Abstract
Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Bacterial Proteins / metabolism
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Biosensing Techniques*
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Brain / cytology
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Brain / metabolism
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Cytosol / metabolism
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Escherichia coli Proteins / metabolism
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Fluorescence Resonance Energy Transfer*
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Glycolysis
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Green Fluorescent Proteins / metabolism
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HEK293 Cells
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Humans
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Luminescent Proteins / metabolism
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Male
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Mammals
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Mice
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Mice, Inbred C57BL
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Mitochondria / metabolism*
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Molecular Imaging / methods*
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Pyruvic Acid / metabolism*
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Repressor Proteins / metabolism
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Single-Cell Analysis / methods*
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Transcription, Genetic
Substances
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Bacterial Proteins
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Cyan Fluorescent Protein
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Escherichia coli Proteins
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Luminescent Proteins
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PdhR protein, E coli
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Repressor Proteins
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yellow fluorescent protein, Bacteria
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Green Fluorescent Proteins
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Pyruvic Acid
Grants and funding
This work was partly funded by a joint grant from CONICYT-Chile and the Deutsche Forschungsgemeinschaft to L. Felipe Barros and Joachim W. Deitmer (DFG-12, DE 231/25-1). The Centro de Estudios Científicos (CECs) is funded by the Chilean Government through the Centers of Excellence Basal Financing Program of CONICYT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.