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. 2001 Feb;80(2):776–788. doi: 10.1016/S0006-3495(01)76057-2

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study.

L M Loura 1, A Fedorov 1, M Prieto 1
PMCID: PMC1301276  PMID: 11159445

Abstract

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).

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Selected References

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  1. Almeida P. F., Vaz W. L., Thompson T. E. Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol lipid bilayers: a free volume analysis. Biochemistry. 1992 Jul 28;31(29):6739–6747. doi: 10.1021/bi00144a013. [DOI] [PubMed] [Google Scholar]
  2. Almeida P. F., Vaz W. L., Thompson T. E. Percolation and diffusion in three-component lipid bilayers: effect of cholesterol on an equimolar mixture of two phosphatidylcholines. Biophys J. 1993 Feb;64(2):399–412. doi: 10.1016/S0006-3495(93)81381-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CHEN R. F., BOWMAN R. L. FLUORESCENCE POLARIZATION: MEASUREMENT WITH ULTRAVIOLET-POLARIZING FILTERS IN A SPECTROPHOTOFLUOROMETER. Science. 1965 Feb 12;147(3659):729–732. doi: 10.1126/science.147.3659.729. [DOI] [PubMed] [Google Scholar]
  4. Chattopadhyay A. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem Phys Lipids. 1990 Mar;53(1):1–15. doi: 10.1016/0009-3084(90)90128-e. [DOI] [PubMed] [Google Scholar]
  5. Chattopadhyay A., London E. Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry. 1987 Jan 13;26(1):39–45. doi: 10.1021/bi00375a006. [DOI] [PubMed] [Google Scholar]
  6. Davenport L., Dale R. E., Bisby R. H., Cundall R. B. Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer. Biochemistry. 1985 Jul 16;24(15):4097–4108. doi: 10.1021/bi00336a044. [DOI] [PubMed] [Google Scholar]
  7. Davenport L. Fluorescence probes for studying membrane heterogeneity. Methods Enzymol. 1997;278:487–512. doi: 10.1016/s0076-6879(97)78026-1. [DOI] [PubMed] [Google Scholar]
  8. Edidin M. Lipid microdomains in cell surface membranes. Curr Opin Struct Biol. 1997 Aug;7(4):528–532. doi: 10.1016/s0959-440x(97)80117-0. [DOI] [PubMed] [Google Scholar]
  9. Ho C., Slater S. J., Stubbs C. D. Hydration and order in lipid bilayers. Biochemistry. 1995 May 9;34(18):6188–6195. doi: 10.1021/bi00018a023. [DOI] [PubMed] [Google Scholar]
  10. Ipsen J. H., Mouritsen O. G., Bloom M. Relationships between lipid membrane area, hydrophobic thickness, and acyl-chain orientational order. The effects of cholesterol. Biophys J. 1990 Mar;57(3):405–412. doi: 10.1016/S0006-3495(90)82557-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kenworthy A. K., Edidin M. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer. J Cell Biol. 1998 Jul 13;142(1):69–84. doi: 10.1083/jcb.142.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lafleur M., Cullis P. R., Bloom M. Modulation of the orientational order profile of the lipid acyl chain in the L alpha phase. Eur Biophys J. 1990;19(2):55–62. doi: 10.1007/BF00185086. [DOI] [PubMed] [Google Scholar]
  13. Lentz B. R., Barrow D. A., Hoechli M. Cholesterol-phosphatidylcholine interactions in multilamellar vesicles. Biochemistry. 1980 Apr 29;19(9):1943–1954. doi: 10.1021/bi00550a034. [DOI] [PubMed] [Google Scholar]
  14. Loura L. M., Fedorov A., Prieto M. Resonance energy transfer in a model system of membranes: application to gel and liquid crystalline phases. Biophys J. 1996 Oct;71(4):1823–1836. doi: 10.1016/S0006-3495(96)79383-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mabrey S., Mateo P. L., Sturtevant J. M. High-sensitivity scanning calorimetric study of mixtures of cholesterol with dimyristoyl- and dipalmitoylphosphatidylcholines. Biochemistry. 1978 Jun 13;17(12):2464–2468. doi: 10.1021/bi00605a034. [DOI] [PubMed] [Google Scholar]
  16. Méléard P., Gerbeaud C., Pott T., Fernandez-Puente L., Bivas I., Mitov M. D., Dufourcq J., Bothorel P. Bending elasticities of model membranes: influences of temperature and sterol content. Biophys J. 1997 Jun;72(6):2616–2629. doi: 10.1016/S0006-3495(97)78905-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pralle A., Keller P., Florin E. L., Simons K., Hörber J. K. Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol. 2000 Mar 6;148(5):997–1008. doi: 10.1083/jcb.148.5.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Reyes Mateo C., Ulises Acuña A., Brochon J. C. Liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid. Biophys J. 1995 Mar;68(3):978–987. doi: 10.1016/S0006-3495(95)80273-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robinson A. J., Richards W. G., Thomas P. J., Hann M. M. Behavior of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study. Biophys J. 1995 Jan;68(1):164–170. doi: 10.1016/S0006-3495(95)80171-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sankaram M. B., Marsh D., Thompson T. E. Determination of fluid and gel domain sizes in two-component, two-phase lipid bilayers. An electron spin resonance spin label study. Biophys J. 1992 Aug;63(2):340–349. doi: 10.1016/S0006-3495(92)81619-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sankaram M. B., Thompson T. E. Cholesterol-induced fluid-phase immiscibility in membranes. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8686–8690. doi: 10.1073/pnas.88.19.8686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Smaby J. M., Momsen M. M., Brockman H. L., Brown R. E. Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol. Biophys J. 1997 Sep;73(3):1492–1505. doi: 10.1016/S0006-3495(97)78181-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smondyrev A. M., Berkowitz M. L. Structure of dipalmitoylphosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation. Biophys J. 1999 Oct;77(4):2075–2089. doi: 10.1016/S0006-3495(99)77049-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Snyder B., Freire E. Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions. Biophys J. 1982 Nov;40(2):137–148. doi: 10.1016/S0006-3495(82)84468-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  27. Tu K., Klein M. L., Tobias D. J. Constant-pressure molecular dynamics investigation of cholesterol effects in a dipalmitoylphosphatidylcholine bilayer. Biophys J. 1998 Nov;75(5):2147–2156. doi: 10.1016/S0006-3495(98)77657-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vanderkooi G. Computation of mixed phosphatidylcholine-cholesterol bilayer structures by energy minimization. Biophys J. 1994 May;66(5):1457–1468. doi: 10.1016/S0006-3495(94)80936-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. WEBER G. Polarization of the fluorescence of macromolecules. I. Theory and experimental method. Biochem J. 1952 May;51(2):145–155. doi: 10.1042/bj0510145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xiang T. X., Anderson B. D. Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory. Biophys J. 1997 Jan;72(1):223–237. doi: 10.1016/S0006-3495(97)78661-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

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