Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

Abstract

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.

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: Studies on TGF-β1 regulation

“…Although this dye has been proposed as specific for H 2 O 2 , it seems that it may be also activated by other ROS (4). Thus, to be sure about changes in H 2 O 2 elicited by extracellular CAT, we transfected the cells with the HyPer plasmid (1), that specifically responds to changes in H 2 O 2 concentrations and we confirmed the significant decrease of intracellular H 2 O 2 by CAT addition. Cells with low intracellular H 2 O 2 concentration did not show any significant toxicity, at least in the intervals that these studies were performed.…”

Oligomeric peroxiredoxin-I is an essential intermediate for p53 to activate MST1 kinase and apoptosis

“…First, we confirmed the requirement of MST1/2 for cisplatin-induced apoptosis in U2OS cells (Supplementary Figure 5). We then performed H 2 O 2 -imaging analysis using GFP-HyPer, which specifically responds to H 2 O 2 by increasing its fluorescent signal intensity (Belousov et al, 2006). As shown in Figure 3a, treatment of COS-7 cells with cisplatin gradually increased the GFP-HyPer signal, which was evident after 4 h. By contrast, we did not observe any significant increase in GFP-HyPer-expressing cells not treated with cisplatin or control GFPexpressing cells treated with cisplatin.…”

Hydrogen peroxide primes heart regeneration with a derepression mechanism

“…To visualize the spatiotemporal dynamics of H 2 O 2 in the intact heart during regeneration, we used Tol2-based transgenesis and zebrafish myosin light chain 7 (myl7) promoter to generate transgenic zebrafish with cardiac-specific expression of Hyper ( Figure 1D), a fluorescent protein-based H 2 O 2 sensor [25,31]. High-resolution confocal imaging was performed in intact adult hearts excised from Tg(myl7:Hyper) transgenic animals, and H 2 O 2 levels were directly measured as the ratio between the fluorescent signals dually excited at 488 and 405 nm (R=F 488 /F 405 ).…”