Abstract 1920 Illuminating compartmentalized AMPK signaling in single cells

AMP activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, with a variety of downstream targets throughout the cell. However, mechanisms behind subcellular AMPK activity are still unclear. We previously developed a single-fluorophore excitation-ratiometric AMPK activity reporter (ExRai AMPKAR), which enables detection of subtle, subcellular AMPK activity. We used ExRai AMPKAR to uncover mechanisms for energy stress-induced subcellular AMPK activity. However, how other forms of stress, including oxidative stress, influence subcellular AMPK activity is largely unknown. Using ExRai AMPKAR, we find that oxidative stress induced by hydrogen peroxide results in location-specific differences in AMPK activity, which has implications for the subcellular regulation of metabolism. Further, we used an adenosine triphosphate (ATP) sensor to observe how cellular energy levels directly change in response to hydrogen peroxide. This work lays the foundation towards understanding how the subcellular environment regulates localized AMPK activity and the subsequent compartmentalized metabolic regulation. The Schmitt Lab and this project is funded by the NIH's Directors New Innovator Award Grant DP2-GM154012.