All-optical spatiotemporal mapping of ROS dynamics across mitochondrial microdomainsin situ

Abstract

AbstractHydrogen peroxide (H2O2) functions as a second messenger to signal metabolic distress through highly compartmentalized production in mitochondria. The dynamics of ROS generation and diffusion between mitochondrial compartments and into the cytosol govern oxidative stress responses and pathology, though our understanding of these processes remains limited. Here, we couple the H2O2biosensor, HyPer7, with optogenetic stimulation of the ROS-generating protein KillerRed targeted into multiple mitochondrial microdomains. Single mitochondrial photogeneration of H2O2demonstrates the spatiotemporal dynamics of ROS diffusion and transient hyperfusion of mitochondria due to ROS. Measurement of microdomain-specific H2O2diffusion kinetics reveals directionally selective diffusion through mitochondrial microdomains. All-optical generation and detection of physiologically-relevant concentrations of H2O2between mitochondrial compartments provide a map of mitochondrial H2O2diffusion dynamicsin situ. These kinetic details of spatiotemporal ROS dynamics and inter-mitochondrial spreading forms a framework to understand the role of ROS in health and disease.