Real-time imaging of the intracellular glutathione redox potential
Real-time imaging of the intracellular glutathione redox potential
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DOI：
10.1038/nmeth.1212
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发表时间：
2008-06-01
期刊：
影响因子：
48
通讯作者：
Dick, Tobias P.
中科院分区：
文献类型：
--
作者：
Gutscher, Marcus;Pauleau, Anne-Laure;Dick, Tobias P.
由于缺乏合适的氧化还原生物传感器，活细胞中基于氧化还原过程的动态分析受到限制。传统的氧化还原敏感性GFP（roGFP）受到不确定的特异性和对氧化还原电位变化反应缓慢的限制。在这项研究中，我们证明了人谷氧还蛋白-1（Grx 1）与roGFP 2的融合促进了传感器蛋白和谷胱甘肽氧化还原对之间的特异性实时平衡。Grx 1-roGFP 2融合蛋白允许动态实时成像的谷胱甘肽氧化还原电位（E-GSH）在不同的细胞隔室具有高灵敏度和时间分辨率。该生物传感器检测纳摩尔的氧化型谷胱甘肽（GSSG）的变化对一个背景下的毫摩尔还原型谷胱甘肽（GSH）的规模为秒至分钟。它有助于观察与生长因子可用性、细胞密度、线粒体去极化、呼吸爆发活动和免疫受体刺激相关的氧化还原变化。
Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential (E-GSH) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.