Monitoring cytosolic H₂O₂ 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.