Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide

Abstract

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H2O2 concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H2O2, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pKa of 5.9 vs. ≥7.6) and oxidized states (pKa of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H2O2 and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H2O2 when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H2O2 and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H2O2 upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H2O2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H2O2 indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.

A guide to genetically encoded tools for the study of H2O2

“…Due to this property, NeonOxIrr was found to be useful for revealing H 2 O 2 levels in fixed biological specimens. The low reduction rate allows for preservation of the redox state of NeonOxIrr by alkylation of thiols during fixation and preparation of the samples by treating with N-ethylmaleimide [50]. This approach enables study of "snapshotted" H 2 O 2 patterns in biological systems when live imaging is complicated or impossible.…”

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

“…Since the LSSmScarlet protein does not bleed through into the red channel (please, see above), we next attempted to demonstrate three-color confocal microscopy with the LSSmScarlet LSSRFP, mCherry RFP and mNeonGreen-derived green-yellow NeonOxIrr indicator for hydrogen peroxide [ 20 ] using a 488/561 nm dual excitation and standard emission filters. We transiently co-expressed dMito-LSSmScarlet, H2B-mCherry and Vimentin-NeonOxIrr fusion proteins in the lumen of mitochondria, in the nucleus and in the cytoskeleton of the HeLa cells.…”

“…In order to construct the pLU-CMV-vimentin-LSSmScarlet plasmid, the LSSmScarlet gene was PCR amplified as the BamHI-BsrGI fragment, using LSSmSc-BamHI/LSSmSc-XbaI-r primers listed in the Table S3 , and swapped with the mCherry gene in the pLU-CMV-vimentin-NeonOxIrr vector [ 20 ].…”

Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

“…For instance, complex signaling networks in intact cells, such as a change in kinase activity and second messenger transient responses to stimulations, were elegantly dissected by multiplexed imaging [32,33]. Moreover, emerging NIR fluorescent proteins, combined with newly developed NIR-FRET technologies, have opened a new window to understanding how complex biological processes, including calcium dynamics, signaling pathways, and/or proteolysis, are spatiotemporally regulated in cells [19,20,[34][35][36][37]. Given that PS/γ-secretase plays a pivotal role in normal development [9,10] and diseases [4,5,12], multiplexed recording of PS/γsecretase utilizing the NIR-C99 720-670 probe [19] along with other biological events will provide significant insights into PS/γ-secretase biology and its related diseases.…”