Characterization of novel FRET sensors.

<p>A. Localization of the FRET-sensors: N1E-115 cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)-^cp173Venus-Venus (Epac-S^H126); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td^cp173Venus (Epac-S^H187); mTurquoise2Δ-Epac(CD, ΔDEP)-td^cp173Dark Venus (Epac-S^H159) and mTurquoise2Δ-Epac(CD, ΔDEP)-^cp174Cit (Epac-S^H147). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S^H105); mTurquoise (Epac-S^H74) or mTurquoise2 (Epac-S^H126) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor ^cp173Venus-Venus (Epac-S^H134); td^cp173Venus (Epac-S^H187) or td^cp173Dark Venus (Epac-S^H189), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of HEK293T cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S^H126 or Epac-S^H134) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S^H187. After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S^H189. A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.