L-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long been generally considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of L-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of L-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically-encoded green fluorescent protein (GFP)-based intracellular L-lactate biosensors with a range of affinities. We evaluated the performance of these biosensors by in vitro and live-cell characterization and demonstrated the utility with imaging applications in several cell lines.
L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of L-lactate, we develop a second-generation green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 (ΔF/F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response (ΔF/F = 8.1 in cultured neurons) with faster response kinetics to extracellular L-lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in L-lactate concentration in vitro (ΔF/F = 15 to 22) and in live mammalian cells (ΔF/F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular L-lactate in cultured mammalian cells.
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