Fig. 3
Demonstration of selective CALI. a Schematic overview of a selective CALI experiment using two indicator constructs with distinct excitation to induce ROS production which then liberates them from a plasma membrane tether. b Images taken at 0 s (before ROS-producing light irradiation), 10 s (immediately after light irradiation) and 15 min after 3 W/cm2 light irradiation for 10 s. Prior to ROS-producing light irradiation, all constructs were localized to the plasma membrane. After inactivation of the PHdomain, fluorescence increase in cytoplasm was seen for EGFP-PH-KillerRed (560 nm irradiation), mNeptune-PH-SNR (560 nm irradiation) and Venus-PH-SNG (440 nm irradiation). c Quantitative measurement of cytoplasm-to-plasma membrane fluorescence ratio increase. Fluorescence ratio of cytoplasm and plasma membrane at each time point was normalized to t0. As negative and positive control respectively, EGFP-PH (440 nm) and EGFP-PH-KillerRed (560 nm) were used (i). Only EGFP-PH-KillerRed showed a significant ratio increase (p < 0.05, one-way ANOVA, Tukey, n = 10 cells). (ii) 560 nm light irradiation to co-transfected cells with Venus-PH-SNG and mNeptune-PH-SNR caused a significant ratio increase of mNeptune fluorescence over time (p < 0.05, one-way ANOVA, Tukey, n = 11 cells) and also when compared to Venus at t15 (p < 0.05, t test, n = 11 cells for each construct). Conversely, 440 nm light irradiation (iii) caused significant ratio increase of Venus fluorescence (p < 0.05, one-way ANOVA, Tukey, n = 10 cells) and when compared to mNeptune at t15 (p < 0.05, t test, n = 10 cells for each construct). (iv) Images of light control taken from cells expressing Venus-PH-SNG and mNeptune-PH-SNR without light irradiation were calculated and showed no significant ratio changes over time. Error bar represents ±SEM