Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 1994 Jan;66(1):120–132. doi: 10.1016/S0006-3495(94)80763-5

Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence.

T Parasassi 1, M Di Stefano 1, M Loiero 1, G Ravagnan 1, E Gratton 1
PMCID: PMC1275671  PMID: 8130331

Abstract

Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.

Full text

PDF
120

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ben-Yashar V., Barenholz Y. The interaction of cholesterol and cholest-4-en-3-one with dipalmitoylphosphatidylcholine. Comparison based on the use of three fluorophores. Biochim Biophys Acta. 1989 Nov 3;985(3):271–278. doi: 10.1016/0005-2736(89)90412-4. [DOI] [PubMed] [Google Scholar]
  2. Ipsen J. H., Karlström G., Mouritsen O. G., Wennerström H., Zuckermann M. J. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta. 1987 Nov 27;905(1):162–172. doi: 10.1016/0005-2736(87)90020-4. [DOI] [PubMed] [Google Scholar]
  3. Keough K. M., Giffin B., Matthews P. L. Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration. Biochim Biophys Acta. 1989 Jul 24;983(1):51–55. doi: 10.1016/0005-2736(89)90379-9. [DOI] [PubMed] [Google Scholar]
  4. Lakowicz J. R., Weber G. Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules. Biochemistry. 1973 Oct 9;12(21):4161–4170. doi: 10.1021/bi00745a020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Levine Y. K., Wilkins M. H. Structure of oriented lipid bilayers. Nat New Biol. 1971 Mar 17;230(11):69–72. doi: 10.1038/newbio230069a0. [DOI] [PubMed] [Google Scholar]
  6. Melchior D. L., Scavitto F. J., Steim J. M. Dilatometry of dipalmitoyllecithin-cholesterol bilayers. Biochemistry. 1980 Oct 14;19(21):4828–4834. doi: 10.1021/bi00562a018. [DOI] [PubMed] [Google Scholar]
  7. Mortensen K., Pfeiffer W., Sackmann E., Knoll W. Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram. Biochim Biophys Acta. 1988 Nov 22;945(2):221–245. doi: 10.1016/0005-2736(88)90485-3. [DOI] [PubMed] [Google Scholar]
  8. Mouritsen O. G. Theoretical models of phospholipid phase transitions. Chem Phys Lipids. 1991 Mar;57(2-3):179–194. doi: 10.1016/0009-3084(91)90075-m. [DOI] [PubMed] [Google Scholar]
  9. Nemecz G., Schroeder F. Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange. Biochemistry. 1988 Oct 4;27(20):7740–7749. doi: 10.1021/bi00420a024. [DOI] [PubMed] [Google Scholar]
  10. Parasassi T., Conti F., Glaser M., Gratton E. Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence. J Biol Chem. 1984 Nov 25;259(22):14011–14017. [PubMed] [Google Scholar]
  11. Parasassi T., De Stasio G., Ravagnan G., Rusch R. M., Gratton E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys J. 1991 Jul;60(1):179–189. doi: 10.1016/S0006-3495(91)82041-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Parasassi T., De Stasio G., d'Ubaldo A., Gratton E. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys J. 1990 Jun;57(6):1179–1186. doi: 10.1016/S0006-3495(90)82637-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parasassi T., Di Stefano M., Ravagnan G., Sapora O., Gratton E. Membrane aging during cell growth ascertained by Laurdan generalized polarization. Exp Cell Res. 1992 Oct;202(2):432–439. doi: 10.1016/0014-4827(92)90096-q. [DOI] [PubMed] [Google Scholar]
  14. Parasassi T., Ravagnan G., Rusch R. M., Gratton E. Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence. Photochem Photobiol. 1993 Mar;57(3):403–410. doi: 10.1111/j.1751-1097.1993.tb02309.x. [DOI] [PubMed] [Google Scholar]
  15. Rubenstein J. L., Smith B. A., McConnell H. M. Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines. Proc Natl Acad Sci U S A. 1979 Jan;76(1):15–18. doi: 10.1073/pnas.76.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sankaram M. B., Thompson T. E. Interaction of cholesterol with various glycerophospholipids and sphingomyelin. Biochemistry. 1990 Nov 27;29(47):10670–10675. doi: 10.1021/bi00499a014. [DOI] [PubMed] [Google Scholar]
  17. Sankaram M. B., Thompson T. E. Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance study. Biochemistry. 1990 Nov 27;29(47):10676–10684. doi: 10.1021/bi00499a015. [DOI] [PubMed] [Google Scholar]
  18. Schroeder F., Jefferson J. R., Kier A. B., Knittel J., Scallen T. J., Wood W. G., Hapala I. Membrane cholesterol dynamics: cholesterol domains and kinetic pools. Proc Soc Exp Biol Med. 1991 Mar;196(3):235–252. doi: 10.3181/00379727-196-43185. [DOI] [PubMed] [Google Scholar]
  19. Shimshick E. J., McConnell H. M. Lateral phase separation in phospholipid membranes. Biochemistry. 1973 Jun 5;12(12):2351–2360. doi: 10.1021/bi00736a026. [DOI] [PubMed] [Google Scholar]
  20. Subczynski W. K., Antholine W. E., Hyde J. S., Kusumi A. Microimmiscibility and three-dimensional dynamic structures of phosphatidylcholine-cholesterol membranes: translational diffusion of a copper complex in the membrane. Biochemistry. 1990 Aug 28;29(34):7936–7945. doi: 10.1021/bi00486a023. [DOI] [PubMed] [Google Scholar]
  21. Tampé R., von Lukas A., Galla H. J. Glycophorin-induced cholesterol-phospholipid domains in dimyristoylphosphatidylcholine bilayer vesicles. Biochemistry. 1991 May 21;30(20):4909–4916. doi: 10.1021/bi00234a011. [DOI] [PubMed] [Google Scholar]
  22. Vist M. R., Davis J. H. Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry. Biochemistry. 1990 Jan 16;29(2):451–464. doi: 10.1021/bi00454a021. [DOI] [PubMed] [Google Scholar]
  23. van Langen H., van Ginkel G., Shaw D., Levine Y. K. The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles. Effects of unsaturation, headgroup and cholesterol on orientational order and reorientational dynamics. Eur Biophys J. 1989;17(1):37–48. doi: 10.1007/BF00257144. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES