GPCR signaling and Arl6 drive assembly of large processive retrograde BBSome trains. (A) Representative kymographs of BBSome train movement. WT or Arl6^−/− IMCD3-[pEF1α-^NG3BBS5; pEF1αΔ-^APSSTR3] cells were treated with vehicle, sst, SAG, or PKI for 40 min before imaging at 4 Hz for 30 s. Bars, 2 µm. (B) Representative kymograph from an IMCD3-[pEF1α-^NG3BBS1, pCMV-^tdTomatoIFT88] cell showing comovement and uncoupling between IFT-B and BBSome trains in untreated cells. Nearly 20% of BBSome trains displayed a distinct pause. n = 25 cilia. Bar, 2 μm. (C and D) WT or Arl6^−/− IMCD3-[pEF1α-^NG3BBS5; pEF1αΔ-^APSSTR3] cells were treated with vehicle, sst, SAG, or PKI for 40 min before imaging. (C) The processivity of retrograde BBSome trains was measured by deconvolving kymographs into anterograde and retrograde components (see Materials and methods). The distance traveled by each retrograde train (normalized to the length of cilia) was estimated by manual inspection of the retrograde kymographs. (D) The fluorescence intensity of ^NG3BBS5 retrograde trains was extracted from deconvolved kymographs, and the total number of BBS5 molecules per train was calculated using the NG calibrator (see Materials and methods). The whiskers represent 1.5× the interquartile range. n = 52–91 cilia from three independent experiments. Asterisks indicate Mann-Whitney U test significance values; ***, P < 0.0001; n.s., P > 0.05. (E) Treatment with sst, SAG, or PKI did not change the frequency of retrograde BBSome trains. (F) Retrograde velocities of IFT trains, BBSome trains, and single SSTR3 molecules. IMCD3-[pEF1α-^NG3IFT88], IMCD3-[pEF1α-^NG3BBS5; pEF1αΔ-^APSSTR3], or IMCD3-[pEF1αΔ-^APSSTR3^NG] were treated with vehicle, sst, SAG, or PKI for 40 min before imaging. IFT and BBSome train velocities were extracted from kymographs (see Materials and methods). ^QDSSTR3 velocities were measured from persistent retrograde movements lasting >6 s. Error bars represent SD. n = 9–18 cilia. Pairwise Mann-Whitney tests failed to show significant differences between any two conditions (P > 0.1). (G) The number of IFT88 molecules per retrograde train was measured in IMCD3-[pEF1α-^NG3IFT88] cells treated with vehicle or SAG. Counting of molecules is detailed in Materials and methods. n = 11 cilia. (H) Arl6 immunofluorescence of cells treated with vehicle, SAG, or PKI. Optical sections were deconvolved, and X/Z projections are shown. The percentages of Arl6-positive tips are indicated below the micrographs. n = 88–118 cilia from four to five microscopic fields. b, base; t, tip. Bar, 2 μm.