Professor, Department of Anatomy & Structural Biology
Green fluorescent protein (GFP) from jellyfish and its fluorescent homologues from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Anthozoa GFP-like proteins are available in colors and with features distinct to those of GFP variants. Several Anthozoa GFP-like proteins have already been developed into biotechnological tools to study cancer, however, their photochemical and oligomeric properties limit their usefulness as molecular probes.
Our analysis and understanding of chromophore formation mechanisms and molecular color determinants suggest that monomeric proteins with novel spectral and photochemical features can be engineered. Utilizing existing and novel red-shifted fluorescent proteins and chromoproteins, our work focuses on developing three types of novel protein labels with applications to biomedical research. The first of which are photoactivatable fluorescent proteins (PAFPs), which are originally dark or fluoresce at one wavelength but become fluorescent at a different wavelength upon irradiation with a specific wavelength. Secondly, monomeric Fluorescent Timers (FTs) that change fluorescent color with time. Lastly, enhanced far-red and near-infrared fluorescent proteins (RFPs) designed for deep-tissue in vivo imaging.
Applications of PAFPs consist of pulse-chase photolabeling and subsequent tracking of cells, organelles and proteins. In contrast to the observation of fluorescently-tagged objects through constant imaging, PAFPs allow objects to be tracked without the need for continual visualization. This feature greatly extends the spatiotemporal limits for the studies of biological dynamics and reduces detrimental photobleaching and phototoxicity during imaging.
PAFPs provide a unique opportunity for non-invasive labeling and tracking of specific types of cells in living organisms and tissues. Obvious examples include the study of tumor formation, metastasis and embryogenesis, the migration of small parasites within a host and the taxis reactions of free unicellular organisms. Various cellular organelles can also be loaded with PAFPs using polypeptide targeting signals or through fusions with proteins with a specific subcellular localization. PAFPs provide the possibility to study the transport, fusion and fission events of individual organelles.
Perhaps the most important use of PAFPs is a kinetic characterization of unique proteins through protein photolabeling. When fused to a protein of interest, PAFPs can provide detailed information about protein localization, turnover, and the direction and rate of trafficking in a living cell. DNA and RNA molecules can also be labeled with PAFPs and tracked. Labeling in this manner involves interaction between a specific DNA/RNA-binding domain fused to a PAFP and the corresponding target sequence that can be introduced into the nucleic acid of interest.
In contrast to irreversibly photoactivatable probes, reversible PAFPs allow for repeated activation events and photolabeling of several subcellular regions one after another. These reversible PAFPs features should confer a novel approach to map protein trafficking pathways and reveal a “topography” of subcellular transportation and signaling.
1. Subach F.M. and Verkhusha V.V. Chromophore transformations in red fluorescent proteins. Chemical Reviews 2012, 112: 4308-4327.
2. Subach O.M., Entenberg D, Condeelis J.S., and Verkhusha V.V. A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics. J. Am. Chem. Soc. 2012, 134: 14789-14799.
3. Shcherbakova D.M., Hink M.A., Joosen L., Gadella T.W.J., and Verkhusha V.V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J. Am. Chem. Soc. 2012, 134: 7913-7923.
4. 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.
5. 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 Biotechnology 2011, 29: 757-761.
6. 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 Methods 2011, 8: 771-777.
7. Piatkevich K.D., Hulit J., Subach O.M., Wu B., Abdulla A., Segall J.E., and Verkhusha V.V. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. USA 2010, 107: 5369-5374.
8. Subach F.V., Malashkevich V.N., Zencheck W.D., Xiao H., Filonov G.S., Almo S.C., and Verkhusha V.V. Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc. Natl. Acad. Sci. USA 2009, 106: 21097-21102.
9. 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.
10. 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.
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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Ullmann Building, Room 1217
Bronx, NY 10461