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Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins

Published online by Cambridge University Press:  16 May 2006

Mark A. Rizzo ,
Gerald Springer ,
Katsuhisa Segawa ,
Warren R. Zipfel  and
David W. Piston
Mark A. Rizzo
Affiliation:
University of Maryland School of Medicine, Baltimore, MD 21201, USA
Gerald Springer
Affiliation:
Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA
Katsuhisa Segawa
Affiliation:
3-204 Murayamadainijutaku, 1-2-46 Fujimicho Higashimurayama-shi, Tokyo 189-0024, Japan
Warren R. Zipfel
Affiliation:
Applied & Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, NY 14853, USA
David W. Piston
Affiliation:
Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA
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Abstract

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.Note: M. Rizzo and K. Segawa performed this research at Vanderbilt University (same address as Piston). W. Zipfel performed research at Cornell University.

Keywords

FRETGFPtwo-photon microscopyconfocal microscopyfluorescence spectral imaginganisotropylifetime imaging
Type
BIOLOGICAL APPLICATIONS
Information
Microscopy and Microanalysis , Volume 12 , Issue 3 , June 2006 , pp. 238 - 254
Copyright
© 2006 Microscopy Society of America

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References

REFERENCES

Agronskaia, A.V., Tertoolen, L., & Gerritsen, H.C. (2004). Fast fluorescence lifetime imaging of calcium in living cells. J Biomed Opt 9, 1230–1237.Google Scholar
Axelrod, D. (1979). Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J 26, 557–573.Google Scholar
Berney, C. & Danuser, G. (2003). FRET or no FRET: A quantitative comparison. Biophys J 84, 3992–4010.Google Scholar
Blackman, S.M., Cobb, C.E., Beth, A.H., & Piston, D.W. (1996). The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy. Biophys J 71, 194–208.Google Scholar
Brunsting, A. & Mullaney, P. (1974). Differential light scattering from spherical mammalian cells. Biophys J 14, 439–453.Google Scholar
Clayton, A.H.A., Hanley, Q.S., Arndt-Jovin, D.J., Subramaniam, V., & Jovin, T.M. (2002). Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). Biophys J 83, 1631–1649.Google Scholar
Clegg, R.M. (1992). Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol 211, 353–388.Google Scholar
Creemers, T.M., Lock, A.J., Subramaniam, V., Jovin, T.M., & Volker, S. (1999). Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning. Nat Struct Biol 6, 557–560.Google Scholar
Erickson, M.G., Alseikhan, B.A., Peterson, B.Z., & Yue, D.T. (2001). Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells. Neuron 31, 973–985.Google Scholar
Förster, T. (1948). Zwischenmolckulare energiewanderung und fluoreszenz. Ann Phys 2, 55–77.Google Scholar
Gordon, G.W, Berry, G., Liang, X.H., Levine, B., & Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74, 2702–2713.Google Scholar
Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A., & Tsien, R.Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276, 29188–29194.Google Scholar
Gurskaya, N.G., Fradkov, A.F., Terskikh, A., Matz, M.V., Labas, Y.A., Martynov, V.I., Yanushevich, Y.G., Lukyanov, K.A., & Lukyanov, S.A. (2001). GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett 507, 16–20.Google Scholar
Hanley, Q.S., Subramaniam, V., Arndt-Jovin, D.J., & Jovin, T.M. (2001). Fluorescence lifetime imaging: Multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry 43, 248–260.Google Scholar
Harpur, A.G., Wouters, F.S., & Bastiaens, P.I. (2001). Imaging FRET between spectrally similar GFP molecules in single cells. Nat Biotechnol 19, 167–169.Google Scholar
Heim, R. & Tsien, R.Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6, 178–182.Google Scholar
Honda, A., Adams, S.R., Sawyer, C.L., Lev-Ram, V., Tsien, R.Y., & Dostmann, W.R. (2001). Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA 98, 2437–2442.Google Scholar
Hoppe, A., Christensen, K., & Swanson, J.A. (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83, 3652–3664.Google Scholar
Jares-Erijman, E.A. & Jovin, T.M. (2003). FRET imaging. Nat Biotechnol 21, 1387–1395.Google Scholar
Karasawa, S., Araki, T., Nagai, T., Mizuno, H., & Miyawaki, A. (2004). Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381, 307–312.Google Scholar
Krishnan, R.V., Masuda, A., Centonze, V.E., & Herman, B. (2003). Quantitative imaging of protein–protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera. J Biomed Opt 8, 362–367.Google Scholar
Lakowicz, J.R. (1999). Fluorescence anisotropy. In Principles of Fluorescence Spectroscopy, 2nd ed. pp. 291–317. New York: Kulwer Academic/Plenum.
Lakowicz, J.R., Gryczynski, I., Gryczynski, Z., Danielsen, E., & Wirth, M.J. (1992). Time-resolved fluorescence intensity and anisotropy decays of 2,5-diphenyloxazole by two-photon excitation and frequency-domain fluorometry. J Phys Chem 96, 3000–3006.Google Scholar
Mattheyses, A.L., Hoppe, A.D., & Axelrod, D. (2004). Polarized fluorescence resonance energy transfer microscopy. Biophys J 87, 2787–2797.Google Scholar
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M., & Tsien, R.Y. (1997). Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.Google Scholar
Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20, 87–90.Google Scholar
Nguyen, A.W. & Daugherty, P.S. (2005). Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23, 355–360.Google Scholar
Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., & Remington, S.J. (1996). Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.Google Scholar
Patterson, G., Day, R.N., & Piston, D. (2001). Fluorescent protein spectra. J Cell Sci 114, 837–838.Google Scholar
Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R., & Piston, D.W. (1997). Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73, 2782–2790.Google Scholar
Patterson, G.H., Piston, D.W., & Barisas, B.G. (2000). Förster distances between green fluorescent protein pairs. Anal Biochem 284, 438–440.Google Scholar
Pearce, L.L., Gandley, R.E., Han, W., Wasserloos, K., Stitt, M., Kanai, A.J., McLaughlin, M.K., Pitt, B.R., & Levitan, E.S. (2000). Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci USA 97, 477–482.Google Scholar
Pepperkok, R., Squire, A., Geley, S., & Bastiaens, P.I. (1999). Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol 9, 269–272.Google Scholar
Rizzo, M.A. & Piston, D.W. (2005). High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. Biophys J 88, L14–L16.Google Scholar
Rizzo, M.A., Springer, G.H., Granada, B., & Piston, D.W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22, 445–449.Google Scholar
Rocheleau, J.V., Edidin, M., & Piston, D.W. (2003). Intrasequence GFP in class I MHC molecules, a rigid probe for fluorescence anisotropy measurements of the membrane environment. Biophys J 84, 4078–4086.Google Scholar
Shagin, D.A., Barsova, E.V., Yanushevich, Y.G., Fradkov, A.F., Lukyanov, K.A., Labas, Y.A., Semenova, T.N., Ugalde, J.A., Meyers, A., Nunez, J.M., Widder, E.A., Lukyanov, S.A., & Matz, M.V. (2004). GFP-like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity. Mol Biol Evol 21, 841–850.Google Scholar
Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., & Tsien, R.Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572.Google Scholar
Swedlow, J.R., Hu, K., Andrews, P.D., Roos, D.S., & Murray, J.M. (2002). Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy. Proc Natl Acad Sci USA 99, 2014–2019.Google Scholar
Ting, A.Y., Kain, K.H., Klemke, R.L., & Tsien, R.Y. (2001). Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci USA 98, 15003–15008.Google Scholar
Van Rheenen, J., Langeslag, M., & Jalink, K. (2004). Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys J 86, 2517–2529.Google Scholar
Wall, M.A., Socolich, M., & Ranganathan, R. (2000). The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Struct Biol 7, 1133–1138.Google Scholar
Xia, Z. & Liu, Y. (2001). Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81, 2395–2402.Google Scholar
Youvan, D.C., Silva, C.M., Bylina, E.J., Coleman, W.J., Dilworth, M.R., & Yang, M.M. (1997). Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnology 3, 1–18.Google Scholar
Zacharias, D.A., Violin, J.D., Newton, A.C., & Tsien, R.Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916.Google Scholar
Zal, T. & Gascoigne, N.R. (2004). Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86, 3923–3939.Google Scholar
Zhang, J., Ma, Y., Taylor, S.S., & Tsien, R.Y. (2001). Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 98, 14997–15002.Google Scholar
Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., & Pepperkok, R. (2002). Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett 531, 245–249.Google Scholar
Zipfel, W.R., Williams, R.M., & Webb, W.W. (2003). Nonlinear magic: Multiphoton microscopy in the biosciences. Nat Biotechnol 21, 1369–1377.Google Scholar