Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.
Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.
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DOI：
10.1042/bj20100946
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发表时间：
2010-12-01
期刊：
影响因子：
--
通讯作者：
Frommer WB
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文献类型：
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作者：
Bermejo C;Haerizadeh F;Takanaga H;Chermak D;Frommer WB
细胞内代谢物水平的精确和动态测量一直受到阻碍的困难，在区分吸附和进口馏分和细胞质，内膜室和线粒体之间的亚细胞分布。在这里，遗传编码的福斯特共振能量转移（FRET）为基础的传感器被部署在不同的外部供应和葡萄糖转运突变体的游离胞质葡萄糖和ATP的动态测量。此外，通过在微流体平台中使用FRET传感器，我们能够监测单个酵母细胞中细胞内游离葡萄糖的体内变化。我们通过证明与野生型和其他hxtΔ菌株相比，hxt 5 Δ己糖转运蛋白突变体中游离细胞内葡萄糖和ATP水平降低，证明了FRET传感器对于获得生理学洞察的适用性。
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. Here, genetically encoded Förster Resonance Energy Transfer (FRET)-based sensors were deployed for dynamic measurements of free cytosolic glucose and ATP at 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 in 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 an hxt5Δ hexose transporter mutant compared to wild type and to other hxtΔ strains.