Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics

Aburto, Camila; Galaz, Alex; Bernier, Angelo; Yohana Sandoval, Pamela; Holtheuer-Gallardo, Sebastian; Ruminot, Ivan; Soto-Ojeda, Ignacio; Hertenstein, Helen; Aurelia Schweizer, Johanna; Schirmeier, Stefanie; Paulina Pastor, Tammy; Antonio Mardones, Gonzalo; Felipe Barros, Luis; San Martin, Alejandro


Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening method-ologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of similar to 300 mu M. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

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Título según WOS: ID WOS:000879524400001 Not found in local WOS DB
Título de la Revista: ACS SENSORS
Volumen: 7
Número: 11
Fecha de publicación: 2022
Página de inicio: 3278
Página final: 3286


Notas: ISI