Monitoring cytosolic H 2 O 2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations. Reliable methods of measuring intracellular H2O2 fluctuations are necessary to advance redox biology. Here the authors design a H2O2 sensor based on the fission yeast peroxiredoxin Tpx1 to sense nanomolar fluctuations of intracellular H2O2 in response to genetic and environmental perturbations.