FAP256A localizes to the tips of cilia and unciliated basal bodies and the ends of A-tubules and CP. (A) The segmental organization of the motile cilium (the blue and green arrows mark the proximal boundary of the distal segment and the CP region, respectively). (B and D–F) TIRFM imaging of live Tetrahymena cells that express FAP256A-GFP under native promoter at several times before and after low pH–induced deciliation (bar, 5 µm). (C) Differential interference contrast (DIC) and TIRFM images of the same cilium (white arrows, FAP256A-GFP dots at the tip). (G) An SR-SIM immunofluorescence image of a Tetrahymena cell expressing FAP256A-2xmNeonGreen-6xMyc-BirA* under native promoter (green, 6xMyc; red, polyE; green arrowheads, unciliated basal bodies; white arrowheads, ciliated basal bodies; white arrows, FAP256A signals at the tip; asterisks mark the gap between the polyE tubulin and FAP256A signals). (H and I) Immunoelectron TEM localization of FAP256A-GFP. Axonemes of wild type (H) and FAP256A-GFP (I) cells were labeled with anti-GFP and gold-conjugated secondary antibodies. Red circles mark the gold particles on the axonemes. The red dots on the left side summarize the approximate positions of gold particles (one red dot denotes one gold particle) found in a total of 14 wild-type and 14 FAP256A-GFP axonemes. The As mark the visible termination points for the A-tubules (note that less than nine ends are visible, some of the ends could be on the nonimaged side of the axoneme or were lost during preparation), and the blue arrows mark the proximal boundary of the distal segment (bar, 200 nm).