Vladislav Verkhusha, Ph.D.

Non-invasive optical imaging, monitoring and manipulation of metabolic processes in living mammals is more feasible within the near-infrared (NIR) optical transparency window (650-900 nm) where hemoglobin and melanin absorbance significantly decreases, and water absorbance is still low. The most red-shifted fluorescent proteins (FPs) of the GFP-like family have excitation and emission spectra outside of the NIR region and suffer from low brightness and modest photostability. Natural bacterial phytochrome photoreceptors (BphPs) utilize an enzymatic product of heme, low-molecular-weight biliverdin, as a chromophore.

BphPs provide many advantages over other natural chromophore-containing proteins. Unlike the chromophores of non-bacterial phytochromes, biliverdin is ubiquitous in mammals. This makes BphP applications in mammalian cells, tissues and whole mammals as easy as conventional GFP-like FPs, without supplying chromophore through an external solution. BphPs exhibit NIR absorbance and fluorescence, which are red-shifted relative to that of any other phytochromes, and lie within the NIR optical window. This makes BphPs spectrally complementary to other existing optical probes and optogenetic tools based on the GFP, flavoprotein and rhodopsin-like protein families. Independent domain architecture and pronounced conformational changes upon biliverdin photoisomerization make BphPs attractive templates to design various photocontrollable genetically-encoded probes.

In our laboratory, we engineer new BphP-based FPs, biosensors and optogenetic tools. These include bright and spectrally resolvable permanently fluorescent NIR FPs, photoactivatable with non-phototoxic NIR light FPs, and reversibly photoswitchable FPs. We also focus on designing NIR reporters for protein interactions and biosensors for intracellular ions and metabolites. Lastly, we engineer BphPs into optogenetic elements allowing us to noninvasively regulate intracellular processes in vivo with NIR light.

We apply various directed protein evolution approaches based on rational structure-based design and random mutagenesis of template BphPs, high-throughput flow cytometry and multiwell plate spectroscopy. These conventional techniques allow screening for standard properties of genetically encoded probes, such as excitation and emission wavelengths, brightness, photostability, pH stability and folding efficiency. We also develop new protein engineering and high-throughput approaches to specifically optimize BphP-based constructs. These include time-resolved fluorescence lifetime measurements, expression in bacterial periplasmic space, screening of mutant libraries in yeast and in mammalian cells using shuttle vectors and inducible somatic hypermutations.

The resulting NIR probes, biosensors and molecular tools are tested in mouse models and applied to various in vivo studies. These NIR constructs extend optical methods to multicolor deep-tissue in vivo imaging, cell and tissue labeling, photoactivation and tracking, and detection of enzymatic activities and protein interactions in cells, tissues and whole mammals. The engineered NIR optogenetic tools allow light-manipulations of cellular processes directly through the skin of living animals.

1. Kasatkina L.A., Ma C., Sheng H., Lowerison M., Menozzi L., Baloban M., Tang Y., Xu Y., Humayun L., Humayun L., Vu T., Song P., Yao J. and Verkhusha V.V. Advanced deep-tissue imaging and optogenetic manipulation enabled by biliverdin reductase knockout. Nature Communications 2025, in press.
2. Leopold A.V. and Verkhusha V.V. Engineering signalling pathways in mammalian cells. Nature Biomedical Engineering 2024, 8: 1523-1539.
3. Barykina N.V., Carey E., Oliinyk O.S., Nimmerjahn A. and Verkhusha V.V. Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation. Nature Communications 2024, 15: 7788.
4. Oliinyk O.S., Ma C., Pletnev S., Baloban M., Taboada C., Sheng H., Yao. J. and Verkhusha V.V. Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein. Nature Methods 2023, 20: 70-74.
5. Pennacchietti F., Alvelid J., Morales R.A., Damenti M., Ollech D., Oliinyk O.S., Shcherbakova D.M., Villablanca E.J., Verkhusha V.V. and Testa I. Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms. Nature Communications 2023, 14: 8402.
6. Leopold AV, Thankachan S, Yang C, Gerashchenko D. and Verkhusha V.V. A general approach for engineering RTKs optically controlled with far-red light. Nature Methods 2022, 19: 871-880.
7. Kasatkina L.A., Ma C., Matlashov M.E., Vu T., Li M., Kaberniuk A.A., Yao J. and Verkhusha V.V. Optogenetic manipulation and photoacoustic imaging using a near-infrared transgenic mouse model. Nature Communications 2022, 13: 2813.
8. Oliinyk O.S., Baloban M., Clark C.L., Carey E., Pletnev S., Nimmerjahn A. and Verkhusha V.V. Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies. Nature Methods 2022, 19: 740-750.
9. Shemetov A.A., Monakhov M.V., Zhang Q., Canton-Josh J.E., Kumar M., Chen M., Matlashov M.M., Li R., Yang W., Nie L., Shcherbakova D.M., Kozorovitskiy Ye., Yao J., Ji N. and Verkhusha V.V. A near-infrared genetically encoded calcium indicator for in vivo imaging. Nature Biotechnology 2021, 39: 368-377.
10. Kaberniuk A.A., Baloban M., Monakhov M.V., Shcherbakova D.M. and Verkhusha V.V. Single-component near-infrared optogenetic systems for gene transcription regulation. Nature Communications 2021, 12: 3859.
11. Manoilov K.Y., Verkhusha V.V., and Shcherbakova D.M. A guide to the optogenetic regulation of endogenous molecules. Nature Methods 2021, 18: 1027–1037.
12. Redchuk T.A., Karasev M.M., Donnelly S.K., Hülsemann M., Virtanen J., Moore H.M., Vartiainen M.K., Hodgson L. and Verkhusha V.V. Optogenetic regulation of endogenous proteins. Nature Communications 2020, 11: 605.
13. Matlashov M.E., Shcherbakova D.M., Alvelid J., Baloban M., Pennacchietti F., Shemetov A.A., Testa I. and Verkhusha V.V. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nature Communications 2020, 11: 239.
14. Leopold A.V., Chernov K.G., Shemetov A.A. and Verkhusha V.V. Neurotrophin receptor tyrosine kinases regulated with near-infrared light. Nature Communications 2019, 10: 1129.
15. Oliinyk O.S., Shemetov A.A., Pletnev S., Shcherbakova D.M. and Verkhusha V.V. Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as a versatile tag for spectral multiplexing. Nature Communications 2019, 10: 279.
16. Shcherbakova D.M., Cammer N.C., Huisman T.M., Verkhusha V.V. and Hodgson L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nature Chemical Biology 2018, 14: 591-600.
17. Pennacchietti F., Serebrovskaya E.O., Faro A.R., Irina I. Shemyakina I.I., Bozhanova N.G., Kotlobay A.A., Gurskaya N.G., Boden A., Dreier J., Chudakov D.M., Lukyanov K.A., Verkhusha V.V., Mishin A.S., and Testa I. Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy. Nature Methods 2018, 15: 601-604.
18. Li L., Shemetov A.A., Baloban M., Hu P., Zhu L., Shcherbakova D.M., Zhang R., Shi J., Yao J., Wang L.V. and Verkhusha V.V. Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo. Nature Communications 2018, 9: 2734.
19. Redchuk T.A., Kaberniuk A.A. and Verkhusha V.V. Near-infrared light-controlled systems for gene transcription regulation, protein targeting and spectral multiplexing. Nature Protocols 2018, 13: 1121-1136.
20. Redchuk T.A., Omelina E.S., Chernov K.G. and Verkhusha V.V. Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nature Chemical Biology 2017, 13: 633-639.
21. Fluegen G., Avivar-Valderas A., Wang Y., Padgen M.R., Williams J.K., Verkhusha V., Cheung J.F., Entenberg D., Castracane J., Keely P., Condeelis J. and Aguirre-Ghiso J. Phenotypic heterogeneity of disseminated tumor cells is preset by primary tumor hypoxic microenvironments. Nature Cell Biology 2017,19: 120-132.
22. Yao J., Kaberniuk A.A., Li L., Shcherbakova D.M., Zhang R., Wang L., Li G., Verkhusha V.V.* and Wang L.H.* Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe. Nature Methods 2016, 13: 67-73. *Co-corresponding authors
23. Shcherbakova D.M., Baloban M., Emelyanov A.V., Brenowitz M., Guo P. and Verkhusha V.V. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nature Communications 2016, 7: 12405.
24. Kaberniuk A.A., Shemetov A.A., and Verkhusha V.V. A bacterial phytochrome-based optogenetic system controllable with near-infrared light. Nature Methods 2016, 13: 591-597.
25. Costantini L.M., Baloban M., Markwardt M.L., Rizzo M., Guo F., Verkhusha V.V. and Snapp E.L. A palette of fluorescent proteins optimized for diverse cellular environments. Nature Communications 2015, 6: 7670.
26. Shcherbakova D.M. and Verkhusha V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 2013, 10: 751-754.
27. Piatkevich, K.D., Subach F.V., and Verkhusha V.V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nature Communications 2013, 4: 2153.
28. Subach O.M., Patterson G.H., Ting L.-M., Wang Y., Condeelis J.S. and Verkhusha V.V. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nature Methods2011, 8: 771-777.
29. Koga H., Martinez-Vicente M., Macian F., Verkhusha V.V., and Cuervo A.M. A photoconvertible fluorescent reporter to track chaperone-mediated autophagy. Nature Communications 2011, 2: 386. 
30. Filonov G.S., Piatkevich K.D., Ting L.-M., Zhang J., Kim K. and Verkhusha V.V. Bright and stable near infra-red fluorescent protein for in vivo imaging.  Nature Biotechnology2011, 29: 757-761.
31. Subach F.V., Piatkevich K.D. and Verkhusha V.V. Directed molecular evolution to design advanced red fluorescent proteins. Nature Methods 2011, 8: 1019-1026.
32. Bogdanov A., Mishin A., Yampolsky I., Belousov V., Chudakov D., Subach F., Verkhusha V.V. and Lukyanov K.A. Green fluorescent proteins are light-induced electron donors. Nature Chemical Biology 2009, 5: 459-461.
33. Subach F.V., Patterson G.H., Manley S., Gillette J.M., Lippincott-Schwartz J. and Verkhusha V.V. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nature Methods 2009, 6: 153-159.
34. Gould T.J., Verkhusha V.V. and Hess S.T. Imaging biological structures with fluorescence photoactivation localization microscopy. Nature Protocols 2009, 4: 291-308.
35. Subach F.V., Subach O.M., Gundorov I.S., Morozova K.S., Piatkevich K.D., Cuervo A.M. and Verkhusha V.V. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nature Chemical Biology 2009, 5: 118-126.
36. Kedrin D., Gligorijevic B., Wyckoff J., Verkhusha V.V., Condeelis J., Segall J.E. and van Rheenen J. Intravital imaging of metastatic behavior through a mammary imaging window. Nature Methods 2008, 5: 1019-1021.
37. Gould T.J., Gunawardene M.S., Gudheti M.V., Verkhusha V.V., Yin S.R., Gosse J.A. and Hess S.T. Nanoscale imaging of molecular positions and anisotropies. Nature Methods 2008, 5: 1027-1030.
38. Kapoor V., Subach F.V., Kozlov V.G., Grudinin A., Verkhusha V.V.* and Telford W.G.* New lasers for flow cytometry: filling the gaps. Nature Methods 2007, 4: 678-679. *Co-corresponding authors
39. Pena P.V., Davrazou F., Shi X., Walter K., Verkhusha V.V., Gozani O., Zhao R. and Kutateladze T.G. Molecular mechanism of histone H3K4Me3 recognition by plant homeodomain of ING2. Nature 2006, 442: 100-103.
40. Gurskaya N.G.,# Verkhusha V.V.,# Shcheglov A.S., Staroverov D.B., Chepurnykh T.V., Fradkov A.F., Lukyanov S. and Lukyanov K.A. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology 2006, 24: 461-465. #Co-first authors
41. Lukyanov K.A., Chudakov D.M., Lukyanov S. and Verkhusha V.V. Photoactivatable fluorescent proteins. Nature Reviews Molecular Cell Biology 2005, 6: 885-891.
42. Chudakov D.M.#, Verkhusha V.V.#, Staroverov D.B., Lukyanov S. and Lukyanov K.A. Photoswitchable fluorescent label for protein tracking. Nature Biotechnology 2004, 22: 1435-1439. #Co-first authors
43. Galperin E.#, Verkhusha V.V.# and Sorkin A. Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells. Nature Methods 2004, 1: 209-217.  #Co-first authors
44. Verkhusha V.V.* and Lukyanov K.A.* The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nature Biotechnology 2004, 22: 289-296. #Co-corresponding authors