Signaling promotes coupling between BBSomes and cargoes. (A) Representative images showing ciliary GPR161^NG3 FRAP. IMCD3-[pCrys-^APGPR161^NG3] cells were treated with vehicle or SAG for 40 min before imaging. White dotted boxes in the first image indicate the photobleaching area that covers >80% of the cilia except the tip. After photobleaching, ciliary GPR161^NG3 images were acquired every 5 s. Bar, 2 µm. B, base; T, tip. (B) Line scans of GPR161^NG3 fluorescence intensities along cilia at 60 s after photobleaching. The gray and green lines mark mean intensities along length-normalized cilia for control and SAG-treated cells, respectively. Error bars represent SD. n = 10–11 cilia. (C) GPR161^NG3 fluorescence at the tip was measured, and the decay of fluorescence signal over time in control and SAG-treated cells was plotted. Data were fitted to a single exponential to calculate the half-life. t_1/2(control) = 5.14 ± 0.95 s; t_1/2(SAG) = 10.47 ± 1.76 s. Error ranges represent error of fit. n = 10–11 cilia. (D) ^NG3BBS5 fluorescence was measured in live cells after 40 min of incubation with WGA, sst, or both. The total number of BBS5 at the tip (right) or axoneme (left) was calculated using the NG3 calibrator and the measured ratio of ^NG3BBS5 to total BBS5. Asterisks indicate Mann-Whitney test significance values. *, P < 0.05; ***, P < 0.0005. n = 20–25 cilia from three independent experiments. (E) Representative kymographs from mSA647-labeled WT or Arl6^−/− IMCD3-[pEF1α-^NG3BBS5; pEF1αΔ-^APSSTR3] cells treated with sst for 1 h before imaging. The cells stably expressed an ER-localized biotin ligase BirA to enable visualization of ^APSSTR3 by mSA647 labeling. Bar, 2 μm. B, base; T, tip.