2008 WileyEnciclopedia Supported Lipid Bilayers

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Article Contents

• Supported Bilayers as Models for Cell Mem-branes

• Interactions Between Lipid and Protein Compo-nents in Supported Bilayers

• Procedures to Prepare Supported Bilayers

• Procedures to Characterize Supported Bilayers

• Conclusion

Supported Lipid Bilayers:Development andApplications in ChemicalBiologyVolker Kiessling, Marta K. Domanska, David Murray,Chen Wan and Lukas K. Tamm, University of Virginia, Charlottesville,

Virginia

doi: 10.1002/9780470048672.wecb663

Lipid bilayers supported on solid substrates were developed almost aquarter century ago as a new model membrane system to studyfundamental properties of biological membranes and their constituent lipidand protein molecules, as well as for numerous practical applications. In thisreview, we summarize the development of supported bilayer-based modelmembrane systems of increasing complexity while always keeping in mindtheir primary purpose, which is to reproduce the complex fluid structure ofbiological membranes to mimic cell membrane-based molecular processesas close to physiological reality as possible. This process must includemaintaining the structure and fluidity of the lipid bilayer, as well as thestructure and function of reconstituted membrane proteins. We start thereview with a general description of a contemporary picture of a genericbiological membrane and proceed with an account of how supportedmembranes recapture different physiological aspects of biologicalmembranes, such as membrane fusion, cell adhesion, or membranepermeability. Supported membranes are particularly good models forreproducing the fluidity of cell membranes even when different liquid lipidphases coexist. They are also superb models for measuring ligand-receptorinteractions between membrane-bound receptors and soluble ormembrane-bound ligands. The chemical biology of these and many otherlipid–lipid, lipid–protein, and protein–protein interactions that have beeninvestigated in supported bilayers are summarized in several sections thatform the main body of this review. The article closes with summaries ofpractical procedures to prepare and characterize supported bilayers.

Since the lipid bilayer was accepted as the basic principle ofdesign for biological membranes more than 40 years ago, re-searchers have developed model systems to study numerousaspects of biomembrane structure and function. Large multi-lamellar liposomes have been used frequently in early X-raydiffraction and calorimetric studies that characterize the struc-ture and thermodynamic properties of lipid bilayers. Sonicationof these structures leads to small unilamellar vesicles, which areexcellent model systems for numerous spectroscopic and bind-ing studies with model membranes. Other experiments requirelarger or even “giant” unilamellar vesicles, produced by extru-sion through filters or swelling from solid lipid deposits with

or without applied electric fields. For some applications, suchas recording electrical currents across membranes or measuringthe mobility and lateral organization of membrane constituents,planar bilayer geometry is advantageous. To this end, singleunsupported planar bilayers are suspended with or without sol-vent in a small orifice in a Teflon septum that separates twochambers that are electrically connected to equipment that canrecord currents from single molecular channels (1, 2). A wealthof information on the molecular properties of ion channels, tox-ins, and other membrane-interactive proteins and substances hasbeen obtained using such planar bilayer recordings.

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc. 1

Supported Lipid Bilayers: Development and Applications in Chemical Biology

A different planar model membrane design was developedin the early 1980s mainly from research conducted in HardenMcConnell’s laboratory at Stanford University—the supportedlipid bilayer. The biological motivation driving these early stud-ies was to provide planar model membranes for observationby epi-fluorescence microscopy and to use them as surrogatetarget membranes to study molecular interactions in immuno-logical synapses between antigen-presenting cells and T-cellreceptor-bearing lymphocytes. A precursor of the supportedlipid bilayer has been the supported lipid monolayer, in which alipid monolayer is deposited on a long-chain alkylated substrate(3). This early design using silane chemistry for alkylation waslater superseded with sulfur chemistry on slides with thin evap-orated gold surfaces. However, because these systems mimiconly half a bilayer and cannot accommodate integral membraneproteins, which provide so many key functions to biologicalmembranes, they are not considered in this article.

In a quest to produce a model system that more faithfullyresembles biological membranes and that also allows the func-tional reconstitution of integral membrane proteins, we havedeveloped the supported bilayer as a new model membrane sys-tem (4, 5). Supported bilayers have since become widely used inmany different areas of chemical, biological, and materials re-search. Applications range from fundamental studies of bilayerand membrane properties to their phase behavior; membraneprotein binding; membrane protein structure; electrophoresisand microanalysis of membrane components; functional stud-ies of membrane receptors, pores and channels; interactions withcomponents of bound cells, viruses, and cellular organelles; andso on. This article is organized in four main sections: We willfirst briefly review current concepts of cell membranes and howsupported bilayers can serve as models for cell membranes. Wewill next describe biophysical properties and chemical interac-tions that govern the structure, stability, and use of supportedbilayers in chemical and biological analysis. The article willclose with two sections on procedures that are commonly usedto prepare and characterize supported bilayers.

Supported Bilayers as Modelsfor Cell MembranesBiological membranes are formed by a lipid bilayer with embed-ded proteins. The bilayer-forming lipids have polar headgroupsthat face the aqueous surroundings of the membrane as well astwo hydrophobic tails that face the interior of the membrane.A key feature of cell membranes is that they are both fluid butstill highly ordered in the membrane plane, as has been capturedin the early conception of the fluid mosaic model of biologicalmembranes (6). Lipids move laterally at a fast rate with a lat-eral diffusion coefficient on the order of 1 µm2/s, but they crossthe membrane by flip-flop only once every few hours (7). Theslow kinetic rate of lipid flip-flop allows the cell to establishasymmetry across the bilayer, which, with the input of energy,can be maintained for the entire lifetime of a cell. Membraneproteins come in different varieties. Some have sequences thatcross the membrane, and therefore they are called integral mem-brane proteins. Other membrane proteins, such as the peripheral

membrane proteins, are attached to the membrane surface eitherelectrostatically or by means of covalent linkage to single ormultiple lipid tails. A third class of membrane proteins dip intoonly one leaflet of the bilayer, where they are stabilized by hy-drophobic interactions with the lipids in the bilayer. Regardlessof the specific nature of membrane attachment and incorpora-tion, membrane proteins can diffuse relatively rapidly in theplane of fluid membranes. However, they never cross the lipidbilayer except during biogenesis, which requires specializedmachines called translocons that translocate newly synthesizedpolypeptide chains across cell membranes in a complicated andstill poorly understood energy-requiring process (8).

The notion of a biological membrane represented by a uni-form sea of lipids with proteins freely floating in this envi-ronment has been significantly revised in the last decade or so(Fig. 1a) (9). It has become increasingly clear that biologicalmembranes are laterally structured even if they are still predom-inantly fluid with regard to in-plane diffusion of protein andlipid components. First and foremost, membranes are highlycrowded with proteins. Although the crowding varies greatlybetween different biological membranes (e.g., being very highfor energy-converting membranes in mitochondria and photo-synthetic organelles and rather low for membranes of myelinsheaths that wrap around and electrically insulate neurons),it is clear that membrane proteins do not act independentlyin membranes but often form clusters of interacting species.In a typical average cell membrane, about a quarter to halfof the total cross-sectional area may be occupied by protein.If one considers proteins that have larger extramembraneousdomains than transmembrane domain cross-sections, the areafraction covered with protein may reach half to three quartersof the entire membrane surface. Thus, membrane proteins andlipids are both solvents and solutes at the same time. An al-loy of hydrophobic proteins and lipids may describe the truenature of biological membranes more accurately than a dilutetwo-dimensional solution of proteins in a vast sea of lipids.Second, unlike commonly prepared model membranes, biolog-ical membranes contain thousands of different lipid species(10). The lipid species distinguish themselves by many differentheadgroup classes and chain compositions as well as an expan-sive combinatorial diversity of these structural elements. A verylarge amount of data has been accumulated over the past fourdecades on the structures, energetics, and phase properties ofpure single and two- or three-component lipid bilayers as sum-marized in two monographs (11, 12). We know from this datathat high- and low-melting lipids tend to separate into differentphases and that the lipid phase properties are often dramaticallymodulated by cholesterol.

Even though cholesterol (substituted by other sterols inplants) is chemically classified as a lipid, the molecule is bet-ter grouped it into a special category, which is distinct frommembrane proteins and (phospho- and sphingo-) lipids, whendiscussing its role in biomembrane structure and function.Cholesterol constitutes between 25% and 40% of the total lipidplus cholesterol fraction of most typical cell membranes. Thesenumbers translate into a 33–66% fraction of the noncholes-terol lipids, or considering its smaller size, about a 10–20%

2 WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.


(a)

(b) (c)

Figure 1 Modeling cell membranes with supported bilayers.(a) Contemporary model of a biological cell membrane. The lipid bilayer isheterogeneous across and in the plane of the membrane. The outer leaflet(bottom pink) is enriched in sphingomyelin and the inner cytoplasmicleaflet (top yellow) is enriched in phosphatidyl-serine andphosphatidyl-ethanolamine. Cholesterol and phosphatidyl-choline aredistributed more evenly between the two leaflets. Cholesterol-rich lipiddomains (‘‘rafts’’) are present in register in both leaflets. Transmembraneand monotopic (partially inserted) membrane proteins (larger structuresgreen) occupy much of the available membrane area and are also clusteredinto functional complexes in many cases. Lines (brown) on the topsymbolize interactions with the cytoskeleton, and branched lines (orange)on the bottom of the membrane symbolize carbohydrates of theglycocalix. (b) Supported bilayer with inserted proteins directly supportedon a hydrophilic inorganic substrate (gray). (c) Supported bilayer withinserted proteins supported on a (tethered) polymer cushion (purple) thatincrease the space between the membrane and the support.

area fraction of the lipid-occupied area of a biological mem-brane. Therefore, biological membranes should be consideredtwo-dimensional liquid alloys of three major component classes:lipids, proteins, and cholesterol.

Although some membrane proteins are known to interactwith cholesterol, cholesterol interactions with other lipids havebeen studied much more thoroughly. Most notably, choles-terol interacts strongly with sphingomyelin, perhaps by form-ing complexes, which results in a liquid–liquid phase separa-tion of cholesterol-rich and cholesterol-poor phases (13). Thecholesterol-rich phases exhibit more chain order and there-fore are referred to as liquid-ordered (l o) phases, whereas thecholesterol-poor phases are called liquid-disordered (l d) phases.

In the cell-biological literature, l o phases have often been re-ferred to as “lipid rafts” mostly because certain membraneproteins that are functionally linked are coextracted by milddetergent treatment from complex cell membranes. Despite thefunctional linkage of membrane proteins that are involved, forexample, in cell signaling and the observation that they appear inthe same biochemical fraction as cholesterol and sphingolipids(14), larger platforms of phase-separated cholesterol-rich lipidphases, or “lipid rafts,” that could assemble these proteins havenever been observed directly in cell membranes. Their exis-tence is mainly based on inference from studies with purelipid-cholesterol systems in which cholesterol-rich l o-phase do-mains can be observed. Therefore, the concept of “lipid rafts” asa means of organizing cell membranes remains highly contro-versial. Several excellent reviews critically illuminate the statusof this sometimes-confusing field of membrane biology (15, 16).

Another important component of cell membrane structure andfunction is not integral to the membrane but is adjacent. Cellmembranes are often linked to elements of the cytoskeletonand the extracellular matrix via specialized embedded proteins.Actin networks on the inside of cell membranes and extracellu-lar proteins on the outside confine diffusive and directed motionsof many integral membrane proteins either by direct and indirectattachment to these structures or simply by putting up fences onthe already crowded membrane surface (17). The effect of theseextramembraneous protein networks is often manifested in ir-regular hop-diffusion or anomalous diffusion when analyzed incell membranes with sufficient time and spatial resolution. Suchmembrane-attached protein networks may be less significant inthe case of many intracellular organelle membranes, althoughfor some of these structures (e.g., for clathrin- or COP-coatedendocytic vesicles), similar restrictions on protein motion mayapply.

Supported bilayers with and without embedded membraneproteins offer new opportunities to study several of the afore-mentioned aspects of biological membranes (Figs. 1b and 1c).Because of their planar geometry and stability provided by thesolid support, lateral diffusion of membrane components canbe measured in a straightforward manner by fluorescence re-covery after photobleaching (FRAP) or single particle tracking(SPT). It has been known since the inception of supported bi-layers that phase transitions of lipids are preserved in thesesystems as evidenced by abrupt changes in lateral diffusioncoefficients and morphological changes (4). Lateral phase sep-arations between l o and l d phases and lipid asymmetry mayalso be conveniently studied in supported bilayers (18, 19).Supported bilayers continue to contribute to the current under-standing of the effect of cholesterol on complex lipid mixtures,and interesting new insights may be expected in the near future.Secondary structures and orientations of secondary structure el-ements of membrane proteins and membrane-bound peptidesare conveniently measured by polarized Fourier-transform in-frared (FTIR) spectroscopy in supported bilayers (20). Morerecently, helix–helix interactions of signaling proteins have beenmeasured in supported membranes (21). Helix interactions, forexample in receptor dimerization, are thought to be important intransmembrane signaling of activated receptors, but relativelyfew techniques can measure such interactions in membrane



environments. Another area where supported bilayers offer dis-tinct advantages is studies of the kinetics of ligand binding tomembrane-bound receptors (22). Supported membranes havealso been used successfully to measure protein-mediated fusionof vesicles, for example in virus entry (23) or exocytosis (24).

On the analytical practical side, there is much interest in us-ing supported membranes with incorporated ion channels asbiosensors for various analytes (25–27). Another interestingapplication is to separate membrane components in situ byelectrophoresis in supported membranes (28, 29). Such tech-niques could have a significant future impact on the proteomicsof membrane proteins, that is, an area that is currently under-developed because of difficulties with appropriately separatingmembrane proteins in complex mixtures by more standard tech-niques. Finally, supported membranes continue to provide veryinteresting engineerable substrates that mimick natural cell sur-faces to trigger the differentiation of adjacent cells such as inthe previously mentioned immunological synapses (30).

Interactions Between Lipidand Protein Componentsin Supported Bilayers

A key feature of biological membranes is their graded fluidity.Any useful membrane model system must strive to preserve thecharacteristic fluidity of lipid and protein components. This con-cept clearly has been a great challenge in this field. Although itis relatively easy to maintain the fluidity of the lipid componentsand peripherally attached protein components as demonstratedalready in the earliest publications on supported bilayers (5, 31),to achieve the same result with integral membrane proteins hasproven to be much more difficult. It is known that the gap be-tween supported membranes and solid glass or quartz supportsis on the order of 1 to 2 nm (32, 33), and this gap is filledwith a layer of water that is sufficient to lubricate the lowerleaflet of the bilayer to permit rapid lipid diffusion (Fig. 1b).However, this gap is not large enough to accommodate in-tegral membrane proteins with significant extramembraneousdomains. Such proteins are generally immobilized by interac-tion with the glass support. Much research activity has beendirected in the last decade to uncouple such unwanted non-physiological interactions, by increasing the gap distance withintercalated water-soluble polymers, either by simple physisorp-tion or covalently attached to one or both surfaces by formingtethers between the substrate and the membrane (Fig. 1c). In thefollowing section, we summarize strategies that have been suc-cessful for creating polymer-supported bilayers. The subsequentsections proceed in turn to brief summaries of lipid–lipid in-teractions, lipid–protein interactions, and protein–protein inter-actions in supported bilayers, and then to studies of membranefusion and cell adhesion using supported membranes as a modelfor one membrane in these membrane–membrane interactions.The final sections summarize recent advances in patterning sup-ported membranes and using supported membranes as novelanalytical tools in chemical biology. Depending on the partic-ular application, bilayers directly supported on solid substrates

or (tethered) polymer-supported bilayers are preferred as willbe mentioned in each case.

Polymer-supported bilayerswith and without tethersThe idea of using polymer-supported bilayers has been aroundfor more than a decade (34), but it became practical for chemicaland biological applications only more recently. Early versionshave used relatively short tethers to link the membranes to thesolid substrate and thereby increase their durability for practicalapplications (25, 35). Because these approaches do not increasethe gap distance between substrate and membrane and thereforehave not been used to reconstitute integral membrane proteinsfunctionally, they will not be discussed here.

Our group has developed a polyethylene glycol (PEG)-basedpolymer support for bilayers on glass, quartz, or oxidized silicon(36). PEG that consists of 77 subunits bridges a phospholipidon one end and a silane group on the other end, which al-lows the covalent tethering of the lipopolymer to free silanolson the silicon dioxide surface. In all, 25% of the reconstitutedintegral membrane protein cytochrome b diffused freely in sup-ported bilayers that contained 3% of the lipopolymer in theproximal leaflet. Integral membrane SNARE proteins were 80%mobile with a diffusion coefficient of 0.8 µm2/s in this sys-tem (37). The distance between the substrate and membraneincreased from 2 to 4 nm without and with the polymer, respec-tively (33). A similar approach was introduced by Naumann’sgroup, who used dioctadecylamine(poly(ethyloxazoline)-8988)and octadecyl(poly(ethyloxazoline)-5822) with 85 and 56monomer units, respectively, as lipopolymers in their bilayers(38). In this case, the lipopolymers were photo-cross-linked tothe glass via a benzophenone silane photocoupling agent. Thetethered polymer obstructs lipid diffusion at high concentrationsbut not at low concentrations (39). The increased stability ofthe monolayer that faces the substrate allows the preparation ofasymmetric supported bilayers that contain separated regions ofliquid-ordered and liquid-disordered phases. Lipid raft mixturesin the substrate-proximal layer induce registered raft domainsin the distal layer (40, 41). Sackmann’s group used lipopoly-mers that contain polymerized 2-methyl-2-oxazoline of differentlengths to integrate the large transmembrane cell receptor inte-grin αIIβ3 (42). In all, 20% of the integrins were mobile witha relatively low diffusion coefficient of 0.03 µm2/s. A differentapproach to form a cushion between the solid support and mem-brane uses the Langmuir-Blodgett technique to transfer severallayers of trimethylsilyl cellulose onto a glass substrate. Whensupported bilayers were formed on this substrate by direct vesi-cle fusion, 25% of the reconstituted integrins were mobile witha diffusion coefficient of 0.6 µm2/s (43).

Studies of raft-like lipid domainsin supported bilayersSubstantial literature has been published on studies of lipid do-mains in supported bilayers. Many have investigated lipid mix-tures with coexisting gel and liquid-crystalline phases by atomicforce (AFM), epifluorescence, and near-field fluorescence mi-croscopy (NSOM) (see, for example, References (44–47)).



Other studies have focused on (“raft”) lipid mixtures that formcoexisting l o and l d phase domains in the presence of cholesterol(19,48–50). AFM studies are usually carried out on mica sub-strates, which have the advantage of being atomically flat but areless hydrated than glass or quartz substrates that are commonlyused in fluorescence microscopic studies. Although higher res-olution is achieved by AFM than by optical microscopy, thebilayers are more tightly coupled to the substrate and are notalways fluid on mica. Fluorescence has the advantage that dif-ferent molecular fluorescent dyes can be used to probe differentaspects of complex lipid bilayers, such as diffusion, order, andphase partitioning, but it has the obvious disadvantage that thebehavior of the probe is observed, which may or may not re-flect the behavior of the host lipids accurately. Judicial choicesmust be made when selecting lipid probes for different purposes.Typical “raft” lipid mixtures contain about equimolar sphin-gomyelin, phosphatidylcholine, and cholesterol. Studies haveshown that the area of l o phases in such bilayers depends lin-early on the cholesterol concentration and that a percolationthreshold exists at about 25 to 30 mol % cholesterol, wherel o domains become connected and l d domains become dis-connected (19). Because this mixture is at about physiologicalconcentrations of cholesterol, cells may use shifts in choles-terol concentration as a switch to connect different groups ofmembrane proteins and thereby regulate their function.

An exciting new development in supported bilayer technol-ogy is that bilayers with asymmetric lipid distributions can beprepared. Although it is not so difficult for gel phase lipidswith very low diffusion coefficients, it is much more challeng-ing with fluid phase bilayers. However, these challenges havebeen solved recently (18). It is now possible to study phasecoupling across the mid-plane of bilayers with coexisting l o

and l d phases (40, 41). The observation that l o phases can beinduced in lipid mixtures that mimick the inner leaflet of cellmembranes that do not exhibit such phases on their own hasimportant potential consequences on signal transduction in cellmembranes. Although they do not prove the raft-hypothesis ofsignal transduction in cells, these experiments provide the firstexperimental evidence that fluid lipid bilayers with physiologi-cal lipid compositions can transmit signals across the mid-planeof membranes by inducing new lipid phases on the other side.

Protein–lipid interactionsin supported bilayers

Protein–lipid interactions and particularly peptide–lipid interac-tions have been studied in supported bilayers by attenuated totalreflection (ATR) FTIR spectroscopy. A slightly dated, but stillvalid comprehensive review on this method applied to supportedbilayers has been published (20). Because IR light probes thevibrational properties of different classes of covalent bonds, thismethod is useful to examine lipids, peptides, and interactionsbetween the two in the same sample. The most common pa-rameter for assessing lipid structure and order is to study thestretching vibrations of the lipid acyl chains, for example as afunction of peptide concentration or temperature. Such studieshave lead to the conclusion that fusion peptides from virusesincrease the lipid chain order of fluid phase bilayers and that

this property correlates with the biological activity of the viralpeptide sequences (51). Another prominent band in the FTIRspectra of lipids develops from the ester carbonyl vibration.This band is sensitive to the hydrogen-bonded structure andthus hydration properties of the bilayer interface. The carbonylester band has been shown to change during interaction withsome fusion peptides (52). Lipids also have an effect on pep-tide structure. For example, influenza and HIV fusion peptidesare induced to form α-helices in bilayers at low concentration,but they transition into β-sheets at higher concentration (53, 54).Such structural transitions are readily followed by monitoringthe amide I band of the peptide bonds, which is highly sensitiveto peptide conformation and secondary structure.

Phospholipases bound to supported bilayers hydrolyze dis-tinct paths through the bilayer substrate as observed by AFM(55). The membrane-embedded portions of integral membraneproteins are protected from amide hydrogen exchange, whichcan be measured by a shift in the amide I and II IR bandswhen the system is changed from an H2O to a D2O buffer (56).Binding of proteins to supported bilayers may be studied quan-titatively by total internal reflection fluorescence microscopy(TIRFM), as shown, for example, in binding studies of antibod-ies to lipid haptens (57, 58). Fast kinetics of antibody fragmentbinding and unbinding to supported bilayers have been studiedin detail by combining fluorescence correlation spectroscopywith TIRFM (59).

Another aspect of lipid–protein interaction that is conve-niently studied in supported bilayers is the lateral diffusion ofproteins and lipids and their influence on each other. The regu-latory lipid phosphatidyl-inositol-biphosphate (PIP2) slows thediffusion of syntaxin in supported bilayers (37). Conversely, in-creasing syntaxin concentrations decrease the diffusion of PIP2

and to a lesser extent that of phosphatidylserine. In anothersystem, in which the transmembrane domain of the fibroblastgrowth factor receptor was incorporated into supported bilay-ers, lipid and protein diffusion were measured (60). Althoughprotein diffusion was slow (0.006 µm2/s), lipid diffusion wasfast (2.6 µm2/s).

Protein–protein interactionsin supported bilayersMuch current interest exists in lateral interactions between twoor more integral membrane proteins. Several membrane-boundreceptors are activated by such interactions during ligand bind-ing. In addition, the interaction between individual transmem-brane helices is key to membrane protein folding and has thusreceived a lot of attention. Although these kinds of interac-tions are more often studied in nonsupported model membranes,the Hristova group has recently used supported bilayer for-mats to examine helix–helix interactions between the transmem-brane domains of the human fibroblast growth factor receptor(21). Fluorescence resonance energy transfer measurements offluorescein- and rhodamine-labeled peptides in supported bilay-ers show that these transmembrane domains dimerize with asequence-specific dimerization energy of ∼3.6 kcal/mol. Mean-ingful measurements of such interaction energies require thatthe proteins are laterally mobile in the supported membranes,which was verified in this system (60).



Membrane fusion with supportedbilayers

Membrane fusion is a key process in cell biology that takescenter stage in membrane biogenesis, fertilization, virus entry,and other events. Our group began studying membrane fusionin supported bilayers 14 years ago. Influenza virus hemagglu-tinin (HA) (i.e., the protein that mediates virus entry into cellsby membrane fusion) was reconstituted into supported bilayers.The physiological pH-dependent fusion of liposomes to the pla-nar HA-containing membranes was demonstrated (23). Severalfusion-related structural transformations of HA were recordedby FTIR spectroscopy (61, 62). The fusion of single virions tosupported bilayers has also been reported recently (63).

Recently, SNARE-mediated fusion (i.e., the process that leadsto neurotransmitter release in synaptic transmission) has beenreconstituted in a supported bilayer format in four differentlaboratories including our own. The general design of theseexperiments is illustrated in Fig. 2. Fix et al. (24) observed that15% of the docked vesicles fused within 15 seconds, whichyielded a fusion rate of ∼7 × 10−5 s−1 in POPC bilayers ata coexpressed syntaxin1/SNAP25 protein/lipid concentration of1:300. The fusion probability increased 40-fold after Ca2+ addi-tion. Bowen et al. (64) observed thermally induced fusion withsyntaxin (protein/lipid ratio 1:14,000) in lipid bilayers composedof eggPC and brainPS. In all, 5–15% of docked vesicles fusedwith a rate of 0.07 s−1 within 120 sec after triggering. Liu et al.(65) showed that 77% of the reconstituted synaptobrevin vesi-cles fused within 100 ms after docking (rate, 40 s−1) to syntaxinor syntaxin1/SNAP25 complexes in POPC/DOPS bilayers at aprotein/lipid ratio of 1:30,000. The results of these three groupsare different from each other, and each of these experimentshad several problems, some of which may be caused by the im-mobile reconstitution of the SNARE proteins in the supportedbilayers (66). The independence of fusion on the presence ofSNAP25 in the reconstitutions of Bowen and Liu is puzzlingbecause it contradicts in vivo as well as in vitro fusion results.Preliminary work from our laboratory is in much better agree-ment with the biological literature on this process.

Cell adhesion and signaling in supportedbilayers

Supported bilayers have been used since their inception assurrogate cell surfaces to stimulate immune cells in artifi-cially created immunological synapses. For example, Brian andMcConnell (67) have reconstituted major histocompatibilitycomplex (MHC) proteins into supported bilayers and used theseplanar membranes to stimulate cytotoxic T-cells via specificT-cell receptor interaction. Antigen presentation and signalingthrough the T-cell receptor have been studied in this systemin several follow-up papers as summarized by Watts and Mc-Connell (68). The approach of these early studies (and severalothers that followed) has experienced a recent renaissance be-cause of improved imaging technologies and because we havelearned in the interim how to manipulate supported mem-branes to form specific controllable spatial patterns (see thenext section). These newer approaches are exciting because they

Figure 2 Studying membrane fusion with supported bilayers. Asupported bilayer is suspended from a quartz substrate (top, graybackground) and illuminated by the evanescent wave of a totally internallyreflected laser beam (angled cylinders red). A membrane vesicle isobserved to approach, hemifuse, and then fully fuse with the supportedmembrane. Vesicle contents, lipids, or proteins may be labeledfluorescently to monitor this process.

have allowed investigators to manipulate spatial patterns in theimmunological synapse and thereby ask biologically importantnew questions with regard to the mechanism of cell-inducedcell signaling. Mossman et al. (30). have used laterally con-strained bilayers to induce novel patterns of T-cell receptorand intracellular adhesion molecule (ICAM) distribution inMHC-stimulated cells. The authors used a GPI-linked versionof the MHC in the supported bilayers to circumvent problemsof lateral mobility of the native integral membrane protein inun-cushioned bilayers. Wu et al. (69) demonstrated the compart-mentalization of IgE-receptors in rat basophilic leukemia cellswhen they stimulated these cells with lipid-hapten bound IgEin nano-fabricated bilayer patches.

Adhesion of different immune cells to one another or to epi-thelial cells has also been studied using planar bilayer models.For example, lymphocyte function-associated protein-1 (LFA-1)promotes cell adhesion in inflammation [i.e., a reaction thatcan be mimicked by binding to purified ICAM-1 in supportedmembranes (70)]. Similarly, purified LFA-3 reconstituted intosupported bilayers mediates efficient CD2-dependent adhesionand differentiation of lymphoblasts (71). This work was fol-lowed by a study in which transmembrane domain-anchoredand GPI-anchored isoforms of LFA-3 were compared (72). Be-cause this research occurred before the introduction of polymercushions and because the bilayers were formed by the simplevesicle fusion technique, the transmembrane domain isoformwas immobile, whereas the GPI isoform was partially mobile.By comparing results with these two isoforms at different pro-tein densities in the supported bilayer, the authors showed thatdiffusible proteins at a sufficient minimal density in the sup-ported membrane were required to form strong cell adhesioncontacts in this system.

In another study, endothelial cells were bound via theirintegrin receptors to supported bilayers that presented the



RGD-peptide, which is the classic integrin ligand (73). Thecells spread on RGD presenting membranes but not on con-trol membranes that lacked this peptide. In a similar approach,a laminin-derived peptide was presented on supported bilayersand shown to mediate the spreading and partial differentiationof neuronal subventricular zone progenitor cells (74). The au-thors observed a strong nonlinear relationship between surfaceconcentrations of the peptides and conclude that this approachmay provide novel conditions for growing stem cells with onlya limited and controlled amount of differentiation induction.

Patterning supported bilayers

To use supported bilayers as platforms for screening assays,different approaches have been taken to pattern the membraneon the surface. One goal is to observe the membrane interac-tion of substances simultaneously with supported bilayers ofdifferent composition. Bilayers can be subdivided into differentareas by diffusion barriers erected on the substrate or by blot-ting patches of membrane with polydimethylsiloxane (PDMS)stamps (75). A combination of these techniques may be usedto form patterned membranes of different composition. Barriersmay be formed from metal or metal oxides by standard litho-graphic techniques or by stamping proteins onto the substrate(76). Originally, homogeneous supported membranes have beenpatterned photolithographically using deep-UV light (77). Us-ing photolithography in combination with a polymer lift-offtechnique, patterned lipid bilayers with patches measuring 1to 76 µm may be formed (78). Another approach uses the flowof vesicle suspensions within microfluidic devices to depositmembranes of different compositions at the same time (75).Supported membranes of different compositions on the samesubstrate are conveniently addressed with different solutions ofanalytes flowed through micro-channels in PDMS (79).

Using supported bilayers as platforms forchemical sensing and analysis

Because of their planar geometry, supported bilayers are pre-destined for biological and chemical sensor applications. Thebasic concept is to couple the high specificity and sensitiv-ity of molecular membrane receptors to substrates that inte-grate opto-electronic circuits (25–27). In combination with thedescribed patterning techniques, supported membranes shouldprovide a nearly ideal physiological environment for high-throughput sensing with biological membrane receptors or chan-nels in chip-based arrays. However, despite their great promise,economically viable commercial applications have so far not yetbeen used because of a series of obstacles that still must be over-come. Nanoscopic and microscopic defects in fluid supportedlipid bilayers most often lead to low electrical resistance andtherefore make them unsuitable for high-sensitivity electronicdetection. In some approaches, this problem has been overcomeby linking the lipid bilayer with very short and dense tethers tothe substrate. However, doing so prevents the incorporation oflarger membrane receptors and channels.

Another approach is to prepare black lipid membranes withsolvent over microscopic or nanoscopic holes in the substrate

(80–82), or by using giant vesicles that adhere to the sub-strate (83). Although high-resistance seals have been obtained,long-term stability has not yet been achieved with these systems.In cases where highly insulating membranes are established, theconductivity of ligand-gated ion channels or larger pores may berecorded (25, 84). Alternatively, the conductivity may be probedby measuring the impedance (26) or with metal-free field-effecttransistors that have the advantage of avoiding electrochemicalperturbations (83). Optical detection is suitable for applicationssuch as immunoassays when the molecules of interest can beaddressed by fluorescent- or gold-labeled antibodies (79). As istrue for electronic biosensors, optical biosensors designed forroutine practical applications must be robust and stable for along time. Lipopolymers that stabilize supported bilayers againstair exposure are also helpful in this case (85).

Procedures to Prepare SupportedBilayers

Supported lipid bilayers with or without reconstituted membraneproteins are prepared by one of three methods described belowand illustrated in Fig. 3. Methods details can be found inReference 86.

Langmuir-Blodgett/Langmuir-Schafer(LB/LS) technique (4, 5)

This technique is historically the first method to prepare sup-ported bilayers. A lipid monolayer is spread from a desiredlipid solution in organic solvent onto a pure water surface in aLangmuir trough. After evaporation of the solvent, the mono-layer is compressed slowly to reach a surface pressure of 32mN/m (thought to be the equivalence pressure of a bilayer) andequilibrated. A carefully cleaned hydrophilic substrate (glass,quartz, oxidized silicon, etc.) is then rapidly submerged into thetrough and slowly withdrawn with a dipper mechanism whilea constant surface pressure is maintained. This step transfersa single monolayer of lipids known as the LB layer onto thesubstrate. A second monolayer known as the LS layer is thenspread and compressed on the trough in the same fashion. TheLB-coated substrate is attached to a suctioning tip, and its faceis gently lowered to contact the LS monolayer at the air/waterinterface for a few seconds. To complete the bilayer, the slideis then pushed through the interface and placed on a coverslip fitted with two spacers of water-resistant double-sided tapethat had been previously placed at the bottom of the trough.After removal of the supported bilayer sandwich from thetrough, the water between the surfaces may be exchanged byflow-through with any desired buffer while always maintainingfull hydration of the bilayer. Some investigators have preparedpeptide-containing supported bilayers by placing peptides in thefirst or second monolayer.

Vesicle fusion (VF) technique (67)

This technique is the simplest method for forming supportedbilayers. Much literature is available on the mechanism and



(a)

(b)

(c)

(d)

Figure 3 Methods for supported bilayer formation and membrane protein reconstitution. (a) and (b) LB/LS method. A lipid monolayer is spread at theair-water interface of a Langmuir trough and transferred to a solid substrate while keeping the surface pressure constant. A second monolayer is transferredby horizontal apposition of the first transferred monolayer and collection of a counter-piece with spacers. (c) Direct VF method. Membrane vesicles areflown into a chamber with a clean surface substrate on top. After about an hour of incubation, they form a supported bilayer on the substrate and excessvesicles are flushed out. (d) LB/VF method. The procedures depicted in panels (a) and (c) are combined leading to an asymmetric bilayer with anasymmetric protein distribution. Although this method can also be performed without a polymer, it is shown here with the polymer transferred during theLB step.

kinetics of vesicle spreading on hydrophilic substrates, whichis not reviewed here. Although it is simple, the method tendsto result in bilayers with more defects, and the orientation ofmembrane proteins cannot be controlled in this method. Smallor large unilamellar pure lipid vesicles or proteoliposomes areprepared by standard liposome preparation or membrane proteinreconstitution methods. A clean hydrophilic substrate is placedin a flow-through chamber, and the vesicles are injected andincubated with the surface for 30–60 minutes. Excess vesiclesare washed out by extensive rinsing with a buffer. This methodhas also been used to make polymer-supported bilayers, inwhich case the surface of the support is pre-treated with thepolymer using conditions that depend on the particular polymerbeing used.

Langmuir-Blodgett/vesicle fusion(LB/VF) technique (87)This method is a combination of the other two methods. Inour opinion, it is the most gentle method to reconstitute mem-brane proteins into supported bilayers and to prepare supportedbilayers with fragile coexisting liquid phases of lipids. A LBmonolayer is prepared on a hydrophilic substrate as describedabove. To prepare tethered-polymer supported bilayers, a suit-able lipopolymer may be included at a concentration of a few

mol % at this stage. With some lipopolymers, it is neces-sary to cure the slide (by light, temperature, zero humidity,etc.) at this stage. The monolayer-coated slide is then placedin a custom-built flow-through chamber, and vesicles or pro-teoliposomes are injected and incubated with the surface for30–60 minutes (pure lipid vesicles) or 60–120 minutes (pro-teoliposomes). Excess vesicles are washed out by extensiverinsing with buffer. Because the second monolayer is com-pletely vesicle derived and because membrane proteins areintroduced as proteoliposomes only in the second step, theytend to be unidirectionally oriented in the supported bilayeras can be verified with quenching antifluorophore antibodies(23, 37) or by FLIC microscopy (33).

Procedures to CharacterizeSupported Bilayers

Microscopy

The simplest way to examine the quality and integrity of sup-ported membranes is to include a fluorescent lipid probe such asnitrobenzoxa-diazol (NBD)-PE or rhodamine-PE and to look for



their uniform appearance on a standard epifluorescence micro-scope. Many artifacts can be detected readily and eliminatedwith this very simple test. Higher-resolution images can beobtained by AFM or NSOM. These techniques are useful to de-tect small defects in the 10 to 500 nm range that might escapedetection by standard wide-field optical microscopy. Becausesupported bilayers are only stable under water, these imagingmodalities must be carried out under water. AFM probes heightprofiles, but NSOM and epifluorescence microscopy permit thelabeling of specific chemical structures or physical propertiesof the structures by using different fluorescent probes. It is of-ten necessary to discriminate surface from bulk fluorescence,for example when measuring the binding of fluorescent ligandsto supported membranes. This result is conveniently achievedwith TIRFM, which has a typical 1/e illumination depth of 50 to100 nm from the surface, which depends on refractive indices,angle of incidence, and wavelength of light. Brewster angle andsurface plasmon microscopies are also surface-selective opticalimaging techniques, which do not require the fluorescent label-ing of the membrane, but rather depend on lateral changes ofrefractive index in the sample.

Lateral diffusionA next and very important level of characterization of supportedbilayers is the measurement of the lateral mobility of theirconstituents. Because biological membranes and their properfunction are defined by their fluidity, to recreate this charac-teristic is imperative for biology-motivated work. In addition,lateral diffusion measurements on supported bilayers can easilydetect many artifacts that may go unnoted by simple micro-scopic inspection. For example, deposited membranes may lookcompletely uniform but may not show any long-range lateraldiffusion when vesicles or membrane fragments that are smallerthan the resolution of the light microscope are densely packedon the substrate surface. Two techniques are common to deter-mine the diffusion of fluorescently labeled lipids or proteins insupported bilayers: FRAP and SPT. The lateral diffusion coeffi-cient and fraction of mobile molecules are obtained from eithermeasurement.

In FRAP, a brief pulse of intense laser light is used tophotobleach fluorophores partially in a small area of the sample.The recovery of fluorescence caused by diffusion of labeledmolecules into the bleached area is then observed over time,while care is taken to minimize additional photobleaching. Inspot photobleaching the light is focused to a circular spot,which reflects the Gaussian beam profile of the laser. Duringrecovery, the half-width of the bleached area decreases whereasthe intensity increases. The diffusion coefficient and mobilefraction are extracted from the time course and the amplitude,respectively, of the recorded recovery curve (88). In a variantcalled periodic pattern photobleaching, the bleach pulse projectsa stripe pattern of a Ronchi ruling onto the sample, whichpermits integration over a larger area and therefore an increasedsignal/noise (89).

SPT is the preferred technique when more detail on differentpopulations of moving particles in a heterogeneous system isrequired. Although the information content of SPT is muchhigher than that of FRAP, it is much more demanding on

instrumentation and statistical evaluation procedures. SPT hasbeen introduced to the characterization of supported bilayers in1995 (90) and has been used frequently since then. In practice,the technique is best used in combination with TIRF microscopyand high-sensitivity charge-coupled devices. Typical labelingratios of the lipid bilayer are 1:108 fluorescent probes:lipids.The reconstructed particle trajectories and appropriate statisticscan be analyzed to distinguish between diffusion, anomalousdiffusion, confined diffusion, and directed motions.

Bilayer structure

Neutron reflectivity has been used to characterize the transverseorganization of supported bilayers structurally (32, 91). The lat-eral structure of lipid bilayers on solid supports may also becharacterized by grazing incidence X-ray diffraction, althoughthis technique has so far been mainly used on monolayers at theair-water interface. Vibrational spectroscopies open interestingwindows to look at details of lipid structure in supported bilay-ers. FTIR spectroscopy has several bands that are characteristicof the state of lipid order and hydration in supported bilayers(20). An interesting relatively recent method to study the orga-nization of supported bilayers, particularly with respect to theirasymmetry, is sum frequency vibrational spectroscopy (SFVS)(92). Signals develop in this nonlinear form of vibrational spec-troscopy only when symmetry is broken (i.e., when the type,structure, and number of lipids is unequal across the mid-planeof the supported bilayer). A complementary technique to studylipid asymmetry, or more specifically fluorescent probe asym-metry, is FLIC microscopy, in which the average distance offluorophores from an oxidized silicon mirror surface is mea-sured interferometrically (18).

Protein secondary structure andorientation

Two methods have been used to determine the secondarystructure and orientation of membrane proteins in supportedbilayers: polarized ATR-FTIR spectroscopy and oriented CDspectroscopy. SFVS may also be applied to study peptide andprotein structures in supported bilayers. Polarized ATR-FTIRspectroscopy is sensitive enough that high-quality spectra canbe obtained from a single bilayer. Beta-sheet structures arereadily distinguished from α-helical and random structures, andthe orientations of α-helices are determined from the lineardichroism of the peptide amide I bands (20). Multiple stacksof supported bilayers have to be used to gain enough sensitivityto determine the structure and orientation of α-helices in lipidbilayers by oriented CD spectroscopy (60, 93).

Conductance and impedance

Pure lipid membranes are electrical insulators with a specificcapacitance of ∼1 µF/cm2, which separate two electrolyticcompartments. The conductance of biological membranes ismainly determined by highly specialized proteins that act asion channels. For supported membranes to mimic the electricalproperties of a biological membrane, it is necessary to measureits electrical characteristics. Even very small defects that are not



visible by microscopy increase the conductance significantly. Ifthe membrane is supported by a conductive substrate like goldor indium tin oxide, then it is possible to measure the impedanceby applying A/C voltages with frequencies up to 100 kHz andby measuring the magnitude and phase of the current. Theconductance and capacitance of the supported membrane isdetermined from the evaluation of the entire electrical circuit,which includes resistance and capacitance of the electrode andif necessary alternative electrical paths around the membrane(94). A similar approach uses metal-free field effect transistorsthat probe the electrical potential at the transistor gates thatface the cleft beneath the supported membrane. The electricalmembrane parameters are extracted from the voltage transferfrom the electrolyte bath to the transistor gates (83).

Conclusion

In conclusion, supported bilayers have evolved into a reli-able model membrane system since their first inception almosta quarter century ago. Numerous basic research questions re-garding the structure and function of biological membranes andapplications that range from biosensing to proteomic analysesof membrane components have been addressed with this sys-tem. We anticipate more growth and an even more prominentrole of this tool in basic and applied membrane research in thedecades to come.

Acknowledgments

The work in our laboratory was supported by grants from theNational Institutes of Health.

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See Also

Membranes, Fluidity ofMembrane Proteins, Properties ofMembrane Fusion, Mechanisms ofLipid Bilayers, Properties ofLipid RaftsBiosensorsBio/Inorganic Interfaces


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