Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.
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.
Centro de Estudios Científicos (CECs), Valdivia, Chile ; Universidad Austral de Chile, Valdivia, Chile.
Alejandro
A
San Martín
Centro de Estudios Científicos (CECs), Valdivia, Chile.
Sebastián
S
Ceballo
Centro de Estudios Científicos (CECs), Valdivia, Chile.
Felipe
F
Baeza-Lehnert
Centro de Estudios Científicos (CECs), Valdivia, Chile ; Universidad Austral de Chile, Valdivia, Chile.
Rodrigo
R
Lerchundi
Centro de Estudios Científicos (CECs), Valdivia, Chile.
Rocío
R
Valdebenito
Centro de Estudios Científicos (CECs), Valdivia, Chile ; Universidad Austral de Chile, Valdivia, Chile.
Yasna
Y
Contreras-Baeza
Centro de Estudios Científicos (CECs), Valdivia, Chile.
Karin
K
Alegría
Centro de Estudios Científicos (CECs), Valdivia, Chile.
L Felipe
LF
Barros
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D001921:Brain / Q000166:cytology / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006019:Glycolysis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008322:Mammals; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D019289:Pyruvic Acid / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; C084955:PdhR protein, E coli; D012097:Repressor Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D019289:Pyruvic Acid