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Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue
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Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Camila Aburto1,2, Alex Galaz1, Angelo Bernier1,2

  • 1Centro de Estudios Científicos (CECs), Avenida Arturo Prat 514, Postal Code 5110466 Valdivia, Chile.

ACS Sensors
|October 28, 2022
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Summary

Researchers developed CanlonicSF, a novel single-fluorophore indicator for tracking cellular lactate. This tool overcomes limitations of previous methods, enabling subcellular lactate dynamics monitoring and high-throughput screening for drug discovery.

Keywords:
AR-C155858AZD3965CanlonicSFGFP-basedTTHA0766endoplasmic reticulumlactate transportphloretin and pCMBs

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Imaging

Background:

  • Lactate serves as an energy substrate and intercellular signal.
  • Genetically encoded FRET indicators like Laconic exist but have limitations.
  • FRET indicators face challenges in subcellular targeting and high-throughput screening due to complexity and small fluorescent changes.

Purpose of the Study:

  • Develop a single-fluorophore indicator for lactate monitoring.
  • Overcome limitations of FRET-based lactate indicators.
  • Enable subcellular lactate dynamics visualization and high-throughput screening.

Main Methods:

  • Engineered a single-fluorophore indicator (CanlonicSF) using the bacterial periplasmic binding protein TTHA0766.
  • Characterized CanlonicSF's fluorescence response, affinity (KD), and specificity.
  • Targeted CanlonicSF to the endoplasmic reticulum for subcellular imaging.
  • Developed a multiwell plate assay for screening monocarboxylate transporter (MCT) inhibitors.
  • Validated CanlonicSF in living tissue (Drosophila melanogaster larvae brain).

Main Results:

  • CanlonicSF demonstrated a 200% maximal fluorescence change and a KD of ~300 μM for lactate.
  • The indicator showed specificity for lactate over other monocarboxylates.
  • Subcellular lactate dynamics were visualized in the endoplasmic reticulum for the first time.
  • A high-throughput screening assay for MCT inhibitors was successfully developed.
  • CanlonicSF proved functional in vivo in Drosophila larvae brain tissue.

Conclusions:

  • CanlonicSF is a robust single-fluorophore indicator for lactate.
  • It enables subcellular lactate dynamics monitoring with improved sensitivity.
  • CanlonicSF facilitates high-throughput screening for pharmaceutical targets like MCTs.
  • The indicator is suitable for exploring lactate dynamics in various intact systems.