PKD2 organizes ciliary mastigonemes. (A) Whole-mount EM of control (CC-5235; a and f), pkd2 (b and g), pkd2 PKD2-GFP (c), and pkd2 PKD2-mNG (d and h) rescue strains and mst1 (e and i). Mastigonemes are indicated by brackets. f, g, h, and i are enlarged images of the boxed areas in a, b, d, and e. Bars = 800 nm. (B) Immunofluorescence staining with anti-MST1 and the corresponding bright-field images of formaldehyde-fixed control (CC-5235; a and b), pkd2^CLIP (c and d), pkd2 PKD2-GFP (e and f), and mst1 (g and h) mutant cells. Note that a pool of MST1 is present near the basal bodies (large arrows) of control cells, but absent in pkd2 and restored in pkd2 PKD2-GFP. In pkd2, anti-MST1 signals were dispersed throughout the cell body (c, small arrows); these were absent in mst1 (g). Arrowheads indicate the position of the cilia. Bar = 2 µm. (C) Western blot analysis of control (CC-5235) and mst1 whole-cell (WC) samples and isolated cilia. Anti-IC2 and Coomassie staining were used as loading controls. (D) Relative amount of PKD2 in mst1 cilia. Ciliary PKD2 protein levels were normalized for anti-IC2 staining. The SD (error bar) and the number of independent biological replicates (n) are indicated. Significance based on two-tailed t test is indicated (**, P ≤ 0.01). (E) PKD2-mNG distribution in pkd2 PKD2-mNG (a–d) and mst1 pkd2 PKD2-mNG (e–h) cells. Bright-field (a and e), TIRF (b and f), merged images (c and g), and the corresponding kymograms (d and h) are shown. Bars = 2 s and 2 µm.