Simultaneous Live Mapping of pH and Hydrogen Peroxide Fluctuations in Autophagic Vesicles

Hydrogen peroxide (H₂O₂) plays a critical role in the regulation and progress of autophagy, an essential recycling process that influences cellular homeostasis and stress response. Autophagy is characterized by the formation of intracellular vesicles analogous to recycle "bags" called autophagosomes, which fuse with lysosomes to form autolysosomes, eventually ending up as lysosomes. We have developed two novel autophagic vesicle-targeted peptide-based sensors, ROSA for H₂O₂ and pHA for pH, to simultaneously track H₂O₂ and pH dynamics within autophagic vesicles as autophagy advances. Since pH values progressively decrease within autophagic vesicles with the progress of autophagy, we utilized information on vesicular pH to identify stages of autophagic vesicles in live cells. Fluorescence intensities of the H₂O₂ sensor, ROSA, within autophagic compartments at different autophagic stages, which were identified by simultaneous pH mapping, revealed that H₂O₂ levels vary significantly within autophagic vesicles as autophagy progresses, with maximum H₂O₂ levels in the autolysosomal stage. This study provides the first detailed observation of H₂O₂ fluctuations within autophagic vesicles throughout the entire process of autophagy in living mammalian cells, offering insights into the oxidative changes associated with this vital cellular process.