Cby protein is enriched at transition fibers and in the proximal compartment of the transition zone of mature cilia. (A) Fully differentiated MTECs at ALId14 were colabeled with antibodies for Cby and the ciliary axoneme marker acetylated (ac) α-tubulin and imaged by superresolution 3D-SIM. Top-down (x-y; single sections) and side (y-z on the right and x-z on the bottom; maximum projections) views are shown. The squared area is shown in higher magnification at the bottom right corner. See also Video 1. (B) Cby clusters as rings smaller than CEP164 rings at the ciliary base. Fully differentiated MTECs were costained with antibodies against Cby and CEP164 and imaged by 3D-SIM. The images are maximum projections from 10 z sections taken at 125-nm intervals. Top-down views and high-magnification views of the square areas are shown. Dashed lines demarcate cell boundaries. See also Fig. S3. (C) Immuno-EM localization of Cby in thin cross sections (80 nm) through the distal (b and c) and proximal (e and f) regions of the transition zone and the distal part of basal bodies (h and i) at the base of cilia in WT MTEC cultures at ALId14. (d, g, and j) CbyKO MTECs were processed in parallel as a negative control. A representative EM image of a cilium in longitudinal section is shown in a. Arrowheads in a denote the transition fiber. CP, cilia proper; BP, basal plate; TZ, transition zone; BB, basal body; TF, transition fiber. (D) Superresolution 3D-STORM images of Cby ring substructure in ALId14 MTEC cultures. Shown are a top-down view (a), intensity profile along the yellow arrow in a (b), and side view with a height map (c). White arrowheads in a point to Cby clusters. Brackets in c indicate the areas in which Cby molecules were highly enriched. The data are from a single representative experiment out of three repeats. (E) Cartoon depicting Cby localization at the base of mature cilia. Bars: (A, main image) 1 µm; (A [bottom right], C, and D) 0.1 µm.