Work

Two-point calibration protocol for the Förster Resonance Energy Transfer indicator Pyronic in neurons

Year: 2025

Type: article

Abstract: SignificancePyruvate is a nodal intermediate in cellular metabolism, positioned at the crossroads between glycolysis and fermentative metabolism. It is exchanged between the intracellular and extracellular compartments through the proton-coupled monocarboxylate transporters and between the cytosol and mitochondria through the mitochondrial pyruvate carrier, where it serves as a primary carbon source for respiration.AimOur goal is to present a detailed protocol for quantifying cytosolic pyruvate concentration in neurons at single-cell resolution using a minimally invasive, two-point calibration approach with the Förster Resonance Energy Transfer (FRET)-based genetically encoded fluorescent indicator Pyronic.ApproachThis protocol is based on a noninvasive pharmacological two-point calibration approach, where Pyronic’s dynamic range (ΔRMAX) is established by withdrawing all extracellular substrates to deplete intracellular pyruvate (RMIN) and by inducing Pyronic saturation (RMAX) through the combination of inhibition of pyruvate export, stimulation of its production, and blockade of its mitochondrial consumption. The protocol also incorporates the previously published KD values for Pyronic obtained from in vitro experiments. This procedure does not require the use of detergents to permeabilize the cells.ResultsImplementing this protocol enables the measurement of absolute cytosolic pyruvate concentrations. This quantitative parameter facilitates comparisons of pyruvate metabolism across different cells, samples, and experimental batches, thereby enabling the comparison between a plethora of experimental conditions.ConclusionThe FRET-based fluorescent indicator Pyronic can be reliably calibrated using a minimally invasive, pharmacology-based two-point calibration protocol in neurons, thus providing a robust and quantitative method to study pyruvate metabolism under various physiological and pathological scenarios.

Source: Neurophotonics

DOI: 10.1117/1.nph.12.s2.s22807

Authors: Felipe Baeza-Lehnert, Yasna Contreras-Baeza, Camila Aburto, Alejandro San Martín

Institutions: Leipzig University, Centro de Estudios Científicos

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,675

Funder: Agencia Nacional de Investigación y Desarrollo