Fig. 7
Multiplexing of NIR PKA and JNK biosensors with optogenetic kinase inhibitors. a Schematic representation of LOV2-domain-based blue-light-regulatable kinase inhibitor in combination with respective fully-NIR kinase biosensor. Upon illumination with blue light, the Jα helix of LOV2 unfolds, resulting in uncaging of a peptide, which inhibits kinase. b HeLa cells stably expressing NIR PKA biosensor co-transfected with optogenetic PKA inhibitor, PA-PKI, tagged with mVenus (top row). Upon simultaneous 460 nm illumination and stimulation with 1 mM dbcAMP, the changes in FRET/miRFP670nano ratio are shown in pseudocolor (bottom row). c FRET/miRFP670nano ratio time courses of HeLa cells expressing NIR PKA biosensor only (red) or NIR PKA biosensor with PA-PKI (green) upon simultaneous 460 nm illumination and stimulation with 1 mM dbcAMP (n = 3 independent experiments). d HeLa cells stably expressing JNK biosensor co-transfected with optogenetic JNK inhibitor, optoJNKi, tagged with EGFP (top row). Upon simultaneous 460 nm illumination and stimulation with 1 μg ml−1 anisomycin, the changes in FRET/miRFP670nano ratio are shown in pseudocolor (bottom row). e FRET/miRFP670nano ratio time courses of HeLa cells expressing NIR JNK biosensor only (red) or NIR JNK biosensor with optoJNKi (green) upon simultaneous 460 nm illumination and stimulation with anisomycin (n = 3 independent experiments). White arrows indicate cells expressing optogenetic regulators. In b–e the miRFP670nano and FRET fluorescence signals were detected at 667 and 725 nm, respectively. Scale bars, 10 μm
