Abstract

Cytosolic pH is a critical regulator of cellular metabolism and signaling, and its perturbation is intrinsically linked to oxidative stress. This study investigates the spatiotemporal generation of **Reactive Oxygen Species (ROS)** in both mitochondrial and cytosolic compartments following **optogenetic cytosol alkalization** in human cells. Using a genetically encoded pH sensor and a light-activated proton pump, we achieved precise, non-invasive control over cytosolic pH, inducing a rapid and controlled alkalization. Our findings reveal a significant, transient increase in mitochondrial ROS production immediately following alkalization, suggesting a direct impact on the electron transport chain, likely through altered proton motive force or matrix enzyme activity. Concurrently, a delayed but sustained increase in cytosolic ROS was observed, potentially mediated by pH-sensitive cytosolic enzymes like NADPH oxidases. This differential ROS response highlights a complex interplay between cytosolic pH and oxidative stress pathways. The results underscore the importance of pH homeostasis in maintaining cellular redox balance and provide a novel optogenetic tool for dissecting the molecular mechanisms linking pH dysregulation to oxidative stress-related pathologies.