Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

Abstract

Abstractl-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics ofl-Lactate, we develop a second-generation green fluorescent extracellularl-Lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellularl-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 extracellularl-Lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes inl-Lactate concentrationin 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 intracellularl-Lactate in cultured mammalian cells.