Quantitative In Vivo Imaging of ATP Dynamics in Drosophila Using the Ratiometric Biosensor QUEEN

Genetically encoded ATP biosensors enable monitoring of cellular energy status, but their application in multicellular organisms remains limited. QUEEN is a ratiometric ATP biosensor consisting of a bacterial ATP-binding protein fused to a circularly permuted fluorescent protein and has been primarily validated in cultured cells. Here, we generated transgenic Drosophila melanogaster lines expressing QUEEN-7μ, enabling tissue-specific expression through the GAL4/UAS system. We characterized its performance in motor neurons and muscles. QUEEN-7μ responses were validated using pharmacological and genetic perturbations of mitochondrial function. Mitochondrial inhibition decreased the QUEEN-7μ ratio, consistent with reduced ATP levels, whereas acute treatment with mitochonic acid-5 (MA-5), a small molecule that enhances mitochondrial ATP synthesis, increased the QUEEN-7μ ratio, with a larger effect at higher concentration. Consistently, genetic manipulations, including the mitochondrial Complex I knockdown and Mitofilin/MIC60 overexpression, produced corresponding decreases and increases in the QUEEN-7μ ratio. These results establish QUEEN-7μ as a reliable ratiometric ATP reporter for quantitative in vivo analysis in Drosophila. This system provides a versatile platform for investigating energy metabolism and mitochondrial function in Drosophila.