Fig. 2: Optimization and characterization of a new conformational CaMKII FRET sensor.

a, Schematic of the improved CaMKIIα sensor 2dV-Camui. mEGFP, monomeric EGFP. b, Representative fluorescence lifetime images of HeLa cells expressing 2dV-Camui (bottom) or original Green Camuiα (1dV-Camui) (top) before, during ionomycin and during EGTA. c,d, Mean time course (c) and summary of the peak (1 min after 3 µM ionomycin application) (d) of fluorescent lifetime changes of 2dV-Camui (2dV) (n = 89 cells over 8 independent experiments) and Green Camuiα (1dV) (n = 66 cells, 8 experiments) in HeLa cells. 2dV showed around twofold higher sensitivity (two-tailed unpaired t-test, (t₁₅₃ = 11.01, P = 2.0 × 10^–21) (d). e, Representative fluorescence lifetime images of CA1 dendrites in hippocampal culture before, during and after glutamate uncaging at 0.49 Hz in zero extracellular Mg²⁺. Asterisk depicts the spine where glutamate was uncaged. Similar activation profiles were observed in more than 95% of stimulated spines over 8 neurons. f,g, Mean time course (f) and summary of the peak (6th–11th uncaging pulses) (g) of fluorescence lifetime changes in stimulated spines. 2dV (n = 31 spines, 10 neurons) showed higher sensitivity than 1dV (n = 12 spines, 5 neurons). Two-tailed unpaired t-test, (t₄₁ = 4.26, P = 1.18 × 10^–4) (g). h,i, Closer views of mean lifetime changes at spines during the first 8 uncaging pulses (h) and the decay kinetics after the last uncaging pulse (i) in wild-type 1dV(WT) (n = 12 spines, 5 neurons), 2dV(WT) (n = 31 spines, 10 neurons), 2dV(T286A) (n = 28 spines, 3 neurons) and 2dV(T305D/T306D) (n = 39 spines, 5 neurons). The fitting curves indicate single exponential fitting (y = Aexp(–t/τ) + B, where the fast time constant (τ) is 7.9, 7.3 and 0.7 s for 1dV(WT), 2dV(WT) and 2dV(T286A), respectively. The data are presented as the mean ± s.e.m. ****P < 0.0001. Scale bars, 2 μm (e) or 50 μm (b).