Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

Figure 4

Short-term effect of quinpirole addition on D_2LR-YFP expression in the plasma membrane. Distribution of D_2LR-YFP on the plasma membrane in one single plane of transfected HEK-293 cells was studied over a period of time of 6 min. Medium or 100 nM quinpirole were added 30 s after the beginning of the recording. Plasma membrane fluorescence intensity measured in a medium- and a quinpirole-treated cell without photobleaching correction. Images of the same cells after photobleaching correction (b and e). Statistical significance plot (𝑃<0.05) of the fluorescence intensity of each pixel from medium- (c) and quinpirole-treated cells (f) normalized with respect to data obtained prior addition of medium or quinpirole (first 30 s). Significant increases and decreases in fluorescence intensity are represented in red and blue, respectively. Data of the normalized fluorescence intensity (g) for all analyzed cells (medium 𝑛=22, quinpirole 𝑛=24) showed significant changes only in agonist-treated cells and not before 70 s after agonist addition. (h) Temporal evolution of the mean value of the normalized fluorescence intensity. Oscillation around 0 indicates a lack of net increase or decrease in D_2LR-YFP surface expression. (i) Photobleaching-corrected plasma membrane magnification at different time points shows regions with evident increases and/or decreases in fluorescence intensity.

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