Imaging Protein‐Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells

Joseph A. Brzostowski1, Tobias Meckel1, Jiang Hong1, Alice Chen1, Tian Jin1

1 NIH/NIAID—LIG Imaging Facility, Rockville, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.5
DOI:  10.1002/0471140864.ps1905s56
Online Posting Date:  April, 2009
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Abstract

This unit describes an acceptor‐sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end‐point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live‐cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided. Curr. Protoc. Protein Sci. 56:19.5.1‐19.5.12. © 2009 by John Wiley & Sons, Inc.

Keywords: FRET; sensitized emission; acceptor photobleaching; Zeiss LSM 510; N‐FRET; Cerulean; Venus

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • HEK 293 (ATCC # CRL‐1573)
  • Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
  • Fetal bovine serum (FBS; HyClone)
  • Phosphate buffered saline with (PBS++) and without (PBS) Ca2+ and Mg2+ (Invitrogen)
  • DNA plasmid constructs (pC5V, pC5A, pC17V, pC17A, pC32V, pC32A, and pV1)
  • Plasmid DNA isolation kit (Qiagen plasmid mini kit, Qiagen)
  • DNA transfection kit (Lipofectamine 2000, Invitrogen)
  • G418 (Sigma‐Aldrich)
  • Kanamycin (Sigma‐Aldrich)
  • Cells of interest
  • Untransfected cells to assess background autofluorescence
  • 25‐cm2 tissue culture dishes
  • 37°C, 5% CO 2 incubator
  • 6‐well culture plates (Nunc, Thermo Fisher Scientific)
  • Chambers for live‐cell imaging, e.g., coverglass‐bottomed Lab‐Tek II multi‐well chambers (Nunc); 35‐mm coverglass‐bottomed dishes (MatTek); or FCS2 environmental chamber (Bioptechs)
  • Zeiss LSM 510 META laser scanning confocal microscope or equivalent
  • Microscope environmental chamber to regulate temperature and CO 2
NOTE: CFP and YFP variants comprise the most widely used FRET pair in live cells. Alternative fluorophore considerations are discussed in Periasamy and Day ( ), Shaner et al. ( ), and Giepmans et al. ( ).
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Figures

Videos

Literature Cited

Literature Cited
   Becker, W. 2005. Advanced Time‐Correlated Single Photon Counting Techniques. Berlin, Springer.
   Förster, T. 1949. Experimental and theoretical investigation of the intermolecular transfer of electronic excitation energy. Z. Naturforsch. A 4:7.
   Giepmans, B.N., Adams, S.R., Ellisman, M.H., and Tsien, R.Y. 2006. The fluorescent toolbox for assessing protein location and function. Science 312:217‐224.
   Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702‐2713.
   Janetopoulos, C., Jin, T., and Devreotes, P. 2001. Receptor‐mediated activation of heterotrimeric G‐proteins in living cells. Science 291:2408‐2411.
   Koushik, S.V., Chen, H., Thaler, C., Puhl, H.L., and Vogel, S.S. 2006. Cerulean, Venus, and VenusY67C FRET reference standards. Biophys. J. 91:L99‐L101.
   Lakowicz, J.R. 1999. Principles of Fluorescence Spectroscopy, 2nd ed. Kluwer Academic/Plenum Publishers, New York.
   Lissandron, V., Terrin, A., Collini, M., D'alfonso, L., Chirico, G., Pantano, S., and Zaccolo, M. 2005. Improvement of a FRET‐based indicator for cAMP by linker design and stabilization of donor‐acceptor interaction. J. Mol. Biol. 354:546‐555.
   Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M., and Tsien, R.Y. 1997. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882‐887.
   Nagai, Y., Miyazaki, M., Aoki, R., Zama, T., Inouye, S., Hirose, K., Iino, M., and Hagiwara, M. 2000. A fluorescent indicator for visualizing cAMP‐induced phosphorylation in vivo. Nat. Biotechnol. 18:313‐316.
   Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell‐biological applications. Nat. Biotechnol. 20:87‐90
   Periasamy, A. and Day, R.N. eds. 2005. Molecular Imaging: FRET Microscopy and Spectroscopy. Oxford, Oxford University Press.
   Rizzo, M.A., Springer, G.H., Granada, B., and Piston, D.W. 2004. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22:445‐449.
   Shaner, N.C., Steinbach, P.A., and Tsien, R.Y. 2005. A guide to choosing fluorescent proteins. Nat. Methods 2:905‐909.
   Sohn, H.W., Tolar, P., Jin, T., and Pierce, S.K. 2006. Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling. Proc. Natl. Acad. Sci. U.S.A. 103:8143‐8148.
   Tolar, P., Sohn, H.W., and Pierce, S.K. 2005. The initiation of antigen‐induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer. Nat. Immunol. 6:1168‐1176.
   van Rheenen, J., Langeslag, M., and Jalink, K. 2004. Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys. J. 86:2517‐2529.
   Wouters, F.S. and Bastiaens, P.I.H. 2001. Imaging protein‐protein interactions by fluorescence energy transfer (FRET) microscopy. Curr. Protoc. Protein Sci. 23:19.5.1‐19.5.15.
   Xia, Z. and Liu, Y. 2001. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81:2395‐2402.
   Xu, X., Meier‐Schellersheim, M., Jiao, X., Nelson, L.E., and Jin, T. 2005. Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium. Mol. Biol. Cell 16:676‐688.
   Xu, X., Brzostowski, J.A., and Jin, T. 2006. Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol. Biol. 346:281‐296.
   Youvan, D.C., Silva, C.M., Bylina, E.J., Coleman, W.J., Dilworth, M.R., and Yang, M.M. 1997. Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnology et alia 3:18.
   Zal, T. and Gascoigne, N.R. 2004. Photobleaching‐corrected FRET efficiency imaging of live cells. Biophys. J. 86:3923‐3939.
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