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.
- 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).
|Name||Position||Dmitry S. Bilan||PhD firstname.lastname@example.org||Julija A. Bogdanova||PhD email@example.com||Yuliya G. Ermakova||PhD firstname.lastname@example.org||Andrew Y. Gorokhovatsky, ph. d.||r. email@example.com||Ilya . Kelmanson, ph. d.||r. firstname.lastname@example.org||Elena I. Kudryavtseva, ph. d.||r. email@example.com||Kseniya N. Markvicheva, ph. d.||j. r. firstname.lastname@example.org||Mikhail E. Matlashov||PhD email@example.com||Nataliya M. Mishina, ph. d.||r. firstname.lastname@example.org||Arina . Shokhina||PhD email@example.com|