GPR161 traverses the transition zone in an Arl6- and signaling-dependent manner. (A) Representative images of a base residence event for ^QDGPR161 after 40 min of SAG treatment. The most base-proximal position of ^QDGPR161 (red) relative to bulk ^APGPR161^NG3 fluorescence (green) is highlighted by a red box, and the enlarged image is shown in B. (B) Line scans of the fluorescence intensities of ^QDGPR161 (red dotted line) and ^APGPR161^NG3 (green line) along the length of cilia. (C) Because the longitudinal profile of ^APGPR161^NG3 fluorescence is highly reproducible and unchanged by fixation (Fig. S5 C), the profile of ^APGPR161^NG3 can be used as a common reference to align the positions of ^QDGPR161, Cep290, and Cep164 with respect to one another. WT or Arl6^−/− IMCD3-[pCrys-^APGPR161^NG3] cells were treated with SAG or vehicle for 40 min before imaging. The most base-proximal positions of the centroid of ^QDGPR161 relative to the profile of ^APGPR161^NG3 were plotted as thin lines, and the means were plotted as thick lines. The same method was used to plot the positions of immunofluorescence-stained Cep290 and Cep164 relative to ^APGPR161^NG3 fluorescence. n = 15–26. (D and E) The lateral displacement between the centroid of ^QDGPR161 at its most base-proximal position and the center axis of ^APGPR161^NG3 fluorescence was box plotted. The lateral displacement informs the half-width of the cilium base (control) or the intermediate compartment (SAG). IMCD3-[pCrys-^APGPR161^NG3] cells were treated with SAG or vehicle for 40 min before imaging. Bars: (A–C) 1 µm; (D) 0.5 µm. n = 11–16 cilia. (F) Two-barrier model for exit from cilia. The intermediate compartment is displayed in tan, the cilium shaft is blue, and the cell is gray. GPCR is green, agonist is red, and BBSome/Arl6 coats are yellow. IC, intermediate compartment; PCB, periciliary barrier; TF, transition fiber; TZ, transition zone. The diagram is not drawn to scale.