Activated GPCRs undergo processive retrograde movements and confinements at base and tip. (A) Diagram of the Qdot labeling strategy. IMCD3-[pEF1αΔ-^APSSTR3^NG] cells stably expressing BirA-ER were first treated with unlabeled mSA to passivate the surface-exposed biotinylated ^APSSTR3. ^SAQdots were then added to the medium to label the GPCRs newly arrived at the surface. Finally, biotin was added to the medium to passivate the excess SA on Qdots. (B) The diffusive properties of SSTR3 are not altered by Qdot labeling. The instantaneous velocities of mSA647-labeled ^APSSTR3^NG and Qdot655-labeled ^APSSTR3^NG were measured by single-molecule tracking in the absence of sst. n = 1,223–3,032 instantaneous velocities. (C) Qdot labeling does not alter the exit rate of SSTR3. IMCD3-[pEF1α-^APSSTR3^GFP] cells were sparsely labeled with Qdot655 as described in Materials and methods and treated with vehicle or sst for 2 h before fixation. The number of Qdots per cilium was counted in both vehicle- and sst-treated conditions. n = 263–303 cilia from three independent experiments. Error bars represent SEM. (D) Representative kymographs showing the movements of ^SAQdot-labeled ciliary ^APSSTR3^NG (^QDSSTR3) in vehicle- or sst-treated cells. Red labels and line coloring highlight four characteristic movement behaviors. Bar, 2 µm. b, base; t, tip. (E) Centroid mapping of ^QDSSTR3. Left: The contour of the cilium was traced as a dotted line that captures all ^QDSSTR3 positions. (i) Diffusive movement, (ii) tip confinement followed by retrograde movement, (iii) retrograde movement followed by base confinement and return into the cilium, and (iv) tip confinement followed by fully processive retrograde movement and base confinement. The time dimension is color coded from red to purple. (F and G) Signaling increases tip confinement and processive retrograde movement of SSTR3. Cells were treated with sst (green, sst) or vehicle (gray, control) for 40 min before imaging was initiated for 10–20 min. (F) Durations of persistent movement events for ^QDSSTR3 in anterograde and retrograde directions. (G) The durations of confinement events for ^QDSSTR3 at ciliary tip or base were binned into two categories. n = 12 cilia for each condition. (H) Representative kymographs showing the comovement between a single ^QDSSTR3 and a BBSome retrograde train. IMCD3-[pEF1α-^NG3BBS5; pEF1αΔ-^APSSTR3] cells were treated with sst for 40 min before imaging. Bar, 2 μm. B, base; T, tip.