Measuring Lipid Asymmetry in Planar Supported Bilayersby Fluorescence Interference Contrast MicroscopyJonathan M. Crane, Volker Kiessling, and Lukas K. Tamm*Department of Molecular Physiology and Biological Physics, University of Virginia,Charlottesville, Virginia 22908-0736Received September 20, 2004. In Final Form: November 12, 2004There is substantial scientific and practical interest in engineering supported lipid bilayers withasymmetric lipid distributions as models for biological cell membranes. In principle, it should be possibleto make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Scha¨fer (LB/LS) orLangmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103,307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never beenexperimentally examined in any of these systems. In the present work, we developed a technique that isbased on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supportedbilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and withoutcholesterol in liquid-ordered (lo) and liquid-disordered (ld) phases. Of five different fluorescent lipid probesthat were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the bestfor studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetricallylabeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed.In LB/LS bilayers of either loor ldphase, cholesterol increased the degree of lipid mixing between theopposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipidasymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetriclabel, with or without the use of a tethered polymer support. Finally, lipid mixing across the center ofLB/LS bilayers was found to have drastic effects on the appearance of ld-lophase coexistence as shownby epifluorescence microscopy.IntroductionNatural cell membranes are composed of lipid bilayerswith asymmetric phospholipid compositions.1In theplasma membrane of the human erythrocyte, for example,>60% of all phosphatidylcholine (PC) and >80% of allsphingomyelin (SM) reside in the outer leaflet, while mostof the phosphatidylethanolamine (PE), phosphatidylserine(PS), and phosphatidylinositol (PI) is found in the innerleaflet.2-4There are several functional reasons andconsequences of lipid asymmetry, which is why cellsemploy ATP-dependent translocases to move specific lipidsto the desired face of the membrane.2-4An increase inextracellular PS and PE has been correlated with celldeath.3,4Lipids involved in signaling events, such as PIP2,are found only in the inner leaflet, where they interactspecifically with tyrosine kinase receptors as part ofG-protein-receptor-mediated signal transduction path-ways.5Several investigators have found evidence that thelipids in the outer leaflet (PC, SM, and cholesterol) of cellmembranes are capable of segregating into domains,sometimes called “rafts”, of a more ordered lipid phase.6-8Recent fluorescence studies in model membranes haveconfirmed this in vitro,9-12but so far attempts at recon-stituting ordered lipid domains in model membranes thatmimic the inner leaflet have failed.13,14Still, others havesuggested that rafts play a role in cell signaling,15andlipid microdomains have also been cited as affectors ofviral assembly.16Cholesterol has been shown to causeclustering of soluble N-ethyl-maleimide-sensitive factor-attachment protein receptor (SNARE) proteins, but it isunclear whether this plays a role in SNARE-mediatedexocytosis.17Can ordered lipid domains in the outer leafletinfluence these events that occur on the inner leaflet ofthe membrane? For PC/SM/cholesterol phase separation* To whom correspondence should be addressed. E-mail:[email protected]. Tel: (434) 982-3578. Fax: (434) 982-1616.(1) Bretscher, M. S. Asymmetrical lipid bilayer structure for biologicalmembranes. Nat. New Biol. 1972, 236 (61), 11-12.(2) Devaux, P. F. Static and dynamic lipid asymmetry in cellmembranes. Biochemistry 1991, 30 (5), 1163-1173.(3) Boon, J. M.; Smith, B. D. Chemical control of phospholipiddistribution across bilayer membranes. Med. Res. Rev. 2002, 22 (3),251-281.(4) Quinn, P. J. Plasma membrane phospholipid asymmetry. Subcell.Biochem. 2002, 36,39-60.(5) Verkleij, A. J.; Post, J. A. Membrane phospholipid asymmetryand signal transduction. J. Membr. Biol. 2000, 178 (1), 1-10.(6) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature1997, 387 (6633), 569-572.(7) Brown, D. A.; London, E. Structure and function of sphingolipid-and cholesterol-rich membrane rafts. J. Biol. Chem. 2000, 275 (23),17221-17224.(8) Anderson, R. G.; Jacobson, K. A role for lipid shells in targetingproteins to caveolae, rafts, and other lipid domains. Science 2002, 296(5574), 1821-1825.(9) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.;Levi, M.; Jacobson, K.; Gratton, E. Lipid rafts reconstituted in modelmembranes. Biophys. J. 2001, 80 (3), 1417-1428.(10) Silvius, J. R. Fluorescence energy transfer reveals microdomainformation at physiological temperatures in lipid mixtures modeling theouter leaflet of the plasma membrane. Biophys. J. 2003, 85 (2), 1034-1045.(11) Baumgart, T.; Hess, T. S.; Webb, W. W. Imaging coexisting fluiddomains in biomembrane models coupling curvature and line tension.Nature 2003, 425, 821-824.(12) Crane, J. M.; Tamm, L. K. Role of cholesterol in the formationand nature of lipid rafts in planar and spherical model membranes.Biophys. J. 2004, 86 (5), 2965-2979.(13) Wang, T. Y.; Silvius, J. R. Cholesterol does not induce segregationof liquid-ordered domains in bilayers modeling the inner leaflet of theplasma membrane. Biophys. J. 2001, 81 (5), 2762-2773.(14) Crane, J. M.; Tamm, L. K. Cholesterol content and phospholipidasymmetry: Effect on raft formation in planar model membranes.Biophys. J. Abstr. 2003, 1811-Pos.(15) Baird, B.; Sheets, E. D.; Holowka, D. How does the plasmamembrane participate in cellular signaling by receptors for immuno-globulin E? Biophys. Chem. 1999, 82 (2-3), 109-119.(16) Freed, E. O. Virology. Rafting with Ebola. Science 2002, 296(5566), 279.1377Langmuir 2005, 21, 1377-138810.1021/la047654w CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 01/15/2005to affect the inner