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Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

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Abstract

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

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References

  • Bastiaens PIH, Jovin TM (1996) Microscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C βI. Proc Natl Acad Sci USA 93:8407–8412

    Article  CAS  PubMed  Google Scholar 

  • Bastiaens PIH, Majoul IV, Verveer PJ, Soling HD, Jovin TM (1996) Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. EMBO J 15:4246–4253

    CAS  PubMed  Google Scholar 

  • Berney C, Danuser G (2003) FRET or no FRET: a quantitative comparison. Biophys J 84:3992–4010

    CAS  PubMed  Google Scholar 

  • Chan SS, Arndt-Jovin D, Jovin TM (1979) Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system. J Histochem Cytochem 27:56–64

    CAS  PubMed  Google Scholar 

  • Clegg RM, Murchie AI, Zechel A, Lilley DM (1993) Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proc Natl Acad Sci USA 90:2994–2998

    CAS  PubMed  Google Scholar 

  • Cohen-Kashi M, Moshikov S, Zurgil N, Deutsch M (2002) Fluorescence resonance energy transfers measurements on cell surfaces via fluorescence polarization. Biophys J 83:1395–1402

    CAS  PubMed  Google Scholar 

  • dos Remedios CG, Moens PD (1995) Fluorescence resonance energy transfer spectroscopy is a reliable “ruler” for measuring structural changes in proteins: dispelling the problem of the unknown orientation factor. J Struct Biol 115:175–185

    Article  PubMed  Google Scholar 

  • Feller M, Richardson C, Behnke WD, Gruenstein E (1977) High and low affinity binding sites for concanavalin A on normal human fibroblasts in vitro. Biochem Biophys Res Commun 76(4):1027–1035

    CAS  PubMed  Google Scholar 

  • Fernandez SM, Berlin RD (1976) Cell surface distribution of lectin receptors determined by resonance energy transfer. Nature 264:411–415

    CAS  PubMed  Google Scholar 

  • Gadella TW Jr, Jovin TM (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

    Article  CAS  PubMed  Google Scholar 

  • Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702–2713

    CAS  PubMed  Google Scholar 

  • Hirschfeld T (1976) Quantum efficiency independence of the time-integrated emission from a fluorescent molecule. Appl Opt 15:3135–3139

    CAS  Google Scholar 

  • Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83:3652–3664

    CAS  PubMed  Google Scholar 

  • Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395

    Article  CAS  PubMed  Google Scholar 

  • Jovin TM (2003) Quantum dots finally come of age. Nat Biotechnol 21:32–33

    Article  CAS  PubMed  Google Scholar 

  • Jovin TM, Arndt-Jovin DJ (1989) FRET microscopy: digital imaging of fluorescence resonance energy transfer. Application in cell biology. In: Kohen E, Hirschberg JG, Ploem JS (eds) Cell structure and function by microspectrofluorometry. Academic Press, London, pp 99–117

  • Kenworthy AK, Edidin M (1998) Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 Å using imaging fluorescence resonance energy transfer. J Cell Biol 142:69–84

    Article  CAS  PubMed  Google Scholar 

  • Klostermeier D, Millar DP (2001–2002) Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids. Biopolymers 61:159–179

    Article  CAS  PubMed  Google Scholar 

  • Kubitscheck U, Schweitzer-Steiner R, Arndt-Jovin DJ, Jovin TM, Pecht I (1993) Distribution of type I Fcε-receptors on the surface of mast cells probed by fluorescence resonance energy transfer. Biophys J 64:110–120

    CAS  PubMed  Google Scholar 

  • Mekler VM, Averbakh AZ, Sudarikov A, Kharitonova OV (1997) Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IrM with streptavidin and aggregates of concanavalin A. J Photochem Photobiol B Biol 40:278–287

    Article  CAS  Google Scholar 

  • Millar DP (2000) Time-resolved fluorescence methods for analysis of DNA-protein interactions. Methods Enzymol 323:442–459

    Article  CAS  PubMed  Google Scholar 

  • Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7:730–734

    Article  CAS  PubMed  Google Scholar 

  • St. Pierre PR, Petersen NO (1990) Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy. Biophys J 58:503–511

    PubMed  Google Scholar 

  • Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726

    CAS  PubMed  Google Scholar 

  • Sykes JA, Moore EB (1959) A new chamber for tissue culture. Proc Soc Exp Biol Med 100:121–127

    PubMed  Google Scholar 

  • Szollosi J, Tron S, Damjanovich S, Helliwell SH, Arndt-Jovin D, Jovin TM (1984) Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady-state fluorometric and flow cytometric methods. Cytometry 5:210–216

    CAS  PubMed  Google Scholar 

  • Szollosi J, Nagy P, Sebestyen Z, Damjanovich S, Park JW, Matyus L (2002) Applications of fluorescence resonance energy transfer for mapping biological membranes. Rev Mol Biotechnol 82:251–266

    Article  CAS  Google Scholar 

  • Wallach DF, Schmidt-Ullrich R (1976) Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles. J Cell Physiol 89(4):771–774

    CAS  PubMed  Google Scholar 

  • Whitehead RH, Macrae FA, St John DJ, Ma J (1985) A colon cancer cell line (LIM1215) derived from a patient with inherited nonpolyposis colorectal cancer. J Natl Cancer Inst 74:759–765

    CAS  PubMed  Google Scholar 

  • Young RM, Arnette JK, Roess DA, BG Barisas (1994) Quantitation of fluorescence resonance energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. Biophys J 67:881–888

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Ludwig Institute for Cancer Research, Royal Melbourne Hospital, P.O. Box 2008, 3050, Parkville, Victoria , Australia

    Andrew H. A. Clayton, Stephen H. Cody & Edouard C. Nice

  2. Department of Biochemistry, La Trobe University, 3086, Melbourne, Victoria , Australia

    Nectarios Klonis

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  1. Andrew H. A. Clayton
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  2. Nectarios Klonis
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  3. Stephen H. Cody
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  4. Edouard C. Nice
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Correspondence to Andrew H. A. Clayton.

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Clayton, A.H.A., Klonis, N., Cody, S.H. et al. Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells. Eur Biophys J 34, 82–90 (2005). https://doi.org/10.1007/s00249-004-0427-y

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  • DOI: https://doi.org/10.1007/s00249-004-0427-y

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