Мишина Наталия Михайловна

Кандидат биологических наук


Научный сотрудник (Лаборатория молекулярных технологий)

Эл. почта: natsafr@yandex.ru

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
2002–2007 Москва МГУ им. М.В. Ломоносова

Избранные публикации

  1. 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.

    ID:1259
  2. 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.

    ID:1093
  3. 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.

    ID:910
  4. 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.

    ID:911
  5. 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.

    ID:912
  6. 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.

    ID:913
  7. 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.

    ID:915
  8. 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.

    ID:917
  9. 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.

    ID:1088