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Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy

Fred S. Wouters1,  Philippe I.H. Bastiaens2

1Imperial Cancer Research Fund, London, United Kingdom, UK
2European Molecular Biology Laboratory, Heidelberg, Germany


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 17.1
DOI: 
10.1002/0471143030.cb1701s07
Online Posting Date: 
May, 2001
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Abstract

FRET microscopy enables the detection of different biochemical states of proteins in cells. The use of fluorescence in the detection of proteins, by chemical modification, by immunofluorescence, or by genetic encoding of a green fluorescent protein fusion protein, provides more information than just the location of the protein in the cell. The properties of the fluorophore can be exploited to extract information on protein-protein interactions. A straightforward, quantitative imaging approach is presented to measure FRET that is based on internal calibration by acceptor photobleaching.

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol: FRET Microscopy of Fixed Cells
  • Support Protocol 1: Nuclear and Cytosolic Microinjection
  • Support Protocol 2: Protein Labeling with Cy3
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol: FRET Microscopy of Fixed Cells

 Materials
  • Cells of interest
  • Plasmid for GFP-tagged protein
  • Transfection reagent (e.g., Fugene 5 from Boehringer Mannheim, Lipofectin from Life Technologies, Effectene from Qiagen, or Superfect from Qiagen)
  • Serum-free medium
  • Low-background fluorescence CO2-independent medium (Life Technologies, or see recipe)
  • Phosphate-buffered saline (PBS; see recipe), pH 7.4
  • 4% (w/v) formaldehyde fixative solution (see recipe)
  • Quench solution: 50 mM Tris×Cl (pH 8.0)/100 mM NaCl (appendix 2A)
  • 0.1% (v/v) Triton X-100 in PBS
  • Antibody (e.g., PY72 monoclonal anti-phosphotyrosine antibody) labeled with Cy3 (see Support Protocol 2)
  • 1% (w/v) bovine serum albumin (BSA, fraction V) in PBS
  • Mowiol mounting medium (see recipe)
  • 6- and 12-well tissue culture plates
  • Coverslips
  • Microscope slides
  • Confocal laser scanning microscope (e.g., Zeiss LSM 510), equipped with argon (488 nm) and He/Ne (543 nm) lasers selected by the HFT 488/543 double dichroic filter, GFP fluorescence selected by the NFT 545 dichroic and BP 505-530 emission filter, and Cy3 fluorescence selected by the LP560 emission filter
  • Imaging software package (e.g., NIH-image or IPLab Spectrum from Scanalytics)
  • Additional reagents and equipment for transfection of mammalian cells (appendix 3A)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.

NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.


Support Protocol 1: Nuclear and Cytosolic Microinjection

 Materials
  • Cells of interest, cultured in MatTek glass-bottom 35-mm dishes (MatTek)
  • DNA (e.g., human EGFR cDNA in the Clontech pEGFP-N1 expression vector; appendix 3A)
  • HPLC-grade water
  • Millex-GV4 0.22-µm filtration unit
  • GELloader tips (Eppendorf)
  • Needles for microinjection (e.g., Femtotip from Eppendorf)
  • Microinjector (e.g., Eppendorf model 5244)
  • Micromanipulator (e.g., Eppendorf model 5170)
  • Inverted microscope with 10× and 40× air objectives
  • Additional reagents and equipment for preparation of DNA (appendix 3A)

Support Protocol 2: Protein Labeling with Cy3

 Materials
  • Antibody (PY72 monoclonal anti-phosphotyrosine antibody)
  • 1 M Tris×Cl, pH 8.0 (appendix 2A)
  • 10 mM and 100 mM Bicine/NaOH, pH 8.0
  • 100 ml citric acid/NaOH, pH 2.8
  • 1 M Bicine/NaOH, pH 9.0
  • 1 M NaCl (appendix 2A)
  • Labeling buffer: 100 mM Bicine/NaOH (pH 8.0)/100 mM NaCl
  • Cy3.29–OSu monofunctional sulfoindocyanine succinimide ester (Amersham Pharmacia Biotech)
  • Dimethylformamide (DMF) dried by addition of 10 to 20 mesh 3-Å pore diameter molecular sieve dehydrate (Fluka)
  • 1-ml Protein G HiTrap columns (Amersham Pharmacia Biotech)
  • Centricon YM30 concentrators (Amicon)
  • Biogel P6DG Econopac prepacked size-exclusion columns (5.5 × 1.5–cm, ~10 ml; Bio-Rad)
  • 1-ml and 10-ml syringes with HPLC Luer-Lok fitted tubing
  • Additional reagents and equipment for spectrophotometric protein determination (appendix 3B)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 17.1.1
    (A) Cy3-PY72 photobleaching releases FRET-quenched EGFR-GFP emission. The histogram shows the distribution of calculated FRET efficiencies in the cell. (B) FRET measured by fluorescence lifetime imaging microscopy. The histogram shows the distribution of measured lifetime (nsec) and corresponding calculated FRET efficiencies prior and after photobleaching of the acceptor.

    View Image

Literature Cited

Literature Cited
    Adams, S.R., Harootunian, A.T., Buechler, J., Taylor, S.S., and Tsien, R.Y. 1991. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694-697.
    Bastiaens, P.I.H. and Jovin, T.M. 1996. Microspectroscopic imaging tracks the intracellular processing of a signal-transduction protein: Fluorescent labeled protein kinase C beta I. Proc. Natl. Acad. Sci. U.S.A. 93:8407-8412.
    Bastiaens, P.I.H. and Jovin, T.M. 1998. Fluorescence resonance energy transfer microscopy. In Cell Biology a Laboratory Handbook, Vol. 3 (J.E. Celis, ed.), pp.136-146. Academic Press, New York.
    Bastiaens, P.I.H. and Squire, A. 1999. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9:48-52.
    Bastiaens, P.I.H., Majoul, I.V., Verveer, P.J., Soling, H.D., and Jovin, T.M. 1996. Imaging the intracellular trafficking and state of the AB(5) quaternary structure of cholera-toxin. EMBO J. 15:4246-4253.
    Clegg, R.M. 1996. Fluorescence resonance energy transfer spectroscopy and microscopy. In Fluorescence Imaging Spectroscopy and Microscopy. (X.F. Wang and B. Herman eds.) pp.179-251. John Wiley & Sons, New York.
    Day, R.N. 1998. Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Mol. Endocrinol. 12:1410-1419.
    Förster, T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys. 2:55-75.
    Gadella, T.W.J. and Jovin, T.M. 1995. Oligomerization of epidermal growth-factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy—a stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129:1543-1558.
    Gadella, T.W.J., Jovin, T.M., and Clegg, R.M. 1993. Fluorescence lifetime imaging microscopy (FLIM)—spatial resolution of microstructures on the nanosecond time-scale. Biophys. Chem. 48:221-239.
    Gordon, G.W., Berry, G., Huan Liang, X., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702-2713.
    Lakowicz, J.R. and Berndt, K., 1991. Lifetime-selective fluorescence imaging using an rf phase-sensitive camera. Rev. Sci. Instrum. 62:1727-1734.
    Mahajan, N.P., Linder, K., Berry, G., Gordon, G.W., Heim, R., and Herman, B. 1998. Bcl-2 and bax interactions in mitochondria probed with green fluorescent protein and fluorescence energy transfer. Nature Biotechnol. 16:547-552.
    Matz, M.V., Fradkov, A.F., Labasv, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., and Lukyanov, S.A. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol. 17:969-973.
    Mittman, S., Flaming, D.G., Copenhagen, D.R., and Belgum, J.H., 1987. Bubble pressure measurement of micropipette tip outer diameter. J. Neurosci. Methods 22:161-166.
    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.
    Ng, T., Squire, A., Hansra, G., Bornancin, F., Prevostel, C., Hanby, A., Harris, W., Barnes, D., Schmidt, S., Mellor, H., Bastiaens, P.I.H., and Parker, P.J. 1999. Imaging protein kinase C alpha activation in cells. Science 283:2085-2089.
    Squire, A. and Bastiaens, P.I.H. 1999. Three dimensional image restoration in fluorescence lifetime imaging microscopy. J. Microsc. 193:36-49.
    Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 76:509-538.
    Wouters, F.S. and Bastiaens, P.I.H. 1999. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Biol. 9:1127-1130.
    Wouters, F.S., Bastiaens, P.I.H., Wirtz, K.W.A., and Jovin, T.M. 1998. FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO J. 17:7179-7189.
     
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