GTPases drive numerous signaling cascades through the dynamic exchange and hydrolysis of guanosine triphosphate (GTP) and guanosine diphosphate (GDP). The intracellular GTP/GDP ratio serves as a key indicator of cellular metabolic state and downstream signaling activity. To understand how cells coordinate these processes, accurate and organelle-specific quantification of this ratio is essential. Here, we developed FLIM-GTP/GDP, a fluorescence lifetime-based biosensor for quantification of intracellular GTP/GDP ratio using fluorescence lifetime imaging microscopy (FLIM). We engineered FLIM-GTP/GDP by fusing a circularly permuted enhanced yellow fluorescent protein (cpEYFP) to a GTP/GDP-binding domain via optimized peptide linkers. The binding of GTP or GDP induces conformational changes that alter the chromophore environment, producing measurable shifts in fluorescence lifetime. We screened 3,000 mutants and identified an optimized construct that exhibited a 0.7 ns fluorescence lifetime difference between the GTP- and GDP-bound states. FLIM-GTP/GDP detected physiological GTP/GDP ratios ranging from 0.1 to 10. We validated FLIM-GTP/GDP in HeLa cells, where inhibition of glycolysis with 2-deoxy-D-glucose decreased the GTP/GDP ratio, while treatment with mycophenolic acid, an inhibitor of inosine-5'-monophosphate dehydrogenase 2, increased it. Furthermore, organelle-targeted variants exhibited compartmental differences, with the highest GTP/GDP ratios observed at the plasma membrane, lower ratios in the cytoplasm, and the lowest ratios in mitochondria. Notably, we observed a nanoscale GTP/GDP gradient progressing from the plasma membrane to the cytoplasm, suggesting a compartmentalized regulation of GTP metabolism on this scale.
Nguyen et al. (Sun,) studied this question.