Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.
We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.
Laboratory of Genes for Regeneration, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street 16/10, Moscow 117997, Russia.
Vsevolod V
VV
Belousov
Arkady F
AF
Fradkov
Konstantin A
KA
Lukyanov
Dmitry B
DB
Staroverov
Konstantin S
KS
Shakhbazov
Alexey V
AV
Terskikh
Sergey
S
Lukyanov
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005815:Genetic Code; D006367:HeLa Cells / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells / Q000378:metabolism / Q000648:ultrastructure; D011387:Prokaryotic Cells / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D009414:Nerve Growth Factors; D012097:Repressor Proteins; D014157:Transcription Factors; C499299:oxyR protein, E coli; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide