Highly responsive single-fluorophoreindicator to explore lactate dynamics in high calcium environments

Abstract

Lactate is an energy substrate and intercellular signaling molecule with multiple bodily functions. Lactate has physiological roles in neurogenesis, axon integrity, memory consolidation, immune response, exercise, adipose tissue lipolysis, etc, and is involved in inflammation, cancer and neurodegeneration. The FRET lactate indicator Laconic has been instrumental in the discovery of mechanisms involved in neurometabolic coupling, and has advanced the understanding of lactate transport, glycolysis and mitochondrial physiology. However, the low fluorescent response and the complex saturation kinetics of Laconic limit its use for high-throughput screening and quantitation. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed the first single-fluorophore indicator for lactate. The sensor exhibited an intensiometric fluorescence increase of {Delta}F/F0 3.0 and a single binding site with a KD of 293 M. The fluorescence is not affected by other monocarboxylates or pH. However, it is sensitive to Ca2+ in the nanomolar range. Targeting of the sensor to the endoplasmic reticulum revealed that this organelle presents a high permeability for lactate. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. This indicator, which we have termed CanlonicSF, is well suited to explore lactate dynamics in environments with micromolar Ca2+ or higher, such as the endoplasmic reticulum and the extracellular space.