Skip to main content
SpringerLink
Log in

Membrane lipid domains and dynamics as detected by Laurdan fluorescence

  • Special Topic Articles
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. C. J. Scandella, P. Devaux, and H. M. McConnell (1972) Rapid lateral diffusion of phospholipids in rabbit sarcoplasma reticulum,Proc. Natl. Acad. Sci. USA 69, 2056–2060.

    Google Scholar 

  2. S. J. Singer and G. L. Nicolson (1972) The fluid mosaic model of the structure of cell membranes,Science 175, 720–731.

    Google Scholar 

  3. E. J. Shimshick and H. M. McConnel (1973) Lateral phase separation in phospholipid membranes,Biochemistry 12, 2351–2360.

    Google Scholar 

  4. M. P. Andrich and J. M. Vanderkooi (1976) Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phospholipid artificial membranes,Biochemistry 15, 1257–1261.

    Google Scholar 

  5. H. K. Kimelberg (1976) Na,K-dependent adenosine triphosphatase activity in reconstituted systems and in cultured cells,Biochem. Soc. Trans. 4, 755–777.

    Google Scholar 

  6. P. L. Chong, P. A. G. Fortes, and D. M. Jameson (1985) Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes,J. Biol. Chem. 27, 14484–14490.

    Google Scholar 

  7. P. M. Macdonald and J. Seelig (1987) Calcium binding to mixed phosphatidylglycerol-phosphatidylcholine bilayers as studied by deuterium nuclear magnetic resonance,Biochemistry 26, 231–240.

    Google Scholar 

  8. V. Luzzati, A. Tardieu, and D. Taupin (1972) An approach to the phase problem in the X-ray diffraction study of biological membranes and model systems,Chem. Phys. Lipids 8, 292–297.

    Google Scholar 

  9. M. R. Morrow and J. H. Davis (1988) Differential scanning calorimetry and2H NMR studies of the phase behaviour of gramicidin-phosphatidylcholine mixtures,Biochemistry 21, 2024–2032.

    Google Scholar 

  10. D. M. Jameson (1984) Fluorescence: Principles, methodologies and applications, in E. Voss, Jr. (Ed.),Fluorescein Hapten: An Immunological Probe, CRC Press, Boca Raton, Florida, pp. 23–48.

    Google Scholar 

  11. B. W. van der Meer (1988) Biomembranes structure and dynamics viewed by fluorescence, in H. J. Hilderson and J. R. Harris (Eds.),Subcellular Biochemistry, Vol. 3, Plenum Press, New York, pp. 1–53.

    Google Scholar 

  12. B. R. Lentz (1989) Membrane “fluidity” as detected by diphenylhexatriene probes,Chem. Phys. Lipids 50, 171–190.

    Google Scholar 

  13. M. Shinitzky, A. C. Dianoux, C. Gitler, and G. Weber (1971) Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescent probes. I. Synthetic micelles,Biochemistry 10, 2106–2113.

    Google Scholar 

  14. T. Parasassi, G. De Stasio, R. M. Rusch, and E. Gratton (1991) A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles,Biophys. J. 59, 466–475.

    Google Scholar 

  15. R. D. Klausner, A. M. Kleinfeld, R. L. Hoover, and M. J. Karnovsky (1980) Lipid domains in membranes,J. Biol. Chem. 255, 1286–1295.

    Google Scholar 

  16. T. Parasassi, F. Conti, M. Glaser, and E. Gratton (1984) Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-l,3,5-hexatriene fluorescence,J. Biol. Chem. 259, 14011–14017.

    Google Scholar 

  17. R. Fiorini, M. Valentino, S. Wang, M. Glaser, and E. Gratton (1987) Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles,Biochemistry 26, 3864–3870.

    Google Scholar 

  18. T. Parasassi, F. Conti, E. Gratton, and O. Sapora (1987) Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry,Biochim. Biophys. Acta 898, 196–201.

    Google Scholar 

  19. T. Parasassi, O. Sapora, A. M. Giusti, G. De Stasio, and G. Ravagnan (1991) Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime,Int. J. Radiat. Biol. 59, 59–69.

    Google Scholar 

  20. T. Parasassi, G. Ravagnan, O. Sapora, and E. Gratton (1992) Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions,Int. J. Radiat. Biol. 61, 791–796.

    Google Scholar 

  21. L. A. Sklar, G. P. Miljanich, and E. A. Dratz (1979) Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases,Biochemistry 18, 1707–1716.

    Google Scholar 

  22. T. Parasassi, F. Conti, and E. Gratton (1984) Study of heterogeneous emission of parinaric acid isomers using multifrequency phase fluorometry,Biochemistry 23, 5660–5664.

    Google Scholar 

  23. C. R. Mateo, J.-C. Brochon, M. P. Lillo, and A. U. Acuña (1993) Lipid clustering in bilayers detected by the fluorescence kinetics and anisotropy of trans-parinaric acid,Biophys. J. 65, 2237–2247.

    Google Scholar 

  24. C. R. Mateo, P. Taue, and J.-C. Brochon (1993) Pressure effect on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay,Biophys. J. 65, 2248–2260.

    Google Scholar 

  25. G. Weber and F. J. Farris (1979) Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-Propionyl-2-(dimethylamino)naphthalene,Biochemistry 18, 3075–3078.

    Google Scholar 

  26. R. B. MacGregor and G. Weber (1981) Fluorophores in polar media: Spectral effects of the Langevin distribution of electrostatic interactions,Ann. N. Y. Acad. Sci. 366, 140–154.

    Google Scholar 

  27. T. Parasassi, G. De Stasio, G. Ravagnan, R. M. Rusch, and E. Gratton (1991) Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence,Biophys. J. 60, 179–189.

    Google Scholar 

  28. T. Parasassi, F. Conti, and E. Gratton (1986) Fluorophores in a polar medium: Time dependence of emission spectra detected by multifrequency phase and modulation fluorometry,Cell. Mol. Biol. 32, 99–102.

    Google Scholar 

  29. T. Parasassi, F. Conti, and E. Gratton (1986) Time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry,Cell. Mol. Biol. 32, 103–108.

    Google Scholar 

  30. T. Parasassi, G. De Stasio, A. d'Ubaldo, and E. Gratton (1990) Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence,Biophys. J. 57, 1179–1186.

    Google Scholar 

  31. T. Parasassi, and E. Gratton (1992) Packing of phospholipid vesicles studied by oxygen quenching of Laurdan fluorescence,J. Fluorescence 2, 167–174.

    Google Scholar 

  32. T. Parasassi, G. Ravagnan, R. M. Rusch, and E. Gratton (1993) Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence,Photochem. Photobiol. 57, 403–410.

    Google Scholar 

  33. S. Mitaku, T. Jippo, and R. Kataoka (1983) Thermodynamic properties of the lipid bilayer transition,Biophys. J. 42, 137–144.

    Google Scholar 

  34. L. Davenport, J. R. Knutson, and L. Brand (1988) Time-resolved fluorescence anisotropy of membrane probes: Rotations gated by packing fluctuations, in J. R. Lakowicz (Ed.),Time-Resolved Laser Spectroscopy in Biochemistry, Proceedings SPIE909, pp. 263–270.

  35. J. H. K. Ipsen, K. Jörgensen, and O. G. Mouritsen (1990) Density fluctuations in saturated phospholipid bilayers increase as the acylchain length decreases,Biophys. J. 58, 1099–1107.

    Google Scholar 

  36. T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora, and E. Gratton (1992) Membrane aging during cells growth ascertained by Laurdan generalized polarization,Exp. Cell Res. 202, 432–439.

    Google Scholar 

  37. T. Parasassi, M. Loiero, M. Raimondi, G. Ravagnan, and E. Gratton (1993) Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types,Biochim. Biophys. Acta 1153, 143–154.

    Google Scholar 

  38. M. Levi, D. Jameson, P. Wilson, and J. Cooper (1993) Effect of lipid-protein and lipid-lipid interactions on lipid dynamics and lipid phases in renal apical membranes,Biophys. J. 64, 165a.

    Google Scholar 

  39. T. Parasassi, M. Di Stefano, M. Loiero, G. Ravagnan, and E. Gratton (1994) Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence,Biophys. J. 66, 120–132.

    Google Scholar 

  40. M. R. Vist and J. H. Davis (1990) Phase equilibria of cholesterol/ dipalmitoyl-phosphatidyl-choline mixtures:2H nuclear magnetic resonance and differential scanning calorimetry,Biochemistry 29, 451–464.

    Google Scholar 

  41. M. B. Sankaram and T. E. Thompson (1990) Interaction of cholesterol with various glycerophospholipids and sphingomyelin,Biochemistry 29, 10670–10675.

    Google Scholar 

  42. M. B. Sankaram and T. E. Thompson (1990) Modulation of phospholipid acyl chain order by cholesterol. A solid-state2H nuclear magnetic resonance study,Biochemistry 29, 10676–10684.

    Google Scholar 

  43. W. K. Subczynski, W. E. Antholine, J. S. Hyde, and A. Kusumi (1990) Microimmiscibility and three-dimensional dynamic structure of phosphatidylcholine-cholesterol membranes: Translational diffusion of a copper complex in the membrane.Biochemistry 29, 7936–7945.

    Google Scholar 

  44. R. Tampé, A. von Lukas, and H.-J. Galla (1991) Glycophorininduced cholesterol-phospholipid domains in dimyristoyl-phos-phatidylcholine bilayer vesicles,Biochemistry 30, 4909–1916.

    Google Scholar 

  45. K. M. Keough, B. Giffin, and P. L. Matthews (1989) Phosphatidylcholine-cholesterol interactions: Bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentrations,Biochim. Biophys. Acta 983, 51–55.

    Google Scholar 

  46. D. L. Melchior, F. J. Scavitto, and J. M. Steim (1980) Dilatometry of dipalmitoyl-lecithin-cholesterol bilayersBiochemistry 19, 4828–4834.

    Google Scholar 

  47. N. K. Mortensen, W. Pfeiffer, E. Sackmann, and W. Knoll (1988) Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: Ripple structure and phase diagram,Biochim. Biophys. Acta 945, 221–245.

    Google Scholar 

  48. F. Schroeder, J. R. Jefferson, A. B. Kier, J. Knittel, T. J. Scallen, W. Gibson Wood, and I. Hapala (1991). Membrane cholesterol dynamics: Cholesterol domains and kinetic pools,Proc. Soc. Exp. Biol. Med. 196, 235–252.

    Google Scholar 

  49. V. Ben-Yasar and Y. Barenholz (1989). The interaction of cholesterol and cholest-4-en-3-one with dipalmitoylphosphatidyl-choline. Comparison based on the use of three fluorophores,Biochim. Biophys. Acta 985, 271–278.

    Google Scholar 

  50. G. Nemecz and F. Shroeder (1988). Time-resolved fluorescence investigation on membrane cholesterol heterogeneity and exchange,Biochemistry 27, 7740–7749.

    Google Scholar 

  51. J. H. Ipsen, G. Karlstrom. O. G. Mouritsen, H. Wennerstrom, and M. H. Zuckermann (1987) Phase equilibria in the phosphatidyl-choline-cholesterol system,Biochim. Biophys. Acta 905, 162–172.

    Google Scholar 

  52. O. G. Mouritsen (1991) Theoretical models of phospholipid phase transitions,Chem. Phys. Lipids 57, 179–194.

    Google Scholar 

  53. P. L. Yeagle (1985) Cholesterol and the cell membrane,Biochim. Biophys. Acta 822, 267–287.

    Google Scholar 

  54. S. A. Simon, T. J. McIntosh, and R. Latorre (1982) Influence of cholesterol on water penetration into bilayers,Science 216, 65–67.

    Google Scholar 

  55. T. Parasassi, M. Di Stefano, M. Loiero, G. Ravagnan, and E. Gratton (1994) Cholesterol modifies water concentration and dynamics in phospholipid bilayers. A fluorescence study using Laurdan probe,Biophys. J., in press.

  56. P. J. Somerharju, J. A. Virtanen, K. K. Eklund, P. Vainio, and P. K. J. Kinnunen (1985). 1-Palmitoyl-2-pyrenedecanoyl glycerophospholipids as membrane probes: Evidence for regular distribution in liquid-crystalline phosphatidylcholine bilayers,Biochemistry 24, 2773–2781.

    Google Scholar 

  57. D. Tang and P. L. Chong (1992). E/M dips. Evidence for lipids regularly distributed into hexagonal super-lattices in pyrene-PC/DMPC binary mixtures at specific concentrations,Biophys. J. 63, 903–910.

    Google Scholar 

  58. J. M. Beechem and E. Gratton (1988) Fluorescence spectroscopy data analysis environment: A second generation global analysis program, inTime-Resolved Laser Spectroscopy in Biochemistry, Proceedings SPIE909, pp. 70–81.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Istituto di Medicina Sperimentale, CNR, Viale Marx 15, 00137, Rome, Italy

    Tiziana Parasassi & Enrico Gratton

  2. Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, 61801, Urbana, Illinois

    Enrico Gratton

Authors
  1. Tiziana Parasassi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Enrico Gratton
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Parasassi, T., Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J Fluoresc 5, 59–69 (1995). https://doi.org/10.1007/BF00718783

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00718783

Key words

Search

Navigation