Reconstitution of signal-dependent retrieval of SSTR3 and GPR161. (A) Diagram of the signal-dependent retrieval systems under study. Left: Addition of sst triggers SSTR3 exit from cilia by directly activating SSTR3. Right: Addition of SAG activates the Hedgehog pathway and promotes GPR161 retrieval. SMO, Smoothened. (B) Kinetics of SSTR3 disappearance from cilia of primary hippocampal neurons and of IMCD3 stably expressing ^APSSTR3^NG under the control of the TATA-less EF1α promoter were estimated by quantitation of immunofluorescence signals after addition of sst. The entire dataset for the sst condition is shown in Fig. S1 B. Data were fitted to a single exponential. Error bars indicate 95% confidence interval (CI). n = 280–424 cilia (neurons) and 57–80 cilia (IMCD3). (C) High-level expression of SSTR3 drives elongation of primary cilia. Top: ^APSSTR3^GFP driven by various promoters or ^APSSTR3^NG driven by EF1αΔ promoter was expressed stably at the FlpIn locus of IMCD3 cells, and ciliary fluorescence levels were measured and compared to a GFP calibrator (Breslow et al., 2013) or an NG calibrator (see Materials and methods). Endogenous SSTR3 levels were estimated by comparative immunostaining (see Materials and methods). A Mann-Whitney test was used for pairwise comparisons of the number of SSTR3 molecules per cilia in neurons and in IMCD3 cells expressing ^APSSTR3^NG or ^APSSTR3^GFP under the control of pEF1αΔ. P > 0.05. n = 10–38 cilia. Error bars represent SD. Bottom: Effect of ^APSSTR3^GFP expression on cilium length. Cilia lengths were measured in the GFP channel by live-cell imaging. n = 10–38 cilia. Error bars represent SD. Cilium lengthening upon GPCR overexpression was previously reported by Guadiana et al. (2013). (D) IMCD3-[pCrys-^APGPR161^NG3] were treated for 2 h with either SAG or vehicle. ^APGPR161^NG3 was visualized by NG fluorescence, and basal bodies of cilia were stained with ninein. All cells were pretreated with the translation inhibitor emetine to eliminate signals from new protein synthesis. Bar, 4 µm. (E) Absolute quantitation of ciliary GPCR abundance. Top: Calibration of single-molecule fluorescence intensity. Bacterially expressed NG3 protein was spotted on glass coverslips (inset), and the fluorescent intensity of each individual NG3 was measured. n = 1,257 particles measured. Bottom: The three-step photobleaching of a representative spot shows that the fluorescence was emitted by a single NG3 molecule. The measured fluorescence intensity of NG3 was used to calibrate NG- and NG3-tagged SSTR3, GPR161, BBS5, and IFT88. Bar, 0.5 μm. (F) IMCD3-[pEF1αΔ-^APSSTR3^NG] cells were treated with vehicle or sst for 2 h. Stable expression of an ER-localized biotin ligase BirA enables the biotinylation of ^APSSTR3 with the biotin existing in the DMEM/F-12 cell culture medium. Ciliary ^APSSTR3 was pulse-labeled by Alexa Fluor 647–conjugated mSA (mSA647) for 5–10 min before imaging (see Materials and methods for details). Bar, 1 μm. The absolute number of ^APSSTR3^NG molecules per cilia at t₀ was calculated by measuring the NG signal and using the NG3 calibrator. For all other time points, the ratio in ciliary mSA647 signal compared with t₀ was used to calculate the absolute number of molecules (see Materials and methods for details). Data were fitted to a single exponential. Error bars indicate 95% CI. n = 14 cilia. (G) IMCD3-[pCrys-GPR161^NG3] cells were treated with SAG or vehicle for 2 h. NG fluorescence was tracked in individual cilia, and the ratio of GPR161^NG3 to endogenous GPR161 was used to calculate the total levels of GPR161 as detailed in Materials and methods. Bar, 1 μm. Data were fitted to a single exponential. Error bars indicate 95% CI. n = 12–20 cilia.