Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
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DOI：
10.1038/nmeth866
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发表时间：
2006-04-01
期刊：
影响因子：
48
通讯作者：
Lukyanov, S
中科院分区：
文献类型：
--
作者：
Belousov, VV;Fradkov, AF;Lukyanov, S
我们开发了一种基因编码的、高度特异的荧光探针，用于检测活细胞内的过氧化氢(H2O2)。这个名为HYPER的探针由插入原核生物过氧化氢敏感蛋白OxyR的调节域的环状排列的黄色荧光蛋白(CpYFP)组成。利用HYPER，我们在单细胞水平上监测了Apo2L/TRAIL处理的HeLa细胞胞浆和线粒体中过氧化氢的产生。我们发现，细胞质中过氧化氢的增加与线粒体跨膜电位(Delta Psi)的下降和细胞形状的改变平行。我们还观察到在凋亡的HeLa细胞中，Delta Psi振荡期间线粒体过氧化氢产生的局部爆发。此外，该探针的灵敏度足以观察到生理刺激下过氧化氢的增加。我们用HYPER检测到神经生长因子刺激的PC-12细胞胞浆中过氧化氢的时间性增加。
We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H2O2) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR. Using HyPer we monitored H2O2 production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/ TRAIL. We found that an increase in H2O2 occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (Delta Psi) and a change in cell shape. We also observed local bursts in mitochondrial H2O2 production during Delta Psi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H2O2 increase upon physiological stimulation. Using HyPer we detected temporal increase in H2O2 in the cytoplasm of PC-12 cells stimulated with nerve growth factor.