Monitoring cellular redox dynamics using newly developedBRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affectthe cellular redox state and regulate various physiological andcellular phenomena. To monitor changes in the redox state, wepreviously developedfluorescent redox sensors named Re-Q,the emissions of which are quenched under reduced condi-tions. However, suchfluorescent probes are unsuitable for usein the cells of photosynthetic organisms because they requirephotoexcitation that may change intracellular conditions andinduce autofluorescence, primarily in chlorophylls. In addition,the presence of various chromophore pigments may interferewithfluorescence-based measurements because of their strongabsorbance. To overcome these problems, we adopted thebioluminescence resonance energy transfer (BRET) mechanismfor the sensor and developed two BRET-based redox sensors byfusing cyanfluorescent protein-based or yellowfluorescentprotein-based Re-Q with the luminescent protein Nluc. Wenamed the resultingredox-sensitiveBRET-basedindicatorprobes"ROBINc"and"ROBINy."ROBINc is pH insensitive,which is especially vital for observation in photosynthetic or-ganisms. By using these sensors, we successfully observed dy-namic redox changes caused by an anticancer agent in HeLacells and light/dark-dependent redox changes in the cells ofphotosynthetic cyanobacteriumSynechocystissp. PCC 6803.Since the newly developed sensors do not require excitationlight, they should be especially useful for visualizing intracel-lular phenomena caused by redox changes in cells containingcolored pigments.