(A) Schematic presentation of the experimental design used to determine the gap in anterograde traffic of sfGFP-tubulin after photobleaching of the basal body. Cells were deflagellated by a pH shock, allowed to initiate regeneration, and mounted for TIRF analysis. Then, the two flagella were partially bleached, followed by spot bleaching of either one or both basal body pools (for the latter the size of the laser beam was extended to ~2 µm), and TIRF analysis of tubulin traffic. (B) Two images from a focal series showing the flagella and the microtubular roots; the latter were used to position the cells with respect to the laser beam. The two images are displayed in red and green in the merged image. Bar = 2 µm. (C) Gallery of kymograms from experiments in which one of the two basal body pools was bleached. The bleached basal bodies are oriented to the top. Bars = 2 s 2 µm (in d for a, c, d; in b for b). (D) Gallery of kymograms in which both basal body pools were bleached. In C and D, trajectories indicating IFT of tubulin are indicated by arrowheads. Bars = 2 s 2 µm. (E) Schematic presentation of the open IFT system. IFT-A proteins (and IFT27 and the motors) are recruited to the pool, cycle once through the flagellum, and return into the cell body pool. IFT-B proteins return to the bb-pool to be either reused in subsequent IFT trains or released into the cell body pool. IFT proteins mix in the cb-pool and are randomly re-deployed into IFT trains. (F) Model of the temporal-spatial organization of IFT proteins in the basal body pool. We propose that IFT trains assemble in distinct positions near the basal body from which they are sequentially released into the cilium upon completion. Assembly commences with the arrival of IFT-A complexes which then combine with the other IFT-subcomplexes and motors as they move toward the distal end of the basal body. For simplicity, only IFT-A and kinesin-2 are depicted. See also Figure 7—figure supplement 1.