Vsevolod V. Belousov

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

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.

Unraveling the Biological Roles of Reactive Oxygen Species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.

Green fluorescent proteins are light-induced electron donors

Proteins of the green fluorescent protein (GFP) family are well known due to their unique biochemistry and extensive use as in vivo markers. Here, we discovered a new feature of GFPs of diverse origins to act as the light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, this process accompanying with green-to-red GFP photoconversion can be observed in living cells without additional treatment.

Hydrogen Peroxide Probes Directed to Different Cellular Compartments

Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.

Genetically encoded fluorescent redox sensors.

BACKGROUND Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes. SCOPE OF REVIEW In this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development. MAJOR CONCLUSIONS Fluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H2O2, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP. GENERAL SIGNIFICANCE Genetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O2 and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

HyPer-3: A Genetically Encoded H 2 O 2 Probe with Improved Performance for Ratiometric and Fluorescence Lifetime Imaging

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.

A genetically encoded sensor for H2O2 with expanded dynamic range

Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicators dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca(2+) uptake.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

BackgroundGenetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).ResultsHere we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons.ConclusionWe believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

The 'mitoflash' probe cpYFP does not respond to superoxide

Arising from E.-Z. Shen et al. 508, 128–132 (2014); doi:10.1038/nature1301210.1038/nature13012Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial; several studies suggest that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al. provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of three-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of mitoflashes as ‘bursts of superoxide radicals’ assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide, but this interpretation has been criticized because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide. Here we show that purified cpYFP is completely unresponsive to superoxide, and that mitoflashes do not reflect superoxide generation or provide a link between mitochondrial free radical dynamics and lifespan. There is a Reply to this Brief Communication Arising by Cheng, H. et al. Nature 514, http://dx.doi.org/10.1038/nature13859 (2014).