Cilia are visualized with GFP-tagged endogenous mutant OSM-3. Scale bar, 5 μm. (E) Representative amphid and phasmid cilia images of osm-3^S96A animals ectopically expressing NEKL-3 fused to PCRG-1. Scale bar, 5 μm. (F) Quantification of phasmid cilium length (mean ± SD) in the animals of indicated genotypes. N = 37–60 animals. Statistical significance, compared with the control with a matching color code, is based on Student’s t test. n.s., not significant.