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 (ΔΨ) and a change in cell shape. We also observed local bursts in mitochondrial H2O2 production during ΔΨ 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.
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We thank D. Zorov (Belozersky Institute of Physical and Chemical Biology, Moscow) and Y. Labas (Bach Institute of Biochemistry, Moscow) for constructive discussions, and M. Gaspariane (Institute of Bioorganic Chemistry, Moscow) for providing Apo2L/TRAIL. This research was supported by grants from the European Commission (FP-6 Integrated Project LSHG-CT-2003-503259), the Russian Academy of Sciences Program in Molecular and Cell Biology, the Russian Foundation for Basic Research (Project 05-04-49316), the US National Institutes of Health (GM070358) and the Howard Hughes Medical Institute grant HHMI 55005618.
V.V.B. and S.L. have applied for a patent that covers the work described in this study. D.B.S. is employed by Evrogen.
pH dependency of excitation ratio 500/420 nm of HyPer. (PDF 104 kb)
Western blotting of Bcl-2. (PDF 452 kb)
OxyR-RD fragments amplification to prepare fluorescent indicators for hydrogen peroxide. (DOC 50 kb)
Image series of fluorescence of HyPer-C (green) and TMRM (red) in HeLa cells during apoptosis induced by Apo2L/TRAIL. HeLa cells expressing the cytosolic form of HyPer (HyPer-C) were loaded with 20 nM TMRM and exposed to 400 ng/ml Apo2L/TRAIL. Scanning was performed using 400 Hz line frequency, 512×512 format. Green fluorescent signal was acquired using 488 nm excitation laser line (4% intensity) and detected at 500-520 nm wavelength range. Red fluorescent signal was acquired using 543 nm excitation laser line (12% intensity) and detected at 600-650 nm. Time series speed was 1 frame per 2 minutes. (MOV 1768 kb)
Image series of fluorescence of HyPer-M (green) and TMRM (red) in mitochondria of HeLa cells during apoptosis induced by Apo2L/TRAIL. HeLa cells expressing the mitochondrial form of HyPer (HyPer-M) were loaded with 20 nM TMRM and exposed to 400 ng/ml Apo2L/TRAIL. Scanning was performed using 400 Hz line frequency, 512×512 format. Green fluorescent signal was acquired using 488 nm excitation laser line (4% intensity) and detected at 500-520 nm wavelength range. Red fluorescent signal was acquired using 543 nm excitation laser line (12% intensity) and detected at 600-650 nm. Time series speed was 1 frame per 2 minutes. (MOV 1314 kb)
Image series of fluorescence of HyPer-C (green) and TMRM (red) in Bcl-2 overexpressing HeLa cells during apoptosis induced by Apo2L/TRAIL. Bcl-2 overexpressing HeLa cells expressing the cytosolic form of HyPer (HyPer-C) were loaded with 20 nM TMRM and exposed to 400 ng/ml Apo2L/TRAIL. Scanning was performed using 400 Hz line frequency, 512×512 format. Green fluorescent signal was acquired using 488 nm excitation laser line (4% intensity) and detected at 500-520 nm wavelength range. Red fluorescent signal was acquired using 543 nm excitation laser line (12% intensity) and detected at 600-650 nm. Time series speed was 1 frame per 2 minutes. (MOV 1815 kb)
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Belousov, V., Fradkov, A., Lukyanov, K. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3, 281–286 (2006). https://doi.org/10.1038/nmeth866
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