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. 2002 Nov;83(5):2625–2633. doi: 10.1016/S0006-3495(02)75273-9

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy.

Chad Leidy 1, Thomas Kaasgaard 1, John H Crowe 1, Ole G Mouritsen 1, Kent Jørgensen 1
PMCID: PMC1302348  PMID: 12414696

Abstract

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.

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Selected References

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  1. Bagatolli L. A., Gratton E. A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study. Biophys J. 2000 Jul;79(1):434–447. doi: 10.1016/S0006-3495(00)76305-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagatolli L. A., Gratton E. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys J. 2000 Jan;78(1):290–305. doi: 10.1016/S0006-3495(00)76592-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown D. A., London E. Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem Biophys Res Commun. 1997 Nov 7;240(1):1–7. doi: 10.1006/bbrc.1997.7575. [DOI] [PubMed] [Google Scholar]
  4. Carlson JM, Sethna JP. Theory of the ripple phase in hydrated phospholipid bilayers. Phys Rev A Gen Phys. 1987 Oct 1;36(7):3359–3374. doi: 10.1103/physreva.36.3359. [DOI] [PubMed] [Google Scholar]
  5. Chen C, Lubensky TC, MacKintosh FC. Phase transitions and modulated phases in lipid bilayers. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1995 Jan;51(1):504–513. doi: 10.1103/physreve.51.504. [DOI] [PubMed] [Google Scholar]
  6. Copeland B. R., McConnel H. M. The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol. Biochim Biophys Acta. 1980 Jun 20;599(1):95–109. doi: 10.1016/0005-2736(80)90059-0. [DOI] [PubMed] [Google Scholar]
  7. Dietrich C., Bagatolli L. A., Volovyk Z. N., Thompson N. L., Levi M., Jacobson K., Gratton E. Lipid rafts reconstituted in model membranes. Biophys J. 2001 Mar;80(3):1417–1428. doi: 10.1016/S0006-3495(01)76114-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feigenson G. W., Buboltz J. T. Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol. Biophys J. 2001 Jun;80(6):2775–2788. doi: 10.1016/S0006-3495(01)76245-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giocondi M. C., Pacheco L., Milhiet P. E., Le Grimellec C. Temperature dependence of the topology of supported dimirystoyl-distearoyl phosphatidylcholine bilayers. Ultramicroscopy. 2001 Jan;86(1-2):151–157. doi: 10.1016/s0304-3991(00)00086-3. [DOI] [PubMed] [Google Scholar]
  10. Heimburg T. A model for the lipid pretransition: coupling of ripple formation with the chain-melting transition. Biophys J. 2000 Mar;78(3):1154–1165. doi: 10.1016/S0006-3495(00)76673-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hicks A., Dinda M., Singer M. A. The ripple phase of phosphatidylcholines: effect of chain length and cholesterol. Biochim Biophys Acta. 1987 Sep 18;903(1):177–185. doi: 10.1016/0005-2736(87)90167-2. [DOI] [PubMed] [Google Scholar]
  12. Hollars C. W., Dunn R. C. Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy. Biophys J. 1998 Jul;75(1):342–353. doi: 10.1016/S0006-3495(98)77518-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hui S. W. Geometry of phase-separated domains in phospholipid bilayers by diffraction-contrast electron microscopy. Biophys J. 1981 Jun;34(3):383–395. doi: 10.1016/S0006-3495(81)84857-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hui S. W., Viswanathan R., Zasadzinski J. A., Israelachvili J. N. The structure and stability of phospholipid bilayers by atomic force microscopy. Biophys J. 1995 Jan;68(1):171–178. doi: 10.1016/S0006-3495(95)80172-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hønger T., Jørgensen K., Biltonen R. L., Mouritsen O. G. Systematic relationship between phospholipase A2 activity and dynamic lipid bilayer microheterogeneity. Biochemistry. 1996 Jul 16;35(28):9003–9006. doi: 10.1021/bi960866a. [DOI] [PubMed] [Google Scholar]
  16. Janiak M. J., Small D. M., Shipley G. G. Nature of the Thermal pretransition of synthetic phospholipids: dimyristolyl- and dipalmitoyllecithin. Biochemistry. 1976 Oct 19;15(21):4575–4580. doi: 10.1021/bi00666a005. [DOI] [PubMed] [Google Scholar]
  17. Janiak M. J., Small D. M., Shipley G. G. Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin. J Biol Chem. 1979 Jul 10;254(13):6068–6078. [PubMed] [Google Scholar]
  18. Jørgensen K., Mouritsen O. G. Phase separation dynamics and lateral organization of two-component lipid membranes. Biophys J. 1995 Sep;69(3):942–954. doi: 10.1016/S0006-3495(95)79968-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jørgensen K., Sperotto M. M., Mouritsen O. G., Ipsen J. H., Zuckermann M. J. Phase equilibria and local structure in binary lipid bilayers. Biochim Biophys Acta. 1993 Oct 10;1152(1):135–145. doi: 10.1016/0005-2736(93)90240-z. [DOI] [PubMed] [Google Scholar]
  20. Kapitza H. G., Rüppel D. A., Galla H. J., Sackmann E. Lateral diffusion of lipids and glycophorin in solid phosphatidylcholine bilayers. The role of structural defects. Biophys J. 1984 Mar;45(3):577–587. doi: 10.1016/S0006-3495(84)84195-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katsaras J., Tristram-Nagle S., Liu Y., Headrick R. L., Fontes E., Mason P. C., Nagle J. F. Clarification of the ripple phase of lecithin bilayers using fully hydrated, aligned samples. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 2000 May;61(5 Pt B):5668–5677. doi: 10.1103/physreve.61.5668. [DOI] [PubMed] [Google Scholar]
  22. Korlach J., Schwille P., Webb W. W., Feigenson G. W. Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8461–8466. doi: 10.1073/pnas.96.15.8461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koynova R., Koumanov A., Tenchov B. Metastable rippled gel phase in saturated phosphatidylcholines: calorimetric and densitometric characterization. Biochim Biophys Acta. 1996 Nov 13;1285(1):101–108. doi: 10.1016/s0005-2736(96)00155-1. [DOI] [PubMed] [Google Scholar]
  24. Leidy C., Wolkers W. F., Jørgensen K., Mouritsen O. G., Crowe J. H. Lateral organization and domain formation in a two-component lipid membrane system. Biophys J. 2001 Apr;80(4):1819–1828. doi: 10.1016/S0006-3495(01)76152-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lentz B. R., Hoechli M., Barenholz Y. Acyl chain order and lateral domain formation in mixed phosphatidylcholine--sphingomyelin multilamellar and unilamellar vesicles. Biochemistry. 1981 Nov 24;20(24):6803–6809. doi: 10.1021/bi00527a010. [DOI] [PubMed] [Google Scholar]
  26. Luna E. J., McConnell H. M. The intermediate monoclinic phase of phosphatidylcholines. Biochim Biophys Acta. 1977 May 2;466(3):381–392. doi: 10.1016/0005-2736(77)90331-5. [DOI] [PubMed] [Google Scholar]
  27. Mabrey S., Sturtevant J. M. Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3862–3866. doi: 10.1073/pnas.73.11.3862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marder M., Frisch H. L., Langer J. S., McConnell H. M. Theory of the intermediate rippled phase of phospholipid bilayers. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6559–6561. doi: 10.1073/pnas.81.20.6559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyer H. W., Bunjes H., Ulrich A. S. Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles. Chem Phys Lipids. 1999 Jun;99(2):111–123. doi: 10.1016/s0009-3084(99)00029-8. [DOI] [PubMed] [Google Scholar]
  30. Meyer H. W. Pretransition-ripples in bilayers of dipalmitoylphosphatidylcholine: undulation or periodic segments? A freeze-fracture study. Biochim Biophys Acta. 1996 Jul 26;1302(2):138–144. doi: 10.1016/0005-2760(96)00054-9. [DOI] [PubMed] [Google Scholar]
  31. Meyer H. W., Richter W. Freeze-fracture studies on lipids and membranes. Micron. 2001 Aug;32(6):615–644. doi: 10.1016/s0968-4328(00)00050-0. [DOI] [PubMed] [Google Scholar]
  32. Muresan A. S., Diamant H., Lee K. Y. Effect of temperature and composition on the formation of nanoscale compartments in phospholipid membranes. J Am Chem Soc. 2001 Jul 18;123(28):6951–6952. doi: 10.1021/ja015792r. [DOI] [PubMed] [Google Scholar]
  33. Möhwald H., Dietrich A., Böhm C., Brezesinski G., Thoma M. Domain formation in monolayers. Mol Membr Biol. 1995 Jan-Mar;12(1):29–38. doi: 10.3109/09687689509038492. [DOI] [PubMed] [Google Scholar]
  34. Nagle J. F., Tristram-Nagle S. Structure of lipid bilayers. Biochim Biophys Acta. 2000 Nov 10;1469(3):159–195. doi: 10.1016/s0304-4157(00)00016-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peters M. W., Mehlhorn I. E., Barber K. R., Grant C. W. Evidence of a distribution difference between two gangliosides in bilayer membranes. Biochim Biophys Acta. 1984 Dec 19;778(3):419–428. doi: 10.1016/0005-2736(84)90389-4. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Rappolt M., Pabst G., Rapp G., Kriechbaum M., Amenitsch H., Krenn C., Bernstorff S., Laggner P. New evidence for gel-liquid crystalline phase coexistence in the ripple phase of phosphatidylcholines. Eur Biophys J. 2000;29(2):125–133. doi: 10.1007/s002490050257. [DOI] [PubMed] [Google Scholar]
  38. Schütz G. J., Kada G., Pastushenko V. P., Schindler H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 2000 Mar 1;19(5):892–901. doi: 10.1093/emboj/19.5.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silvius J. R., del Giudice D., Lafleur M. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry. 1996 Dec 3;35(48):15198–15208. doi: 10.1021/bi9615506. [DOI] [PubMed] [Google Scholar]
  40. Stamatoff J., Feuer B., Guggenheim H. J., Tellez G., Yamane T. Amplitude of rippling in the P beta phase of dipalmitoylphosphatidylcholine bilayers. Biophys J. 1982 Jun;38(3):217–226. doi: 10.1016/S0006-3495(82)84551-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sun W. J., Tristram-Nagle S., Suter R. M., Nagle J. F. Structure of the ripple phase in lecithin bilayers. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7008–7012. doi: 10.1073/pnas.93.14.7008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tardieu A., Luzzati V., Reman F. C. Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J Mol Biol. 1973 Apr 25;75(4):711–733. doi: 10.1016/0022-2836(73)90303-3. [DOI] [PubMed] [Google Scholar]
  43. Tenchov B. G., Yao H., Hatta I. Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases. Biophys J. 1989 Oct;56(4):757–768. doi: 10.1016/S0006-3495(89)82723-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trandum C., Westh P., Jorgensen K. Slow relaxation of the sub-main transition in multilamellar phosphatidylcholine vesicles. Biochim Biophys Acta. 1999 Oct 15;1421(2):207–212. doi: 10.1016/s0005-2736(99)00142-x. [DOI] [PubMed] [Google Scholar]
  45. Tsuchida K., Hatta I. ESR studies on the ripple phase in multilamellar phospholipid bilayers. Biochim Biophys Acta. 1988 Nov 3;945(1):73–80. doi: 10.1016/0005-2736(88)90364-1. [DOI] [PubMed] [Google Scholar]
  46. Varma R., Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998 Aug 20;394(6695):798–801. doi: 10.1038/29563. [DOI] [PubMed] [Google Scholar]
  47. Verkleij A. J., Ververgaert P. H., van Deenen L. L., Elbers P. F. Phase transitions of phospholipid bilayers and membranes of Acholeplasma laidlawii B visualized by freeze fracturing electron microscopy. Biochim Biophys Acta. 1972 Nov 2;288(2):326–332. doi: 10.1016/0005-2736(72)90253-2. [DOI] [PubMed] [Google Scholar]
  48. Ververgaert P. H., Verkleij A. J., Elbers P. F., van Deenen L. L. Analysis of the crystallization process in lecithin liposomes: a freeze-etch study. Biochim Biophys Acta. 1973 Jul 6;311(3):320–329. doi: 10.1016/0005-2736(73)90313-1. [DOI] [PubMed] [Google Scholar]
  49. Viola A., Schroeder S., Sakakibara Y., Lanzavecchia A. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science. 1999 Jan 29;283(5402):680–682. doi: 10.1126/science.283.5402.680. [DOI] [PubMed] [Google Scholar]
  50. Vladkov R., Teuchner K., Leupold D., Koynova R., Tenchov B. Detection of the metastable rippled gel phase in hydrated phosphatidylcholine by fluorescence spectroscopy. Biophys Chem. 2000 Apr 14;84(2):159–166. doi: 10.1016/s0301-4622(00)00107-1. [DOI] [PubMed] [Google Scholar]
  51. Woodward J. T., 4th, Zasadzinski J. A. High-resolution scanning tunneling microscopy of fully hydrated ripple-phase bilayers. Biophys J. 1997 Feb;72(2 Pt 1):964–976. doi: 10.1016/s0006-3495(97)78731-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zasadzinski J. A. Effect of stereoconfiguration on ripple phases (P beta') of dipalmitoylphosphatidylcholine. Biochim Biophys Acta. 1988 Dec 22;946(2):235–243. doi: 10.1016/0005-2736(88)90398-7. [DOI] [PubMed] [Google Scholar]

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