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

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 (K_D), 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 K_D 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.