Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street., Stanford, CA 94305, USA.
Biochem J. 2010 Dec 1;432(2):399-406. doi: 10.1042/BJ20100946.
Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. In the present study, genetically encoded FRET (Förster resonance energy transfer)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP with varying external supply and in glucose-transport mutants. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells. We demonstrate the suitability of the FRET sensors for gaining physiological insight by demonstrating that free intracellular glucose and ATP levels are reduced in a hxt5Δ hexose-transporter mutant compared with wild-type and other hxtΔ strains.
细胞内代谢物水平的精确和动态测量一直受到区分吸附和导入部分以及细胞质、内质网隔室和线粒体之间亚细胞分布的困难的阻碍。在本研究中,基于遗传编码的 FRET(Förster 共振能量转移)的传感器被用于动态测量具有不同外部供应的游离细胞质葡萄糖和 ATP,以及葡萄糖转运突变体。此外,通过在微流控平台中使用 FRET 传感器,我们能够监测单个酵母细胞内游离细胞内葡萄糖的体内变化。我们通过证明与野生型和其他 hxtΔ 菌株相比,hxt5Δ 己糖转运蛋白突变体中的游离细胞内葡萄糖和 ATP 水平降低,证明了 FRET 传感器在获得生理见解方面的适用性。