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. 2016 Jun 16;113(27):E3950–E3959. doi: 10.1073/pnas.1601747113

Fig. 2.

Fig. 2.

Evaluation of FRET in GluA2-CFP/YFP receptors. (A) FRET spectra of suspensions of HEK cells transfected with GluA2-CFP/YFP constructs (●) and their GluA2-CFP/amber (○) equivalents are shown. Spectra show normalized emission relative to the peak of the CFP emission at 480 nm obtained using CFP excitation (430 nm). An emission spectrum for cells expressing CFP (○) alone is shown for comparison. A, amber; C, CFP; Y, YFP. (B) Confocal images of CFP emission (CFP intensity) and fluorescence lifetime (CFP lifetime) in HEK293 cells expressing the GluA2-6Y-10C construct or the FRET-deficient amber equivalent GluA2-6A-10C were obtained by confocal microscopy as described in Materials and Methods. Donor fluorescence lifetimes (τCFP) were acquired at each pixel by fitting the time course of fluorescence decay obtained by TCSPC analysis (Materials and Methods), and used to construct fluorescence lifetime images where pixel color corresponds to the measured CFP lifetime as indicated by scale bars. (C) Determination of τCFP in the presence and absence of acceptor YFP in a single representative pixel in the lifetime images of the GluA2-6Y-10C– or GluA2-6A-10C–expressing cells shown in B. Decay of GluA2-6A-10C (black circles) represents the donor fluorescence lifetime in the absence of acceptor, whereas GluA2-6Y-10C (red circles) represents the donor lifetime in the presence of YFP. Both decays follow a single exponential time course. (D) Frequency distribution histograms of lifetime values from individual pixels in the FLIM images (Right Insets) within the surface membrane region (demarcated with red and black punctuated lines) of the cells expressing GluA2-6Y-10C (Upper Inset) and GluA2-6A-10C (Lower Inset). The lifetime distributions for CFP in the representative cells expressing GluA2-6Y-10C (red) and GluA2-6A-10C (black) were fitted to a single binomial (Materials and Methods) to determine the mean ± SD of the lifetime values of CFP in the absence (3.54 ± 0.09 ns) and presence (2.98 ± 0.07 ns) of acceptor, respectively. Summaries of CFP lifetimes obtained from cells expressing GluA2-6Y-10C (E), the CTZ-insensitive mutant GluA2-6Y-10C-S754Q (F), the amber equivalent GluA2-6A-10C (G), and the donor-alone GluA2-10C (H) obtained under different ligand conditions are shown. **P < 0.01, compared with no ligand condition.