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 recycle 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 and pHA, to simultaneously track H2O2 and pH dynamics, respectively, 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 oxidative changes associated with this vital cellular process.