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

  • Mark A. Rizzo (a1), Gerald Springer (a2), Katsuhisa Segawa (a3), Warren R. Zipfel (a4) and David W. Piston (a2)...
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.

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Corresponding author. E-mail: dave.piston@vanderbilt.edu
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Agronskaia, A.V., Tertoolen, L., & Gerritsen, H.C. (2004). Fast fluorescence lifetime imaging of calcium in living cells. J Biomed Opt9, 12301237.

Axelrod, D. (1979). Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J26, 557573.

Berney, C. & Danuser, G. (2003). FRET or no FRET: A quantitative comparison. Biophys J84, 39924010.

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 J71, 194208.

Brunsting, A. & Mullaney, P. (1974). Differential light scattering from spherical mammalian cells. Biophys J14, 439453.

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 J83, 16311649.

Clegg, R.M. (1992). Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol211, 353388.

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 Biol6, 557560.

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. Neuron31, 973985.

Gordon, G.W, Berry, G., Liang, X.H., Levine, B., & Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J74, 27022713.

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 Chem276, 2918829194.

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 Lett507, 1620.

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. Cytometry43, 248260.

Harpur, A.G., Wouters, F.S., & Bastiaens, P.I. (2001). Imaging FRET between spectrally similar GFP molecules in single cells. Nat Biotechnol19, 167169.

Heim, R. & Tsien, R.Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol6, 178182.

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 USA98, 24372442.

Hoppe, A., Christensen, K., & Swanson, J.A. (2002). Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J83, 36523664.

Jares-Erijman, E.A. & Jovin, T.M. (2003). FRET imaging. Nat Biotechnol21, 13871395.

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 J381, 307312.

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 Opt8, 362367.

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 Chem96, 30003006.

Mattheyses, A.L., Hoppe, A.D., & Axelrod, D. (2004). Polarized fluorescence resonance energy transfer microscopy. Biophys J87, 27872797.

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 Biotechnol20, 8790.

Nguyen, A.W. & Daugherty, P.S. (2005). Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol23, 355360.

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. Science273, 13921395.

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 J73, 27822790.

Patterson, G.H., Piston, D.W., & Barisas, B.G. (2000). Förster distances between green fluorescent protein pairs. Anal Biochem284, 438440.

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 USA97, 477482.

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 Biol9, 269272.

Rizzo, M.A. & Piston, D.W. (2005). High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. Biophys J88, L14L16.

Rizzo, M.A., Springer, G.H., Granada, B., & Piston, D.W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol22, 445449.

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 J84, 40784086.

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 Evol21, 841850.

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 Biotechnol22, 15671572.

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 USA99, 20142019.

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 USA98, 1500315008.

Van Rheenen, J., Langeslag, M., & Jalink, K. (2004). Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys J86, 25172529.

Xia, Z. & Liu, Y. (2001). Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J81, 23952402.

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. Science296, 913916.

Zal, T. & Gascoigne, N.R. (2004). Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J86, 39233939.

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 USA98, 1499715002.

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 Lett531, 245249.

Zipfel, W.R., Williams, R.M., & Webb, W.W. (2003). Nonlinear magic: Multiphoton microscopy in the biosciences. Nat Biotechnol21, 13691377.

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Microscopy and Microanalysis
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