Fig. 5.

GFP–β-tubulin rapidly enters cilia by diffusion. (A) Still images (a, b) of a wild-type cell expressing GFP–β-tubulin with full-length cilia prior (a) and during bleaching (b). The corresponding kymogram (c) shows how diffusion (arrows) and IFT (arrowhead) of GFP–β-tubulin become apparent as axonemal GFP–β-tubulin is bleached. Scale bars: 2 s (horizontal) and 2 µm (vertical). The movies were recorded at 40 f.p.s. (B) Gallery of kymograms showing diffusion (white arrows) and IFT (arrowheads) of GFP–β-tubulin in bleached full-length (a–d) and regenerating (e–g) cilia. A subset of GFP–β-tubulin diffusion occurs rapidly and almost unidirectional along the cilia (red arrows). At the ciliary tip, diffusing GFP–tubulin becomes transiently immobilized (indicated by white brackets); more stable association is visible in panels e and g (indicated by red brackets). Scale bars: 2 s (horizontal) 2 µm (vertical). (C) Kymograms of bleached full-length (a) and regenerating (b) cilia showing the entry of GFP–β-tubulin into cilia by diffusion (arrows) and IFT (arrowheads). Note accumulation of GFP–tubulin at the tip of growing cilia. Scale bars: 2 s (horizontal) 2 µm (vertical). (D) Frequency of GFP–β-tubulin entry by IFT and diffusion in regenerating (reg) and full-length (FL) cilia. The standard deviation, number of cilia analyzed and probability value (P) based on a two-tailed t-test are indicated. (E) Kymograms of GFP–β-tubulin moving by IFT in a growing cilium. GFP photobleaching events are marked by green arrowheads; bleaching appears to occur in one (a,b) or two steps (c,d). Loading (filled arrowhead in f) and unloading (open arrowheads in e and f) of GFP–β-tubulin from IFT is indicated. Based on movies recorded at 20 f.p.s. Scale bars: 2 s (horizontal) 2 µm (vertical). In B,C,E: T, position of cilia tip; B, position of cilia base.