Figure 5. BcLOV4 and BcLOV-SOScat in zebrafish embryos and Drosophila cells.
A) Blue-light induced membrane translocation of BcLOV-mCherry in a zebrafish embryo (24 hours post fertilization [hpf]). Scale bar = 50 μm, inset scale bar = 20 μm. B) BcLOV-mCh translocation is sustained over 90 min in zebrafish embryos. Data represent mean +/− SD of 10 cells. C) Schematic of ErkKTR activity. ErkKTR is nuclear when Erk signaling is off and translocates to the cytoplasm when Erk is activated. D) The Ras/Erk pathway can be reversibly stimulated over multiple cycles in zebrafish embryos (24 hpf) that co-express express BcLOV-SOScat and ErkKTR-BFP, as measured by ErkKTR-BFP translocation. White arrows highlight nuclei where ErkKTR translocation is evident. Scale bar = 10 μm. E) Sustained illumination of BcLOV-SOScat permits sustained elevated Erk activity in 24 hpf zebrafish embryos. Plot shows ErkKTR cytoplasmic/nuclear ratios of 12 single cells (light grey; blue trace represents mean) measured over two experiments. Trajectories are normalized between 0 and 1 to permit comparison between experiments. For (A-E) stimulation was performed using 1.45 W/cm2 488 nm light at 1.5% duty cycle. F) BcLOV-mCh membrane translocation in Drosophila S2 cells stimulated with blue light (1.45 W/cm2 at 3% duty cycle) for 90 min. Scale bar = 10 μm. G) Quantification of (F) shows sustained membrane translocation in S2 cells. Data represent mean +/− SD of 10 cells. H) Sustained stimulation of BcLOV-SOScat in S2 cells shows sustained elevated ppErk levels over 90 min, measured by immunofluorescence. Data is the mean +/− SD of three biologically independent samples, with each replicate representing the mean of ~100-200 cells.. Stimulation was performed at (160 mW/cm2 at 20% duty cycle). All experiments shown in Figure 5 were performed at room temperature.