(A) Schematic for local labeling of D1-type dopamine receptor (D1R) in a cilium using PA-GFP. IMCD3 cells expressing Flag-D1-PAGFP were labeled with anti-Flag antibody conjugated to Alexa555 to visualize the overall surface receptor pool. A point-focused 405-nm laser spot was used to locally photoactivate receptors on the mid-portion of the cilium. Non-fluorescent PA-GFP is depicted in gray, fluorescent state in green. (B) Live cell confocal images of a representative cilium showing the frame immediately before the photoactivation pulse (left column), and frames acquired 1 s (middle column) and 10 s (right column) after local photoactivation. The Flag-Alexa555 signal labeling the entire surface receptor pool (top row) was present throughout the cilium at all time points. PA-GFP fluorescence representing the photoactivated pool was non-uniformly distributed at 1 s and uniformly distributed along the cilium within 10 s. (C) Line scan analysis of PA-GFP fluorescence along the cilium from the example in panel B. (D) Integrated PA-GFP fluorescence signal in the cilium as a function of time after the 405-nm laser pulse. The PA-GFP fluorescence at time = 0 was set at 100%. Points represent the mean fraction of PA-GFP fluorescence present in the cilium over an 80-s imaging interval. Error bars represent SD from analysis of n = 6 cilia. There was no detectable loss of ciliary PA-GFP signal quantified over an 80-s interval. (E) Confocal images of a representative cilium acquired immediately after (0 min) and 10 min after photoactivation, showing that the locally photoactivated receptor pool was largely retained in the cilium even after this longer interval. (F) Assessing new D1R delivery to the cilium by saturation photoactivation and the sequential ‘image-photoactivate-image’ scheme described in the ‘Materials and methods’. Bars represent mean fractional increase in ciliary PA-GFP fluorescence elicited by the subsequent test pulse. Error bars represent SD for n = 7 cilia. (G) Schematic for modifying the saturation photoactivation method to assess source of newly delivered D1Rs, based on the ratio of integrated PA-GFP/Alexa555 fluorescence (PA/555) measured in the cilium as a function of time. The initial condition is depicted on the left (‘0 min’) with the fluorescence ratio (PA/555) arbitrarily set to 1. If new receptors enter the cilium from an internal membrane pool during the 30-min incubation period (depicted in center, ‘30 min’), they contribute neither Alexa555 nor PA-GFP signal, so the fluorescence ratio is unchanged from the initial condition (PA/555 = 1). After the subsequent 405-nm test pulse (depicted at right, ‘30 min + PA’), the PA-GFP signal increases without any change in Alexa555 signal, elevating the fluorescence ratio above the initial condition (PA/555 > 1). If new receptors enter the cilium from the extra-ciliary plasma membrane pool, they contribute Alexa555 but not PA-GFP signal during the 30-min incubation, reducing the fluorescence ratio from the initial condition (PA/555 < 1). The subsequent 405-nm pulse restores the fluorescence ratio to the initial value (PA/555 = 1). (H) Experimental results from the strategy depicted in panel G. Bars represent the mean ratio of integrated PA-GFP/Alexa555 fluorescence measured in the cilium. Error bars represent SD from n = 6 cilia. (***) p < 0.001. Scale bars, 5 μm.