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. 2002 Dec;83(6):3380–3392. doi: 10.1016/S0006-3495(02)75338-1

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach.

Timothy V Ratto 1, Marjorie L Longo 1
PMCID: PMC1302413  PMID: 12496105

Abstract

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.

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Selected References

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  1. Abney J. R., Scalettar B. A. Fluctuations and membrane heterogeneity. Biophys Chem. 1995 Dec;57(1):27–36. doi: 10.1016/0301-4622(95)00042-v. [DOI] [PubMed] [Google Scholar]
  2. Abney J. R., Scalettar B. A., Owicki J. C. Mutual diffusion of interacting membrane proteins. Biophys J. 1989 Aug;56(2):315–326. doi: 10.1016/S0006-3495(89)82678-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Almeida P. F., Vaz W. L., Thompson T. E. Lateral diffusion and percolation in two-phase, two-component lipid bilayers. Topology of the solid-phase domains in-plane and across the lipid bilayer. Biochemistry. 1992 Aug 11;31(31):7198–7210. doi: 10.1021/bi00146a024. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Balcom B. J., Petersen N. O. Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules. Biophys J. 1993 Aug;65(2):630–637. doi: 10.1016/S0006-3495(93)81106-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Balgavý P., Dubnicková M., Kucerka N., Kiselev M. A., Yaradaikin S. P., Uhríková D. Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: a small-angle neutron scattering study. Biochim Biophys Acta. 2001 May 2;1512(1):40–52. doi: 10.1016/s0005-2736(01)00298-x. [DOI] [PubMed] [Google Scholar]
  8. Brown D. A., London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem. 2000 Jun 9;275(23):17221–17224. doi: 10.1074/jbc.R000005200. [DOI] [PubMed] [Google Scholar]
  9. Bussell S. J., Koch D. L., Hammer D. A. Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: tracer diffusion in organelle and reconstituted membranes. Biophys J. 1995 May;68(5):1828–1835. doi: 10.1016/S0006-3495(95)80359-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cherry R. J., Smith P. R., Morrison I. E., Fernandez N. Mobility of cell surface receptors: a re-evaluation. FEBS Lett. 1998 Jun 23;430(1-2):88–91. doi: 10.1016/s0014-5793(98)00595-x. [DOI] [PubMed] [Google Scholar]
  11. Chou T., Kim K. S., Oster G. Statistical thermodynamics of membrane bending-mediated protein-protein attractions. Biophys J. 2001 Mar;80(3):1075–1087. doi: 10.1016/S0006-3495(01)76086-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ditaranto K., Tekirian T. L., Yang A. J. Lysosomal membrane damage in soluble Abeta-mediated cell death in Alzheimer's disease. Neurobiol Dis. 2001 Feb;8(1):19–31. doi: 10.1006/nbdi.2000.0364. [DOI] [PubMed] [Google Scholar]
  13. Eze M. O. Membrane fluidity, reactive oxygen species, and cell-mediated immunity: implications in nutrition and disease. Med Hypotheses. 1992 Apr;37(4):220–224. doi: 10.1016/0306-9877(92)90191-e. [DOI] [PubMed] [Google Scholar]
  14. Feder T. J., Brust-Mascher I., Slattery J. P., Baird B., Webb W. W. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys J. 1996 Jun;70(6):2767–2773. doi: 10.1016/S0006-3495(96)79846-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garver T. D., Ren Q., Tuvia S., Bennett V. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin. J Cell Biol. 1997 May 5;137(3):703–714. doi: 10.1083/jcb.137.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gheber L. A., Edidin M. A model for membrane patchiness: lateral diffusion in the presence of barriers and vesicle traffic. Biophys J. 1999 Dec;77(6):3163–3175. doi: 10.1016/S0006-3495(99)77147-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harroun T. A., Heller W. T., Weiss T. M., Yang L., Huang H. W. Experimental evidence for hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin. Biophys J. 1999 Feb;76(2):937–945. doi: 10.1016/S0006-3495(99)77257-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heller W. T., Waring A. J., Lehrer R. I., Harroun T. A., Weiss T. M., Yang L., Huang H. W. Membrane thinning effect of the beta-sheet antimicrobial protegrin. Biochemistry. 2000 Jan 11;39(1):139–145. doi: 10.1021/bi991892m. [DOI] [PubMed] [Google Scholar]
  19. Ho C., Williams B. W., Stubbs C. D. Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores. Biochim Biophys Acta. 1992 Mar 2;1104(2):273–282. doi: 10.1016/0005-2736(92)90041-j. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Johnson M. E., Berk D. A., Blankschtein D., Golan D. E., Jain R. K., Langer R. S. Lateral diffusion of small compounds in human stratum corneum and model lipid bilayer systems. Biophys J. 1996 Nov;71(5):2656–2668. doi: 10.1016/S0006-3495(96)79457-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jähnig F. Critical effects from lipid-protein interaction in membranes. I. Theoretical description. Biophys J. 1981 Nov;36(2):329–345. doi: 10.1016/S0006-3495(81)84735-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kucik D. F., Elson E. L., Sheetz M. P. Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion. Biophys J. 1999 Jan;76(1 Pt 1):314–322. doi: 10.1016/S0006-3495(99)77198-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ladha S. Lipid heterogeneity and membrane fluidity in a highly polarized cell, the mammalian spermatozoon. J Membr Biol. 1998 Sep 1;165(1):1–10. doi: 10.1007/s002329900415. [DOI] [PubMed] [Google Scholar]
  26. Lee SB, Torquato S. Monte Carlo study of correlated continuum percolation: Universality and percolation thresholds. Phys Rev A. 1990 May 15;41(10):5338–5344. doi: 10.1103/physreva.41.5338. [DOI] [PubMed] [Google Scholar]
  27. Martins J., Melo E. Molecular mechanism of lateral diffusion of py(10)-PC and free pyrene in fluid DMPC bilayers. Biophys J. 2001 Feb;80(2):832–840. doi: 10.1016/S0006-3495(01)76062-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mc Kiernan A. E., MacDonald R. I., MacDonald R. C., Axelrod D. Cytoskeletal protein binding kinetics at planar phospholipid membranes. Biophys J. 1997 Oct;73(4):1987–1998. doi: 10.1016/S0006-3495(97)78229-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McKiernan A. E., Ratto T. V., Longo M. L. Domain growth, shapes, and topology in cationic lipid bilayers on mica by fluorescence and atomic force microscopy. Biophys J. 2000 Nov;79(5):2605–2615. doi: 10.1016/S0006-3495(00)76499-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mesquita R. M., Melo E., Thompson T. E., Vaz W. L. Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes. Biophys J. 2000 Jun;78(6):3019–3025. doi: 10.1016/S0006-3495(00)76840-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minton A. P. Lateral diffusion of membrane proteins in protein-rich membranes. A simple hard particle model for concentration dependence of the two-dimensional diffusion coefficient. Biophys J. 1989 Apr;55(4):805–808. doi: 10.1016/S0006-3495(89)82880-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Toole P. J., Wolfe C., Ladha S., Cherry R. J. Rapid diffusion of spectrin bound to a lipid surface. Biochim Biophys Acta. 1999 Jun 9;1419(1):64–70. doi: 10.1016/s0005-2736(99)00048-6. [DOI] [PubMed] [Google Scholar]
  33. Owen J. S. Extrahepatic cell membrane lipid abnormalities and cellular dysfunction in liver disease. Drugs. 1990;40 (Suppl 3):73–83. doi: 10.2165/00003495-199000403-00008. [DOI] [PubMed] [Google Scholar]
  34. Periasamy N., Verkman A. S. Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys J. 1998 Jul;75(1):557–567. doi: 10.1016/S0006-3495(98)77545-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Picard F., Paquet M. J., Dufourc E. J., Auger M. Measurement of the lateral diffusion of dipalmitoylphosphatidylcholine adsorbed on silica beads in the absence and presence of melittin: a 31P two-dimensional exchange solid-state NMR study. Biophys J. 1998 Feb;74(2 Pt 1):857–868. doi: 10.1016/S0006-3495(98)74009-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sako Y., Kusumi A. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J Cell Biol. 1995 Jun;129(6):1559–1574. doi: 10.1083/jcb.129.6.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saxton M. J. Anomalous diffusion due to obstacles: a Monte Carlo study. Biophys J. 1994 Feb;66(2 Pt 1):394–401. doi: 10.1016/s0006-3495(94)80789-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saxton M. J. Lateral diffusion and aggregation. A Monte Carlo study. Biophys J. 1992 Jan;61(1):119–128. doi: 10.1016/S0006-3495(92)81821-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Saxton M. J. Lateral diffusion in a mixture of mobile and immobile particles. A Monte Carlo study. Biophys J. 1990 Nov;58(5):1303–1306. doi: 10.1016/S0006-3495(90)82470-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saxton M. J. Lateral diffusion in an archipelago. Distance dependence of the diffusion coefficient. Biophys J. 1989 Sep;56(3):615–622. doi: 10.1016/S0006-3495(89)82708-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saxton M. J. Single-particle tracking: the distribution of diffusion coefficients. Biophys J. 1997 Apr;72(4):1744–1753. doi: 10.1016/S0006-3495(97)78820-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schram V., Lin H. N., Thompson T. E. Topology of gel-phase domains and lipid mixing properties in phase-separated two-component phosphatidylcholine bilayers. Biophys J. 1996 Oct;71(4):1811–1822. doi: 10.1016/S0006-3495(96)79382-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schram V., Tocanne J. F., Lopez A. Influence of obstacles on lipid lateral diffusion: computer simulation of FRAP experiments and application to proteoliposomes and biomembranes. Eur Biophys J. 1994;23(5):337–348. doi: 10.1007/BF00188657. [DOI] [PubMed] [Google Scholar]
  44. Schwille P., Korlach J., Webb W. W. Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry. 1999 Jul 1;36(3):176–182. doi: 10.1002/(sici)1097-0320(19990701)36:3<176::aid-cyto5>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
  45. Simson R., Yang B., Moore S. E., Doherty P., Walsh F. S., Jacobson K. A. Structural mosaicism on the submicron scale in the plasma membrane. Biophys J. 1998 Jan;74(1):297–308. doi: 10.1016/S0006-3495(98)77787-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Singer S. J., Nicolson G. L. The fluid mosaic model of the structure of cell membranes. Science. 1972 Feb 18;175(4023):720–731. doi: 10.1126/science.175.4023.720. [DOI] [PubMed] [Google Scholar]
  47. Soumpasis D. M. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J. 1983 Jan;41(1):95–97. doi: 10.1016/S0006-3495(83)84410-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tocanne J. F., Cézanne L., Lopez A., Piknova B., Schram V., Tournier J. F., Welby M. Lipid domains and lipid/protein interactions in biological membranes. Chem Phys Lipids. 1994 Sep 6;73(1-2):139–158. doi: 10.1016/0009-3084(94)90179-1. [DOI] [PubMed] [Google Scholar]
  49. Vaz W. L., Hallmann D., Clegg R. M., Gambacorta A., De Rosa M. A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. Eur Biophys J. 1985;12(1):19–24. doi: 10.1007/BF00254091. [DOI] [PubMed] [Google Scholar]
  50. Webb W. W., Barak L. S., Tank D. W., Wu E. S. Molecular mobility on the cell surface. Biochem Soc Symp. 1981;(46):191–205. [PubMed] [Google Scholar]
  51. de Almeida Rodrigo F. M., Loura Luís M. S., Fedorov Aleksandre, Prieto Manuel. Nonequilibrium phenomena in the phase separation of a two-component lipid bilayer. Biophys J. 2002 Feb;82(2):823–834. doi: 10.1016/S0006-3495(02)75444-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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