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

  • 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; 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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Literature Cited

Literature Cited
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