A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells
Fig 2
Performance of the Gαi-sensors in single cell GPCR signaling assays.
(A) FRET ratio-imaging experiments in HeLa cells transfected with the Gαi1-sensor. Rapid loss of FRET, observed by a decreased YFP/CFP ratio, after stimulation of the cells with 10μM UK-14,304, an α2AR specific agonist, addition of 60μM Yohimbine returns the FRET ratio towards baseline levels. Overnight treatment with (100ng/mL) PTX abolishes the response on the Gαi1-sensor in UK-14304 stimulated cells. (B) FRET ratio-imaging experiments in Huvecs transfected with the Gαi1-sensor. A sustained loss of FRET is observed after stimulation with 500nM S1P (Sphingosine-1-phosphate). Overnight treatment with (100ng/mL) PTX abolishes the response on the Gαi1-sensor in S1P stimulated cells. (C) FRET ratio-imaging experiments of HeLa cells transfected with the Gαi1-sensor and BK2B (top-right), LPA2 (top-left), M3 (bottom-right) and β2AR-2A2-mCherry (bottom-left) were stimulated with 1μM bradykinin (BK2B), 1μM lysophosphatidic acid (LPA2), 100μM carbachol and 10μM atropine (M3) or 10μM isoproterenol and 10μM propranolol (β2AR). HeLa cells transfected with BK2B, LPA2 and M3 receptors show a clear change in YFP/CFP FRET ratio upon addition of their respective agonists, whereas stimulation of the β2AR does not alter the FRET ratio of the Gαi1-sensor. In the control conditions, HeLa cells with only the Gαi1-sensor transfected received identical stimulations. (D) FRET ratio-imaging experiments in HeLa cells transfected with the Gαi2-sensor or Gαi3-sensor. Rapid loss of FRET is observed after stimulation of the cells with 10μM UK-14,304, subsequent addition of 60μM Yohimbine returns the FRET ratio towards baseline levels. Overnight treatment with (100ng/mL) pertussis toxin (PTX) abolishes the response on the Gαi2 and Gαi3 biosensors in UK-14304 stimulated cells. HeLa cells were stimulated with an agonist at t = 32s and an antagonist was added at t = 152s where indicated. Huvec cells were stimulated with S1P at t = 55s. Time traces show the average ratio change of YFP/CFP fluorescence (±s.e.m). Average curves consist of data from at least 3 independent experiments, conducted on different days, with the indicated number of cells (n) per condition.