FAP256, CHE12, and ARMC9 affect the dimensions of distal segment and FAP256 is needed for assembly or stability of the CMC. (A–D) Images of negatively stained wild-type and KO axonemes. Arrows point to the ends of the CP microtubules (the location of the CMC), and arrowheads mark the proximal and distal ends of the DFs. The percentage of axonemes with ciliary caps are shown below each image (wild-type CMC score n = 48, DFs n = 19; FAP256-KO CMC n = 76, DFs n = 42; CHE12-KO CMC n = 11, DFs n = 7; and ARMC9-KO CMC n = 10, DFs n = 10). (E–J) Representative electron microscopy images of negatively stained whole mount wild-type and KO cilia (blue and green brackets mark the A-tubule and CP region, respectively; the blue and green arrows mark the proximal boundary of the distal segment and CP region, respectively; the red line indicates the region of decreased stain on the axoneme that corresponds to the termination of dynein arms and radial spokes on the A-tubules; see Fig. S5 [I–K] for the cross sections along the transitional region). (K) Cilium length was quantified using electron microscopic images of negatively stained isolated cilia (WT: 6.9 µm, ±0.97 n = 51; C-KO: 5.8 µm, ±0.57, n = 39; F-KO: 6.7 µm, ±0.79, n = 49; A-KO: 7.8 µm, ±0.82 n = 70; and AC-KO: 6.9 µm, ±0.57 n = 50, asterisks show significant differences compared with wild type; two-sided t test, P < 0.05). (L) Quantifications of the length of distal segment, A-tubule and CP region (averages ± standard deviations, n = 40–70 cilia, asterisks show significant differences as compared with the wild type; two-sided t test, P < 0.05; * at the top of the column shows a significant difference in the distal segment; * on the side of each column shows a significant difference in either the CP [top] or the A-tubule region [bottom]).