Detecting Protein‐Protein Interactions In Vivo with FRET using Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM)

David Llères1, Samuel Swift1, Angus I. Lamond1

1 Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, United Kingdom
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.10
DOI:  10.1002/0471142956.cy1210s42
Online Posting Date:  October, 2007
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Abstract

Protein interactions are critical for many processes in mammalian cells. Such interactions include the stable association of proteins within multi‐subunit complexes and the transient association of regulatory proteins. Information about protein interactions in cells has previously come from either in vitro analyses using recombinant expressed proteins, or from yeast 2–hybrid studies. A limitation of this approach is that the protein interaction is studied in isolation, without regard to the many competing protein interactions that can occur within cells. This unit presents a light microscopy approach for detecting protein‐protein interactions in vivo based on the measurement of FRET using the multiphoton fluorescence lifetime imaging microscopy (FLIM) technique. By using the FLIM‐FRET technique, the spatial organization and quantification of such interactions in a living cell can be characterized. A detailed protocol describing the complete microscope procedure and the choice of the appropriate experimental controls as well as the FRET calculations is also included. Curr. Protocol. Cytom. 42:12.10.1‐12.10.19. © 2007 by John Wiley & Sons, Inc.

Keywords: fluorescence lifetime; GFP; mCherry; FRET; FLIM; multiphoton; protein interaction

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measuring FLIM‐FRET in Live Mammalian Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring FLIM‐FRET in Live Mammalian Cells

  Materials
  • Mammalian cultured cell lines (e.g., HeLa; ATCC #CCL‐2)
  • Enhanced GFP‐C1 (EGFP‐C1; Clontech), monomeric Cherry red variant (mCherry‐C1; a gift from R.Y. Tsien at http://www.tsienlab.ucsd.edu; also available from Clontech; Shaner et al., )
  • Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen Life Technologies; also see appendix 3B) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 100 U/ml penicillin‐streptomycin (Invitrogen)
  • 20 mM HEPES
  • CO 2‐independent phenol red–free DMEM medium (Invitrogen)
  • Effectene transfection reagent (Qiagen)
  • 35‐mm glass‐bottom dish (e.g., WillCo‐dish, Intracel)
  • 35‐mm coverslips
  • Laser scanning confocal microscope (e.g., Bio‐Rad Radiance 2100MP or similar system) with:
    • Argon ion laser (488‐nm laser line)
    • Green HeNe laser (543‐nm laser line)
    • Band‐pass emission filter 528/50 for EGFP
    • 570‐nm long‐pass emission filter for mCherry
    • Photomultiplier tube (PMTs)
    • Acquisition software (e.g., LaserSharp2000, Zeiss)
  • Multiphoton excitation laser, e.g., Coherent Chameleon diode‐pumped laser, 720‐930 nm, Verdi‐pumped ultrafast laser that produces modelocked, sub‐200‐fsec pulses at 90 MHz repetition rate with an output power of ∼1.4 W at the peak of the tuning curve (800 nm)
    • Dichroic filter 560 LP
    • Band‐pass emission filter 528/50 for EGFP
    • Two‐channel direct detectors suitable for lifetime imaging e.g., Hammamatsu 5783P
  • Fluoresence lifetime imaging system consisting of:
    • Black‐walled environmental chamber (e.g., Solent Scientific)
    • Two‐channel direct detectors (e.g., Hammamatsu 5783P) with a fast response for FLIM
    • TCSPC acquisition card SPCM/SPC830 and software for time‐correlated single‐photon counting (Becker & Hickl), or comparable software enabling time‐correlated single‐photon counting
    • Imaging software, e.g., SPCImage software (Becker & Hickl)
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Figures

Videos

Literature Cited

Literature Cited
   Becker, W. 2005. The BH TCSPC Handbook, Becker and Hickl GmbH.
   Chusainow, J., Ajuh, P.M., Trinkle‐Mulcahy, L., Sleeman, J.E., Ellenberg, J., and Lamond, A.I. 2005. FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self‐interaction of U2AF35. RNA 11:1201‐1214.
   Dittrich, P.S. and Schwille, P. 2001. Photobleaching and stabilization of fluorophores used for single‐molecule analysis with one‐ and two‐photon excitation. Appl. Phys. B 73:829‐837.
   Gratton, E., Breusegem, S., Sutin, J., Ruan, Q., and Barry, N. 2003. Fluorescence lifetime imaging for the two‐photon microscope: Time‐domain and frequency‐domain methods. J. Biomed. Opt. 8:381‐390.
   Herman, B. 1989. Resonance energy transfer microscopy. Methods Cell Biol. 30:219‐243.
   Karpova, T.S., Baumann, C.T., He, L., Wu, X., Grammer, A., Lipsky, P., Hager, G.L., and McNally, J.G. 2003. Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J. Microsc. 209:56‐70.
   Köllner, M. and Wolfrum, J. 1992. How many photons are necessary for fluorescence‐lifetime measurements? Phys. Chem. Lett. 200:199‐204.
   Lakowicz, J.R. 1999. Principles of Fluorescence Spectrocopy 2nd editon. Springer, New York.
   Miyawaki, A. 2003. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4:295‐305.
   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.
   Patterson, G.H., Piston, D.W., and Barisas, B.G. 2000. Forster distances between green fluorescent protein pairs. Anal. Biochem. 284:438‐440.
   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.
   Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Bio. 37:1.8.1‐1.8.10.
   Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22:1567‐1572.
   Tramier, M., Zahid, M., Mevel, J.C., Masse, M.J., and Coppey‐Moisan, M. 2006. Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsc. Res. Tech. 69:933‐939.
   Trinkle‐Mulcahy, L., Chusainow, J., Lam, Y.W., Swift, S., and Lamond, A. 2006. Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions. Methods Mol. Biol. 365:133‐154.
   Wallrabe, H. and Periasamy, A. 2005. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotechnol. 16:19‐27.
   Yasuda, R., Harvey, C.D., Zhong, H., Sobczyk, A., van Aelst, L., and Svoboda, K. 2006. Supersensitive Ras activation in dendrites and spines revealed by two‐photon fluorescence lifetime imaging. Nat. Neurosci. 9:283‐291.
   Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science 296:913‐916.
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