Abstract
Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. 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. 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. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. 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.
Highlights
Mitochondria are the chief energy generators of animal cells, accounting for over 90% of ATP production, and they generate building blocks for the synthesis of sugars, amino acids, nucleic acids and prosthetic groups, essential elements for tissue growth, plasticity and regeneration
The main finding of this study is that in cultured astrocytes mitochondrial pyruvate lies in the low micromolar range, endowing the mitochondrial pyruvate carrier (MPC) with the potential capability of ultrasensitive modulation of anaplerosis
Because the fluorescence ratio of the sensor Pyronic is insensitive to pH in the physiological range (San Martın et al, 2014a), we attribute the effect of pH on PyronicSF to the pH-sensitivity typical of circularly-permuted version of GFP (cpGFP) (Marvin et al, 2013; Cambronne et al, 2016)
Summary
Mitochondria are the chief energy generators of animal cells, accounting for over 90% of ATP production, and they generate building blocks for the synthesis of sugars, amino acids, nucleic acids and prosthetic groups, essential elements for tissue growth, plasticity and regeneration. In addition to their metabolic functions, mitochondria are involved in diverse physiological and pathophysiological processes, including Ca2+ signaling, the production of reactive oxygen species, aging and degeneration, cell death and oncogenesis. 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. Experiments were carried out in fruit fly larvae to demonstrate pyruvate dynamics in living tissue
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