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

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Camila Aburto - (Author)
  • Alex Galaz - (Author)
  • Angelo Bernier - (Author)
  • Pamela Yohana Sandoval - (Author)
  • Sebastián Holtheuer-Gallardo - (Author)
  • Iván Ruminot - (Author)
  • Ignacio Soto-Ojeda - (Author)
  • Helen Hertenstein - , Chair of Zoology and Animal Physiology (Author)
  • Johanna Aurelia Schweizer - (Author)
  • Stefanie Schirmeier - , Chair of Zoology and Animal Physiology (Author)
  • Tammy Paulina Pástor - (Author)
  • Gonzalo Antonio Mardones - (Author)
  • Luis Felipe Barros - (Author)
  • Alejandro San Martín - (Author)

Abstract

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 methodologies. 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 ∼300 μM. 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.

Details

Original languageEnglish
Pages (from-to)3278 - 3286
Number of pages9
JournalACS Sensors
Volume7
Issue number11
Publication statusPublished - 25 Nov 2022
Peer-reviewedYes

External IDs

Scopus 85141518628

Keywords