Fig. 1.

IFT-A, IFT-B and IFT dynein proteins dwell for ∼2 s at the flagellar tip. (A) Schematic of the tip-FRAP approach. (a) The bleaching laser was moved from the flagellar tip toward the base, photobleaching all trains in the distal region of the flagellum. Alternatively, we used a stationary larger-diameter beam and flash-bleached the flagellar tip. (b,c) Fluorescent anterograde trains re-enter the distal flagellum. (d) The first unbleached anterograde train arrives at the tip. (e) The first retrograde train containing unbleached IFT material exits the tip. (f) The signal of the IFT pool at the tip is fully recovered. Line A, pause between arrival of the first unbleached anterograde train and departure of the first unbleached retrograde train. Line B, time between the arrival of the first unbleached anterograde train and the recovery of the tip signal. (B) Kymograms from tip-FRAP photobleaching experiments using tagged IFT strains. The time between the arrival of the first post-bleach anterograde train and the departure of the first unbleached retrograde train is marked by brackets; red bars at the top indicate the bleaching steps. Images are representative of three or more experiments for each strain. T, tip; B, base. Scale bars: 2 µm (vertical) and 2 s (horizontal). (C) Distribution of dwell times at the tip for mNG–IFT54 (n=37 particles from three experiments). (D) Mean±s.d. dwell time of the various tagged IFT proteins at the tip. *P<0.001; n.s., not significant (paired two-tailed t-test).