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

Hydrogen peroxide (HO) 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, for HO and for pH, to simultaneously track HO 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 HO sensor, , within autophagic compartments at different autophagic stages, which were identified by simultaneous pH mapping, revealed that HO levels vary significantly within autophagic vesicles as autophagy progresses, with maximum HO levels in the autolysosomal stage. This study provides the first detailed observation of HO 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.