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Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes

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Abstract

The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (<200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.

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Abbreviations

NBD-PC:

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

NBD-PE:

1-Palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine

DPH:

1,6-Diphenyl-1,3,5-hexatriene

References

  • Angelova MI, Dimitrov DS (1988) A mechanism of liposome electroformation. Prog Colloid Polym Sci 76:59–67

    Article  CAS  Google Scholar 

  • Ariola FS, Li Z, Cornejo C, Bittman R, Heikal AA (2009) Membrane fluidity and lipid order in ternary giant unilamellar vesicles using a new bodipy-cholesterol derivative. Biophys J 96:2696–2708

    Article  CAS  PubMed  Google Scholar 

  • Barrow DA, Lentz BR (1985) Membrane structural domains. Resolution limits using diphenylhexatriene fluorescence decay. Biophys J 48:221–234

    Article  CAS  PubMed  Google Scholar 

  • Chattopadhyay A, London E (1987) Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26:39–45

    Article  CAS  PubMed  Google Scholar 

  • de Almeida RF, Fedorov A, Prieto M (2003) Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys J 85:2406–2416

    Article  PubMed  Google Scholar 

  • de Almeida RF, Loura LM, Fedorov A, Prieto M (2005) Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study. J Mol Biol 346:1109–1120

    Article  PubMed  Google Scholar 

  • de Almeida RF, Loura LM, Prieto M (2009) Membrane lipid domains and rafts: current applications of fluorescence lifetime spectroscopy and imaging. Chem Phys Lipids 157:61–77

    Article  PubMed  Google Scholar 

  • Duggan J, Jamal G, Tilley M, Davis B, McKenzie G, Vere K, Somekh MG, O’Shea P, Harris H (2008) Functional imaging of microdomains in cell membranes. Eur Biophys J 37:1279–1289

    Article  CAS  PubMed  Google Scholar 

  • Florine-Casteel K (1990) Phospholipid order in gel- and fluid-phase cell-size liposomes measured by digitized video fluorescence polarization microscopy. Biophys J 57:1199–1215

    Article  CAS  PubMed  Google Scholar 

  • Frazier ML, Wright JR, Pokorny A, Almeida PF (2007) Investigation of domain formation in sphingomyelin/cholesterol/POPC mixtures by fluorescence resonance energy transfer and Monte Carlo simulations. Biophys J 92:2422–2433

    Article  CAS  PubMed  Google Scholar 

  • Greiner AJ, Pillman HA, Worden RM, Blanchard GJ, Ofoli RY (2009) Effect of hydrogen bonding on the rotational and translational dynamics of a headgroup-bound chromophore in bilayer lipid membranes. J Phys Chem B 113:13263–13268

    Article  CAS  PubMed  Google Scholar 

  • Haluska CK, Schroder AP, Didier P, Heissler D, Duportail G, Mely Y, Marques CM (2008) Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles. Biophys J 95:5737–5747

    Article  CAS  PubMed  Google Scholar 

  • Huster D, Muller P, Arnold K, Herrmann A (2001) Dynamics of membrane penetration of the fluorescent 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) group attached to an acyl chain of phosphatidylcholine. Biophys J 80:822–831

    Article  CAS  PubMed  Google Scholar 

  • Hwang J, Gheber LA, Margolis L, Edidin M (1998) Domains in cell plasma membranes investigated by near-field scanning optical microscopy. Biophys J 74:2184–2190

    Article  CAS  PubMed  Google Scholar 

  • Kahya N, Scherfeld D, Bacia K, Schwille P (2004) Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy. J Struct Biol 147:77–89

    Article  CAS  PubMed  Google Scholar 

  • Lentz BR (1989) Membrane fluidity as detected by diphenylhexatriene probes. Chem Phys Lipids 50:171–190

    Article  CAS  Google Scholar 

  • Loura LM, Ramalho JP (2007) Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study. Biochim Biophys Acta 1768:467–478

    Article  CAS  PubMed  Google Scholar 

  • Loura LM, de Almeida RF, Silva LC, Prieto M (2009) FRET analysis of domain formation and properties in complex membrane systems. Biochim Biophys Acta 1788:209–224

    Article  CAS  PubMed  Google Scholar 

  • Margineanu A, Hotta J, Vallee RA, Van der Auweraer M, Ameloot M, Stefan A, Beljonne D, Engelborghs Y, Herrmann A, Mullen K, De Schryver FC, Hofkens J (2007) Visualization of membrane rafts using a perylene monoimide derivative and fluorescence lifetime imaging. Biophys J 93:2877–2891

    Article  CAS  PubMed  Google Scholar 

  • Pike LJ (2006) Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J Lipid Res 47:1597–1598

    Article  CAS  PubMed  Google Scholar 

  • Pike LJ (2009) The challenge of lipid rafts. J Lipid Res 50(Suppl):S323–S328

    Article  PubMed  Google Scholar 

  • Silvius JR (2003) Fluorescence energy transfer reveals microdomain formation at physiological temperatures in lipid mixtures modeling the outer leaflet of the plasma membrane. Biophys J 85:1034–1045

    Article  CAS  PubMed  Google Scholar 

  • Silvius J (2005) Lipid microdomains in model and biological membranes: how strong are the connections? Q Rev Biophys 38:373–383

    Article  CAS  PubMed  Google Scholar 

  • Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572

    Article  CAS  PubMed  Google Scholar 

  • Stockl M, Plazzo AP, Korte T, Herrmann A (2008) Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues. J Biol Chem 283:30828–30837

    Article  PubMed  Google Scholar 

  • Straume M, Litman BJ (1987) Influence of cholesterol on equilibrium and dynamic bilayer structure of unsaturated acyl chain phosphatidylcholine vesicles as determined from higher order analysis of fluorescence anisotropy decay. Biochemistry 26:5121–5126

    Article  CAS  PubMed  Google Scholar 

  • Wesolowska O, Michalak K, Maniewska J, Hendrich AB (2009) Giant unilamellar vesicles—a perfect tool to visualize phase separation and lipid rafts in model systems. Acta Biochim Pol 56:33–39

    PubMed  Google Scholar 

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Acknowledgments

We are grateful to Dr. Axel Bergman of Becker & Hickl GmbH for help with the analysis, to Prof. Tony Parker for help and support, and to the Lasers for Science Facility (CLF, STFC Rutherford Appleton laboratory) for access.

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Authors and Affiliations

  1. Lasers for Science, Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, OX11 0QX, UK

    Stanley W. Botchway, Amanda M. Lewis & Christopher D. Stubbs

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  1. Stanley W. Botchway
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  2. Amanda M. Lewis
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  3. Christopher D. Stubbs
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Correspondence to Christopher D. Stubbs.

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Botchway, S.W., Lewis, A.M. & Stubbs, C.D. Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes. Eur Biophys J 40, 131–141 (2011). https://doi.org/10.1007/s00249-010-0631-x

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  • DOI: https://doi.org/10.1007/s00249-010-0631-x

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