TY - JOUR
T1 - Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate
AU - San Martín, Alejandro
AU - Ceballo, Sebastián
AU - Baeza-Lehnert, Felipe
AU - Lerchundi, Rodrigo
AU - Valdebenito, Rocío
AU - Contreras-Baeza, Yasna
AU - Alegría, Karin
AU - Barros, L. Felipe
PY - 2014/1/21
Y1 - 2014/1/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84907304289&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0085780
DO - 10.1371/journal.pone.0085780
M3 - Article
C2 - 24465702
AN - SCOPUS:84907304289
SN - 1932-6203
VL - 9
JO - PLoS ONE
JF - PLoS ONE
IS - 1
M1 - e85780
ER -