Molecular technologies laboratory

The central direction of our research is using synthetic biology approaches for stydying redox regulation of neurons and other cells and for development of novel optical and enzymatic molecular tools for neurobiology.

Life is a directed flow of electrons via highly organized redox couples. In every cell in every tissue a flow of electrons from primary sources (food) to end acceptors (molecular oxygen or cellular building blocks) occurs via redox pairs of chemical substances. Some of these substances, like NADH/NAD+ or NADPH/NADP+, serve as "electron currencies" driving oxidative and reductive reactions. In turn, molecular oxygen is not only a final acceptor of electrons in the respiratory chain giving energy for ATP synthesis, but also a global regulator of metabolic and signaling cascades acting via reactive oxygen species. It would not be overstating to say that the state of cellular redox pairs and redox messengers regulate all processes in cells.

One of our main research question is how the state of key redox pairs and redox messengers (for example H2O2, NADP+/NADPH) regulate neuronal function. We are especially interested in selective monitoring and modulating redox states of synapses in defined neuronal populations.

The first goal is to direct genetically encoded fluorescent sensors for H2O2, glutathione, NAD(P) and other molecules to neurons, from dissociated cultures to zebrafish and mouse brain in vivo, in order to monitor redox state of neurons upon various physiological and pathological conditions. Moreover, as synapses are functionally and metabolically distinctive compartments, we will use pre- and postsynaptic targeting of the probes to measure redox state of the neurons.    

The second goal is to modulate selectively key redox pairs in neurons at the level of compartments and sub-compartments in order to understand how their redox state changes neuronal functions. Our approach, "metabolic engineering", is to engineer enzymatic systems that utilize substrates normally not present in mammalian cells to selectively manipulate neuronal metabolism, signaling and function via key redox pairs in a number of models, from cultured neurons to living animals (zebrafish, mouse). This approach in combination with imaging using genetically encoded fluorescent probes would allow detailed understanding of key redox metabolism regulation mechanisms in neurons.

Another area of our research is a development of novel optogenetic molecular instruments. Current optogenetic instruments, like channelrhodopsins, have several disadvantages, like low single channel current and poor tissue permeability of the activating light. We work on optogenetic implementation of TRPA cation channels from thermosensing snakes activated with true infrared light. These channels have much greater conductivity compared to ChRs. IR irradiation penetrates tissues much better than visible light. Our results demonstrate the utility of these channels for robust IR-driven activation of cultured neurons and somatosensory neurons of zebrafish in vivo.

One more area of our research is a development of novel super-resolution imaging techniques to enable subdiffraction monitoring of dynamic processes in the living cells, particularly in neurons. Currently, subdiffraction imaging is mainly used to study fine structure of filaments and other supramolecular complexes. Our aim is to apply super-resolution imaging for monitoring dynamic processes, like second messengers dynamics and enzymatic activities. As a first step in this direction, we have shown the utility of a genetically encoded biosensor HyPer2 as a fluorophore for STED. Using HyPer2 we were able to study H2O2 cellular microdomains with subdiffractional resolution (Mishina et al, Nano Letters 2015). Currently we are testing a number of approaches to extracting dynamic information from various super-resolution modalities.

Main results


- a first application of a biosensor in subdiffraction microscopy (Mishina at al, 2015);

- novel tools and instrumentation for thermogenetics (Fedotov et al, 2015; Safronov et al, 2015);

- SypHer2, a genetically encoded pH sensor allowing synaptic pH imaging (Matlashov et al, 2015);

- development of the red fluorescent genetically encoded H2O2 sensor (Ermakova et al, 2014);

- development of genetically encoded H2O2 generation/detection system based on fusion of D-amino acid oxidase and HyPer (Matlashov et al, 2014);

- development of the genetically encoded fluorescent sensor for intracellular NAD+/NADH ratio (Bilan et al, 2014);

- development of HyPer-3, the high dynamic range and fast-responding H2O2 probe for ratiometric and lifetime imaging (Bilan et al, 2013);

- first records of H2O2 production by phagocytosing macrophages at the level of single cell (Mishina et al, 2013);

- discovery of polarized H2O2 production in the immunological synapse (Mishina et al, 2012)

- discovery of H2O2 microdomains in the cells stimulated with growth factors, the first direct demonstration of local H2O2 signaling (Mishina et al., 2011);

- development of HyPer-2, improved version of HyPer with expanded dynamic range (Markvicheva et al., 2011);

- development of HyPer, sensor for H2O2 (Belousov et al, 2006).


Dmitry S. BilanPhD stud.
Yulia A. BogdanovaPhD stud.
Yuliya G. ErmakovaPhD stud.
Ilya . Kelmanson, ph. d.r. f.
Elena I. Kudryavtseva, ph. d.r. f.
Mikhail E. MatlashovPhD stud.
Nataliya M. Mishina, ph. d.r. f.
Arina . ShokhinaPhD stud.

Selected publications

  1. Bilan D.S., Belousov V.V. (2016). HyPer Family Probes: State of the Art. Antioxid. Redox Signal. , [+]

    Hydrogen peroxide (H2O2) is not only a key mediator of oxidative stress but also one of the most important cellular second messengers. This small short-lived molecule is involved in the regulation of a wide range of different biological processes, including regulation of cellular signaling pathways. Studying the role of H2O2 in living systems would be challenging without modern approaches. A genetically encoded fluorescent biosensor, HyPer, is one of the most effective tools for this purpose. Recent Advances: HyPer has been used by many investigators of redox signaling in various models of different scales: from cytoplasmic subcompartments and single cells to tissues of whole organisms. In many studies, the results obtained using HyPer have enabled a better understanding of the roles of H2O2 in these biological processes. However, much remains to be learned.

  2. Santos C.X., Hafstad A.D., Beretta M., Zhang M., Molenaar C., Kopec J., Fotinou D., Murray T.V., Cobb A.M., Martin D., ZehSilva M., Anilkumar N., Schröder K., Shanahan C.M., Brewer A.C., Brandes R.P., Blanc E., Parsons M., Belousov V., Cammack R., Hider R.C., Steiner R.A., Shah A.M. (2016). Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling. EMBO J. , [+]

    Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.

  3. Erapaneedi R., Belousov V.V., Schäfers M., Kiefer F. (2016). A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level. EMBO J. 35 (1), 102–13 [+]

    Hypoxia is an intensively investigated condition with profound effects on cell metabolism, migration, and angiogenesis during development and disease. Physiologically, hypoxia is linked to tissue homeostasis and maintenance of pluripotency. Hypoxia also contributes to pathologies including cardiovascular diseases and cancer. Despite its importance, microscopic visualization of hypoxia is largely restricted to the detection of reductively activated probes by immunostaining. Here, we describe a novel family of genetically encoded fluorescent sensors that detect the activation of HIF transcription factors reported by the oxygen-independent fluorescent protein UnaG. It comprises sensors with different switching and memory behavior and combination sensors that allow the distinction of hypoxic and reoxygenated cells. We tested these sensors on orthotopically transplanted glioma cell lines. Using a cranial window, we could visualize hypoxia intravitally at cellular resolution. In tissue samples, sensor activity was detected in regions, which were largely devoid of blood vessels, correlated with HIF-1α stabilization, and were highly heterogeneous at a cellular level. Frequently, we detected recently reoxygenated cells outside hypoxic areas in the proximity of blood vessels, suggestive of hypoxia-promoted cell migration.

  4. Fedotov I.V., Safronov N.A., Ermakova Y.G., Matlashov M.E., SidorovBiryukov D.A., Fedotov A.B., Belousov V.V., Zheltikov A.M. (2015). Fiber-optic control and thermometry of single-cell thermosensation logic. Sci Rep 5, 15737 [+]Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen-vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels. ID:1328
  5. Shirmanova M.V., Druzhkova I.N., Lukina M.M., Matlashov M.E., Belousov V.V., Snopova L.B., Prodanetz N.N., Dudenkova V.V., Lukyanov S.A., Zagaynova E.V. (2015). Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2. Biochim. Biophys. Acta 1850 (9), 1905–11 [+]

    Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2.

  6. Matlashov M.E., Bogdanova Y.A., Ermakova G.V., Mishina N.M., Ermakova Y.G., Nikitin E.S., Balaban P.M., Okabe S., Lukyanov S., Enikolopov G., Zaraisky A.G., Belousov V.V. (2015). Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology. Biochim. Biophys. Acta 1850 (11), 2318–2328 [+]


    SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use.



    Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer.



    Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.



    SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.



    The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.


  7. Mishin A.S., Belousov V.V., Solntsev K.M., Lukyanov K.A. (2015). Novel uses of fluorescent proteins. Curr Opin Chem Biol 27, 1–9 [+]The field of genetically encoded fluorescent probes is developing rapidly. New chromophore structures were characterized in proteins of green fluorescent protein (GFP) family. A number of red fluorescent sensors, for example, for pH, Ca(2+) and H2O2, were engineered for multiparameter imaging. Progress in development of microscopy hardware and software together with specially designed FPs pushed superresolution fluorescence microscopy towards fast live-cell imaging. Deeper understanding of FPs structure and photophysics led to further development of imaging techniques. In addition to commonly used GFP-like proteins, unrelated types of FPs on the base of flavin-binding domains, bilirubin-binding domains or biliverdin-binding domains were designed. Their distinct biochemical and photophysical properties opened previously unexplored niches of FP uses such as labeling under anaerobic conditions, deep tissue imaging and even patients' blood analysis. ID:1293
  8. Mishina N.M., Mishin A.S., Belyaev Y., Bogdanova E.A., Lukyanov S., Schultz C., Belousov V.V. (2015). Live-Cell STED Microscopy with Genetically Encoded Biosensor. Nano Lett. 15 (5), 2928–2932 [+]

    Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.

  9. Schwarzländer M., Wagner S., Ermakova Y.G., Belousov V.V., Radi R., Beckman J.S., Buettner G.R., Demaurex N., Duchen M.R., Forman H.J., Fricker M.D., Gems D., Halestrap A.P., Halliwell B., Jakob U., Johnston I.G., Jones N.S., Logan D.C., Morgan B., Müller F.L., Nicholls D.G., Remington S.J., Schumacker P.T., Winterbourn C.C., Sweetlove L.J., Meyer A.J., Dick T.P., Murphy M.P. (2014). The 'mitoflash' probe cpYFP does not respond to superoxide. Nature 514 (7523), E12–4 ID:1094
  10. Ermakova Y.G., Bilan D.S., Matlashov M.E., Mishina N.M., Markvicheva K.N., Subach O.M., Subach F.V., Bogeski I., Hoth M., Enikolopov G., Belousov V.V. (2014). Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. Nat Commun 5, 5222 [+]

    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.

  11. Bilan D.S., Matlashov M.E., Gorokhovatsky A.Y., Schultz C., Enikolopov G., Belousov V.V. (2013). Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments. Biochim. Biophys. Acta 1840 (3), 951–957 [+]

    The ratio of NAD(+)/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD(+)/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD(+)/NADH are fundamentally new approach for studying the NAD(+)/NADH dynamics.

  12. Matlashov M.V., Belousov V.V., Enikolopov G. (2013). How Much H2O2 Is Produced by Recombinant D-Amino Acid Oxidase in Mammalian Cells? Antioxid. Redox Signal. , [+]

    Abstract Yeast D-amino acid oxidase (DAO) can serve as a genetically encoded producer of reactive oxygen species (ROS) in redox signaling studies. However, dynamics of hydrogen peroxide production and its sensitivity to externally added D-alanine (D-Ala) in cells have not been determined. Here we show that DAO, fused to a genetically encoded H2O2 indicator HyPer, can be used for controlled production of ROS in living eukaryotic cells. We found a clear heterogeneity in ROS production dynamics between individual cells. Moreover, different cell lines demonstrated distinct sensitivity to added D-Ala. Finally, by comparing signals generated by the HyPer-DAO fusion protein versus coexpressed HyPer and DAO proteins, we show that the fusion system is more sensitive to hydrogen peroxide production. Our results show the utility of the HyPer-DAO genetically encoded system for redox signaling studies and suggest that H2O2 produced by DAO in the cytoplasm acts locally in close proximity to the enzyme. Antioxid. Redox Signal. 00, 000-000.

  13. Lukyanov K.A., Belousov V.V. (2013). Genetically encoded fluorescent redox sensors. Biochim. Biophys. Acta , [+]

    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.

  14. Bilan D.S., Pase L., Joosen L., Gorokhovatsky A.Y., Ermakova Y.G., Gadella T.W., Grabher C., Schultz C., Lukyanov S., Belousov V.V. (2013). HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem. Biol. 8 (3), 535–42 [+]

    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.

  15. Mishina N.M., Markvicheva K.N., Fradkov A.F., Zagaynova E.V., Schultz C., Lukyanov S., Belousov V.V. (2013). Imaging H2O2 microdomains in receptor tyrosine kinases signaling. Meth. Enzymol. 526, 175–87 [+]

    HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.

  16. Mishina N.M., Markvicheva K.N., Bilan D.S., Matlashov M.E., Shirmanova M.V., Liebl D., Schultz C., Lukyanov S., Belousov V.V. (2013). Visualization of intracellular hydrogen peroxide with HyPer, a genetically encoded fluorescent probe. Meth. Enzymol. 526, 45–59 [+]

    The fluorescent sensor HyPer allows monitoring of intracellular H2O2 levels with a high degree of sensitivity and specificity. Here, we provide a detailed protocol of ratiometric imaging of H2O2 produced by cells during phagocytosis, including instructions for experiments on different commercial confocal systems, namely, Leica SP2, Leica SP5, and Carl Zeiss LSM, as well as wide-field Leica 6000 microscope. The general experimental scheme is easily adaptable for imaging H2O2 production by various cell types under a variety of conditions.

  17. Mishina N.M., Bogeski I., Bolotin D.A., Hoth M., Niemeyer B.A., Schultz C., Zagaynova E.V., Lukyanov S., Belousov V.V. (2012). Can we see PIP(3) and hydrogen peroxide with a single probe? Antioxid. Redox Signal. 17 (3), 505–12 [+]

    A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H(2)O(2)) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H(2)O(2). The dynamics of PIP(3) and H(2)O(2) generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts.

  18. Murphy M.P., Holmgren A., Larsson N.G., Halliwell B., Chang C.J., Kalyanaraman B., Rhee S.G., Thornalley P.J., Partridge L., Gems D., Nyström T., Belousov V., Schumacker P.T., Winterbourn C.C. (2011). Unraveling the biological roles of reactive oxygen species. Cell Metab. 13 (4), 361–6 [+]

    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.

  19. Markvicheva K.N., Bilan D.S., Mishina N.M., Gorokhovatsky A.Y., Vinokurov L.M., Lukyanov S., Belousov V.V. (2011). A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg. Med. Chem. 19 (3), 1079–84 [+]

    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 indicator's 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.

  20. Mishina N.M., TyurinKuzmin P.A., Markvicheva K.N., Vorotnikov A.V., Tkachuk V.A., Laketa V., Schultz C., Lukyanov S., Belousov V.V. (2011). Does cellular hydrogen peroxide diffuse or act locally? Antioxid. Redox Signal. 14 (1), 1–7 [+]

    Understanding of redox signaling requires data on the spatiotemporal distribution of hydrogen peroxide (H(2)O(2)) within the cell. The fluorescent reporter HyPer is a powerful instrument for H(2)O(2) imaging. However, rapid diffusion of HyPer throughout the nucleocytoplasmic compartment does not allow visualization of H(2)O(2) gradients on the micrometer scale. Here we dramatically improved the spatial resolution of H(2)O(2) imaging by applying subcytoplasmic targeting of HyPer. The membrane-attached reporters identified "microdomains" of elevated H(2)O(2) levels within the cytoplasm of the cells exposed to growth factors. We demonstrate that diffusion of H(2)O(2) across the cytoplasm was strongly limited, providing evidence that H(2)O(2) acts locally inside cells.

  21. Bogdanov A.M., Mishin A.S., Yampolsky I.V., Belousov V.V., Chudakov D.M., Subach F.V., Verkhusha V.V., Lukyanov S., Lukyanov K.A. (2009). Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol.  (5), 459–461 [+]

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

  22. Markvicheva K.N., Gorokhovatskiĭ A.I.u., Mishina N.M., Mudrik N.N., Vinokurov L.M., Lukianov S.A., Belousov V.V. (2009). Signaling function of phagocytic NADPH oxidase: activation of MAP kinase cascades in phagocytosis. Bioorg. Khim. 36 (1), 133–8 [+]

    Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.

  23. Belousov V.V., Enikolopov G.N., Mishina N.M. (2009). [Compartmentalization of ROS-mediated signal transduction]. Bioorg. Khim. 39 (4), 383–99 [+]

    The localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion ofthe same components. ROS-generating complexes have been suggested to follow this principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model ofredox signaling compartmentalization. Nevertheless, evidences of local ROS production and restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH-oxidase isoforms, their adapter proteins and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant system which prevents uncontrolled ROS diffusion from the sites of generation to the adjacent subcellular compartments; the current data of the specific localization NADPH-oxidases activity and its influence on intracellular processes; the recent evidences of the ROS diffusion restriction.

  24. Markvicheva K.N., Bogdanova E.A., Staroverov D.B., Lukyanov S., Belousov V.V. (2008). Imaging of intracellular hydrogen peroxide production with HyPer upon stimulation of HeLa cells with epidermal growth factor. Methods Mol. Biol. 476, 79–86 [+]

    Reactive oxygen species (ROS) regulate both normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress. For many years since the discovery of ROS in biological systems, there were no adequate methods of detection and quantification of these molecules inside the living cells. We developed the first genetically encoded fluorescent indicator for the intracellular detection of hydrogen peroxide, HyPer, that can be used for imaging of H2O2 production by cells under various physiological and pathological conditions. Unlike most known ROS indicators, HyPer allows the generation of a real-time image series that give precise information about the time course and intensity of H2O2 changes in any compartment of interest. In this chapter, we describe the method of confocal imaging of hydrogen peroxide production in HeLa cells upon stimulation with epidermal growth factor. The technique described may be accepted with minimal variations for the use in other cell lines upon various conditions leading to H2O2, production.

  25. Belousov V.V., Fradkov A.F., Lukyanov K.A., Staroverov D.B., Shakhbazov K.S., Terskikh A.V., Lukyanov S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3 (4), 281–6 [+]

    A unique fluorescent sensor HyPer was introduced for in vivo monitoring of concentration of hydrogen peroxide — one of the major regulators of biological processes. Being a protein, HyPer can be expressed in cells or targeted specifically to a particular cell compartment. Due to its high specificity and sensitivity HyPer can be used for monitoring fluctuations of hydrogen peroxide concentration in a single cell or cell organelle.

  26. Zorov D.B., Bannikova S.Y., Belousov V.V., Vyssokikh M.Y., Zorova L.D., Isaev N.K., Krasnikov B.F., Plotnikov E.Y. (2005). Reactive oxygen and nitrogen species: friends or foes? Biochemistry Mosc. 70 (2), 215–21 [+]

    Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".


Head of the laboratory

Vsevolod Belousov