Fig. 9: All-optical control of endogenous RAS in steady state.

a RAS regulation by light in cells expressing EKAR2G2, in serum. Epifluorescence microscopy; scale bar, 20 µm. Pseudocolor scale: black = 1.0 and white = 2.05. b Normalized whole-cell average FRET/donor ratio in HeLa cells expressing the optogenetically enhanced iB(RAS) and EKAR2G2 FRET biosensor. n = 3, error bars represent SEM. Source data are provided as a Source Data file. c Illumination scheme used for the experiments shown in Fig. 9. d RAS-Akt signaling regulation in cells expressing optogenetically controlled iB(RAS). Representative time-lapse panels of AktAR2 FRET/donor ratio, imaged in HeLa cells in serum. Epifluorescence microscopy; scale bar, 20 µm. Pseudocolor scale: black = 1.0; white = 2.88. e Normalized whole-cell average FRET/donor ratio as a function of time, in HeLa cells expressing the AktAR2 FRET biosensor. Error bars represent SEM, n = 3 independent experiments. Source data are provided as a Source Data file. b, e Black: control cells without light-activation. Green: cells with 740 nm illumination starting at t = 300 s time point. Magenta: cells were irradiated with 740 nm light for 2400 s prior to imaging. f Schematic representation of all-optical control of RAS signaling using iB(RAS). Signaling nodes are shown in gray, optogenetic tools and biosensors are shown in light gray. Fast negative feedback loops acting via phosphorylation are shown as red dotted lines. Slow feedback loops acting via transcription inhibition are shown in gray.