IFT particles carry more tubulin during ciliary growth. (A) Schematic presentation of the experimental design. Cells were deciliated by a pH shock and allowed to partially regrow cilia. Then, the cilia were bleached and the entry and assembly of unbleached GFP–α-tubulin was analyzed by TIRF. (B) Individual frames from videos captures before (T0), during (bleach), and at various time points (T1–T7 in min) after bleaching of the cilia. Brackets: unbleached GFP-tubulin added at the ciliary tip as cilia elongate. Bar, 2 µm. Bottom: kymogram of the same cell showing numerous IFT-like GFP-tubulin tracks. Bar, 2 µm and 2 s. (C) Mean frequency of GFP-tubulin transport by anterograde IFT in steady-state (ss), regenerating (reg), and fully regenerated (fr) cilia. Error bars indicate the standard deviation. (D) Analysis of GFP-tubulin transport frequency by anterograde IFT in regenerating cilia of various lengths. Error bars indicate SEM. (E and F) Segments of the cilia of cells coexpressing GFP–α-tubulin and IFT20-mCherry were bleached using a focused laser beam and protein traffic in the bleached region was analyzed by two-color TIRF microscopy. The kymogram of a cell with fully regrown flagella (E) shows numerous IFT20-mCherry trajectories, whereas transport of GFP–α-tubulin was not observed. In contrast, most IFT20-mCherry trajectories align with GFP–α-tubulin trajectories in the regenerating cilium (F). Bars, 2 µm and 5 s. (G) Scatter plot of the occupancy rate (the percentage of IFT20-mCherry particles moving together with GFP–α-tubulin) in cilia of different length. Cells with regenerating (white marks) or nongrowing (steady-state and fully regenerated, black marks) cilia were analyzed (n = 32 and 13 cilia, respectively).