Non-invasive monitoring of intracellular ATP dynamics in living mammalian cells remains technically challenging, and it is still unclear whether ATP availability is heterogeneous at the single-cell level. To address this, we developed AMPfret, a genetically encoded, FRET-based nanosensor derived from AMP-activated protein kinase (AMPK). Because AMPK is intrinsically sensitive to physiologically relevant ATP: AMP and ATP:ADP ratios, AMPfret provides a readout with direct biological significance. We engineered HEK293T cells to stably express AMPfret in distinct subcellular compartments, including the plasma membrane, cytosol, and mitochondrial surface or intermembrane space. Using this system, we detected pronounced heterogeneity in baseline energy states across cell populations, as well as during energy stress induced by the ROS generator tert-butyl hydroperoxide (TBHP, Luperox) and the mitochondrial uncoupler trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP). Spatiotemporal analysis further revealed compartment-specific differences in the kinetics and amplitude of energy stress responses within single cells, suggesting a wave-like propagation of energy stress signals. Together, these findings provide new insights into the subcellular heterogeneity of ATP:AMP and ATP:ADP ratios.
Nahed et al. (Sun,) studied this question.