San Martín Alejandro, Ceballo Sebastián, Baeza-Lehnert Felipe, Lerchundi Rodrigo, Valdebenito Rocío, Contreras-Baeza Yasna, Alegría Karin, Barros L Felipe
Centro de Estudios Científicos (CECs), Valdivia, Chile ; Universidad Austral de Chile, Valdivia, Chile.
Centro de Estudios Científicos (CECs), Valdivia, Chile.
PLoS One. 2014 Jan 21;9(1):e85780. doi: 10.1371/journal.pone.0085780. eCollection 2014.
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.
目前，线粒体通量只能在低分辨率下进行检测。在此，我们介绍一种用于丙酮酸的基因编码荧光共振能量转移（FRET）传感器，以及在完整的单个细胞中以高时间分辨率定量测量丙酮酸转运、丙酮酸生成和线粒体丙酮酸消耗的方法。在人胚肾293细胞（HEK293）、神经元和星形胶质细胞中，线粒体丙酮酸摄取在生理水平达到饱和，这表明代谢率由细胞器的内在特性决定，而非底物可用性。该传感器的潜力在神经元中得到进一步证明，在由短θ波爆发触发的钙瞬变后数秒内，线粒体通量增加了300%，而葡萄糖水平保持不变。相比之下，星形胶质细胞的线粒体对细胞外ATP引发的类似钙瞬变不敏感。我们预计，丙酮酸传感器提供的更高分辨率将对关注线粒体功能的基础研究和应用研究人员具有实际意义。
