Resultsįigure 1A illustrates PyronicSF (Single Fluorophore), in which the complete sequence of the bacterial transcription factor PdhR ( Quail and Guest, 1995) has been linked to a circularly-permuted version of GFP (cpGFP) ( Nagai et al., 2001) (DNA sequence in Figure 1-figure supplement 1). Experiments were also carried out in fruit fly larvae to demonstrate pyruvate dynamics in living tissue. The main finding of this study is that in cultured astrocytes mitochondrial pyruvate lies in the low micromolar range, endowing the MPC with the potential capability of ultrasensitive modulation of anaplerosis. To demonstrate the usefulness of the probe, we measured cytosolic, nuclear and mitochondrial pyruvate concentration, MPC-mediated permeability and determined the metabolic flux of small groups of mitochondria. This article introduces a new version of Pyronic that improves on the original sensor in terms of dynamic range and facility of use, as it is imaged with the 488 nm Argon laser of standard confocal microscopes. Pyronic, the first genetically-encoded sensor for pyruvate ( San Martín et al., 2014a), has permitted the measurement of cytosolic pyruvate in several organisms with high temporal resolution, see for example ( Mächler et al., 2016 Compan et al., 2015 Plaçais et al., 2017 Delgado et al., 2018 Baeza-Lehnert et al., 2019 Hasel et al., 2017 Rusu et al., 2017).
Carbon is either shed to generate ATP via oxidative phosphorylation or alternatively, carbon is accrued to generate oxaloacetate, a process termed anaplerosis, which provides building blocks for biosynthesis and constitutes the first step of gluconeogenesis. Once in the matrix, the flux of pyruvate is split. Pyruvate enters mitochondria through the mitochondrial pyruvate carrier (MPC) ( Halestrap and Denton, 1975 Herzig et al., 2012 Bricker et al., 2012).
Extracellular pyruvate may also reach the cytosol via monocarboxylate transporters (MCTs) ( Halestrap and Price, 1999). Are mitochondria within a given cell metabolically diverse? A major mitochondrial substrate for mammalian cells is pyruvate, a 3-carbon organic acid produced from glucose, lactate and amino acids. Some open issues in mitochondrial physiology are the regulation of intermediate metabolism, the coordination between cytosolic and mitochondrial pathways, the decision between catabolism and anabolism, and the crosstalk between mitochondria and other organelles like plasma membrane and endoplasmic reticulum. In addition to their metabolic functions, mitochondria are involved in diverse physiological and pathophysiological processes, including Ca 2+ signaling, the production of reactive oxygen species, aging and degeneration, cell death and oncogenesis. Mitochondria are the chief energy generators of animal cells, accounting for over 90% of ATP production, and they also generate building blocks for the synthesis of sugars, amino acids, nucleic acids and prosthetic groups, essential elements for tissue growth, plasticity and regeneration. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate.