BA SE Biotechnol. Agron. Soc. Environ. 2010 14(4), 719-736 Focus on: From biological membranes to biomimetic model membranes Marc Eeman, Magali Deleu Univ. Liege - GemblouxAgro-Bio-Tech. Department of Biological Industrial Chemistry. Passage des Déportés, 2. B-5030 Gembloux (Belgium). E-mail: [email protected]Received on June 4, 2009; accepted on December 9, 2009. Biological membranes play an essential role in the cellular protection as well as in the control and the transport of nutrients. Many mechanisms such as molecular recognition, enzymatic catalysis, cellular adhesion and membrane fusion take place into the biological membranes. In 1972, Singer et al. provided a membrane model, called fluid mosaic model, in which each leaflet of the bilayer is formed by a homogeneous environment of lipids in a fluid state including globular assembling of proteins and glycoproteins. Since its conception in 1972, many developments were brought to this model in terms of composition and molecular organization. The main development of the fluid mosaic model was made by Simons et al. (1997) and Brown et al. (1997) who suggested that membrane lipids are organized into lateral microdomains (or lipid rafts) with a specific composition and a molecular dynamic that are different to the composition and the dynamic of the surrounding liquid crystalline phase. The discovery of a phase separation in the plane of the membrane has induced an explosion in the research efforts related to the biology of cell membranes but also in the development of new technologies for the study of these biological systems. Due to the high complexity of biological membranes and in order to investigate the biological processes that occur on the membrane surface or within the membrane lipid bilayer, a large number of studies are performed using biomimicking model membranes. This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition, membrane dynamic and molecular organization, as well as at describing the most common biomimicking models that are frequently used for investigating biological processes such as membrane fusion, membrane trafficking, pore formation as well as membrane interactions at a molecular level. Keywords. Biological membranes, nanoscale membrane organization, model membranes, lipid monolayers, lipid vesicles, supported lipid bilayers. Des membranes biologiques aux modèles membranaires biomimétiques. Les membranes biologiques jouent un rôle essentiel dans la protection cellulaire ainsi que dans le contrôle et le transport des éléments nutritifs. Elles sont le lieu de nombreux mécanismes biologiques tels que la reconnaissance moléculaire, la catalyse enzymatique, l’adhésion cellulaire ou encore la fusion membranaire. En 1972, Singer et al. ont proposé un modèle membranaire, appelé modèle de la mosaïque fluide. Selon ce modèle, les membranes biologiques consistent en des bicouches lipidiques dynamiques renfermant des protéines et des glycoprotéines. Les protéines membranaires forment des icebergs globulaires dans une mer homogène de lipides à l’état fluide. Depuis sa conception, le modèle de la mosaïque fluide a fortement évolué notamment suite aux travaux de Simons et al. (1997) et de Brown et al. (1997). Ces chercheurs ont montré que les lipides membranaires sont en réalité organisés en microdomaines (rafts lipidiques) dont la composition et la dynamique moléculaire sont bien spécifiques et différentes de celles de la phase liquide cristalline formée par les phospholipides environnants. Une telle découverte a entrainé un intérêt sans cesse croissant pour l’étude des membranes biologiques et a provoqué par la même occasion une émergence de nouvelles technologies de pointe pour améliorer nos connaissances sur ces systèmes biologiques extrêmement complexes. Aussi, en raison de la grande complexité des membranes biologiques, la plupart des études ciblant les mécanismes biologiques qui se déroulent à la surface des membranes ou au sein de leur bicouche lipidique sont réalisées en utilisant des modèles membranaires biomimétiques. Cette synthèse a donc pour principaux objectifs de revisiter les propriétés de base des membranes biologiques en termes de composition membranaire, de dynamique membranaire et d’organisation moléculaire, ainsi que de décrire les modèles membranaires les plus couramment utilisés pour étudier des mécanismes biologiques tels que la fusion membranaire, le transport membranaire, la formation de pores membranaires, ainsi que les interactions membranaires à l’échelle moléculaire. Mots-clés. Membranes biologiques, organisation membranaire à l’échelle nanométrique, modèles membranaires, monocouches lipidiques, vésicules lipidiques, bicouches lipidiques supportées.
Transcript
BASE Biotechnol. Agron. Soc. Environ.201014(4),719-736 Focus on:
Biologicalmembranesplayanessentialroleinthecellularprotectionaswellasinthecontrolandthetransportofnutrients.Manymechanismssuchasmolecularrecognition,enzymaticcatalysis,cellularadhesionandmembranefusiontakeplaceintothebiologicalmembranes.In1972,Singeretal.providedamembranemodel,calledfluidmosaicmodel,inwhicheachleafletofthebilayerisformedbyahomogeneousenvironmentoflipidsinafluidstateincludingglobularassemblingofproteinsandglycoproteins.Sinceitsconceptionin1972,manydevelopmentswerebroughttothismodelintermsofcompositionandmolecularorganization.ThemaindevelopmentofthefluidmosaicmodelwasmadebySimonsetal.(1997)andBrownetal.(1997)whosuggestedthatmembranelipidsareorganizedintolateralmicrodomains(orlipidrafts)withaspecificcompositionandamoleculardynamicthataredifferenttothecompositionandthedynamicofthesurroundingliquidcrystallinephase.Thediscoveryofaphaseseparationintheplaneofthemembranehasinducedanexplosionintheresearcheffortsrelatedtothebiologyofcellmembranesbutalsointhedevelopmentofnewtechnologiesforthestudyofthesebiologicalsystems.Duetothehighcomplexityofbiologicalmembranesandinordertoinvestigatethebiologicalprocessesthatoccuronthemembranesurfaceorwithinthemembranelipidbilayer,alargenumberofstudiesareperformedusingbiomimickingmodelmembranes.This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition,membranedynamicandmolecularorganization,aswellasatdescribing themostcommonbiomimickingmodels thatarefrequentlyusedforinvestigatingbiologicalprocessessuchasmembranefusion,membranetrafficking,poreformationaswellasmembraneinteractionsatamolecularlevel.Keywords.Biologicalmembranes,nanoscalemembraneorganization,modelmembranes, lipidmonolayers, lipidvesicles,supportedlipidbilayers.
Des membranes biologiques aux modèles membranaires biomimétiques. Les membranes biologiques jouent un rôleessentieldans laprotectioncellulaireainsiquedans lecontrôleet le transportdesélémentsnutritifs.Ellessont le lieudenombreuxmécanismesbiologiquestelsquelareconnaissancemoléculaire,lacatalyseenzymatique,l’adhésioncellulaireouencorelafusionmembranaire.En1972,Singeretal.ontproposéunmodèlemembranaire,appelémodèledelamosaïquefluide.Seloncemodèle,lesmembranesbiologiquesconsistentendesbicoucheslipidiquesdynamiquesrenfermantdesprotéinesetdesglycoprotéines.Lesprotéinesmembranairesformentdesicebergsglobulairesdansunemerhomogènedelipidesàl’étatfluide.Depuissaconception,lemodèledelamosaïquefluideafortementévoluénotammentsuiteauxtravauxdeSimonsetal. (1997)etdeBrownetal. (1997).Ceschercheursontmontréque les lipidesmembranairessonten réalitéorganisésenmicrodomaines(raftslipidiques)dontlacompositionetladynamiquemoléculairesontbienspécifiquesetdifférentesdecellesdelaphaseliquidecristallineforméeparlesphospholipidesenvironnants.Unetelledécouverteaentrainéunintérêtsanscessecroissantpourl’étudedesmembranesbiologiquesetaprovoquéparlamêmeoccasionuneémergencedenouvellestechnologiesdepointepouraméliorernosconnaissancessurcessystèmesbiologiquesextrêmementcomplexes.Aussi,enraisondelagrandecomplexitédesmembranesbiologiques,laplupartdesétudesciblantlesmécanismesbiologiquesquisedéroulentàlasurfacedesmembranesouauseindeleurbicouchelipidiquesontréaliséesenutilisantdesmodèlesmembranairesbiomimétiques.Cettesynthèseadoncpourprincipauxobjectifsderevisiterlespropriétésdebasedesmembranesbiologiquesentermesdecompositionmembranaire,dedynamiquemembranaireetd’organisationmoléculaire,ainsiquededécrirelesmodèlesmembranaireslespluscourammentutiliséspourétudierdesmécanismesbiologiquestelsquelafusionmembranaire,letransportmembranaire,laformationdeporesmembranaires,ainsiquelesinteractionsmembranairesàl’échellemoléculaire.Mots-clés.Membranesbiologiques,organisationmembranaireàl’échellenanométrique,modèlesmembranaires,monocoucheslipidiques,vésiculeslipidiques,bicoucheslipidiquessupportées.
Biological membranes play an essential role in thecellular protection as well as in the control and thetransport of nutrients. Many mechanisms such asmolecular recognition, enzymatic catalysis, cellularadhesion and membrane fusion take place into thebiological membranes. The detailed organization ofthesemembranesatamolecularleveliscurrentlynotyet fully determined even ifmany experimentswereconductedinthelastcenturytoachievesuchagoal.
The concept that biological membranes arecomposedoftwooppositelayersoflipidswasalreadyfoundoutin1925byGorteretal.whoobservedusingtheLangmuirtroughtechniquethatthemolecularareaoflipidsextractedfromredbloodcellswastwotimestheareaoftheredbloodcellsmeasuredbymicroscopy.The first membrane model including proteins datesfrom1935andwasproposedbyDaniellietal.Theseresearchers postulated that a protein layer is tightlyassociatedtothepolarheadsoflipidscomposingthecellmembranes.Itwasforcedtowaitmorethanthirtyyears tofindout thatproteinsmayalsospanthroughmembranes.Suchdiscoveryledto theso-calledfluidmosaic model proposed by Singer et al. in 1972.According to this model, each leaflet of the bilayeris formed by a homogeneous environment of lipidsin a fluid state incorporating globular assembling ofproteinsandglycoproteins.Singeretal.alsoassumedthat the lipidcompositionwithin thebilayers ismostlikelyasymmetric.Sinceitsconceptionin1972,somedevelopmentsandrefinementswerebroughttothefluidmosaicmodelespeciallyintermsofcompositionandmolecularorganization.Themostimportantevolutionofthismodelwasobtainedin1997withtheworksofSimonsetal.andofBrownetal.TheseauthorsshowedthatbiologicalmembranesdonotformahomogeneousfluidlipidphaseaspredictedbySingeretal.Incontrast,theysuggestedthatmembranelipidsareorganizedintophase-separatedmicrodomains,calledlipidrafts,withbothaspecificcompositionandamoleculardynamicthat are different to the ones of the surroundingliquidcrystallinephase.Thediscoveryofsuchphaseseparation in theplaneof themembranehas inducedinthelastdecadeanexplosionintheresearcheffortsrelatedtothebiologyofthecellmembraneaswellasinthedevelopmentofnewtechnologiesfordetectinglateral heterogeneities in biological membranes.Nowadays,whilethereisnodoubtaboutthepresenceof phase separation in the plane of the membrane,the existence of lipid rafts,which are believed to beenrichedinsphingolipidsandcholesterol,topresentahighmobilityintheplaneofthemembraneandtobeinvolved inmanybiologicalprocessessuchassignaltransduction,membrane transportandproteinsorting(Simons et al., 1997) is still controversial. A more
detailed discussion about this hot and controversialissue is given later in this paper (see “Membranecomplexityatthenanometrescale”,p697).Figure 1depictstheactualviewofbiologicalmembranes,whichexhibit lateral heterogeneities, cluster and domainformationwithinthemembraneplane.
2. LIpID coMposItIon oF MeMbranes
Biological membranes display a very complexcompositionintermsoflipidsandproteins.Membranelipids are amphiphilic, i.e. they are constituted of ahydrophilicheadgroupandahydrophobicregion.Thelatteroneisprincipallycomposedofaliphaticchains,aromatic groups or polycyclic structures (Heleniuset al., 1975; Lichtenberg et al., 1983). Due to theiramphipathicity and to their geometric constraints,membranelipidsself-associateintobilayersinaqueousmedium.Membranelipidsareclassifiedintothreemaingroups,namelyphospholipids,glycolipidsandsterols.
The main phospholipids found in biologicalmembranesareglycerophospholipids(40-60mol%ofthetotallipidfraction)(Figure 2a).Thesecompoundsare composed of a glycerol backbone onwhich twofatty acid chains are esterified in position sn-1 andsn-2,respectively.Thethirdcarbonatomoftheglycerolbackbone (position sn-3) supports the phospholipidpolar head group, which is composed of an alcoholmolecule (choline, ethanolamine, serine, glycerol orinositol) linked to a negatively charged phosphategroup. The phospholipid polar head group can bezwitterionicornegativelycharged.Thefattyacidchaininposition sn-1 is generally saturated and composedof16or18carbonatomswhilethefattyacidchaininpositionsn-2islongerandusuallyunsaturated(oneorseveraldoublebondsincisconfiguration)(McElhaneyetal.,1971).
Figure 1.Modern viewof biologicalmembranes (PicturegeneratedbyH.SeegerfromMonteCarlosimulationsandkindlyprovidedbyT.Heimburg,NBICopenhagen)—Vue actuelle des membranes biologiques (figure générée par H. Seeger à partir de simulations Monte Carlo et aimable-ment fournie par T. Heimburg, NBI Copenhagen).
Sphingolipids are another important class ofmembrane lipids and are believed to be involved inthe formation of lateral microdomains in biologicalmembranes.Theselipidsarecomposedofasphingosine(or phytosphingosine) base on which is linked arelativelylong(upto24carbonatoms)saturatedfattyacid chain.Acylated sphingosines are referred to asceramides. Sphingomyelin and glycosphingolipids(Figure 2b) result from the attachment of a cholinemoleculeandanoligosaccharidetothehydroxylgroupofceramides,respectively.
Sterols are a particular class ofmembrane lipids.While thehydrophobicmoietyofmostofmembranelipidsisconstitutedofrelativelylongaliphaticchains,theoneofsterolsiscomposedofpolycyclicstructures.The most abundant sterol in mammal is cholesterol(Figure 2c). This compound is very abundant inerythrocytemembranes,otherplasmamembranesandvarioussub-cellularcompartments ineukaryotes(30-50mol%ofthetotallipidfraction).Itcomprisesfourfusedcyclesin transconfiguration,ahydroxylgroupinposition3,adoublebondbetweenthecarbon5and
6,aswellasaniso-octyl lateralchaininposition17.Thehydroxylgroupisresponsiblefortheamphiphilicnature of cholesterol and consequently for itsorientation inbiologicalmembranes (Tanford,1980).Ergosterolandlanosterolaretwootherrepresentativesofthesterolclass.Thesecompoundsexhibitasimilarstructuretotheoneofcholesterol.Ergosterolisfoundin the membranes of fungi, yeasts and protozoans,(Brennan et al., 1974) while lanosterol is the sterolof prokaryotes and the chemical precursor of bothcholesterolandergosterol(Henriksenetal.,2006).
The shape of a membrane lipid depends on theeffective area of its polar head group compared tothe dimension of its hydrophobic moiety (Cullisetal., 1979; Chernomordik, 1996).Membrane lipidsdisplayacylindrical shape (e.g.phosphatidylcholine,phosphatidylserine), a conical shape (e.g. phospha-tidylethanolamine) or an inverted conical shape (e.g.lysophosphatidylcholine). Such a polymorphisminfluences the localization of lipid molecules withinthe biological membranes. The lipid compositionof biological membranes is qualified as asymmetric
Figure 2. Chemical structure of some lipids found in biological membranes— Structure chimique de certains lipides représentatifs des membranes biologiques.
(Bretscher, 1973;OpdenKamp,1979), i.e. the lipidcomposition is different within the two leaflets ofthe samemembrane. Phosphatidylethanolamines andphosphatidylserinesaremainlyfoundintheinnerleafletof theplasmamembrane,whilephosphatidylcholinesand sphingomyelins are essentially located in theouterleaflet(Rothmanetal.,1977).Duetoitsabilitytoundergoafastflip-flopbetweentheouterandinnerleaflets of the lipid bilayers (Muller et al., 2002),cholesterol is assumed to be equally distributed onthe two leaflets of biological membranes. The lipidasymmetry across the membranes is responsible formembranecurvature,whichisessentialforbiologicalprocesses such as vesicle budding and membranefusion (Zimmerberg et al., 1999), and contributesalso to membrane potential, which is a key playerin many membrane-mediated phenomena such asbinding of drugs or proteins to membrane surface,insertionofintegralproteins,andmembranetransport(McLaughlin,1989).Whileitisgenerallyacceptedthatthetransmembranepotentialdroparisesfromachargeimbalanceofsaltionsacrosstheplasmamembrane,ithasbeenrecentlyshownbyGurtovenkoetal.(2007;2008; 2009) that the electrostatic transmembranepotential can be nonzero even in the absence of saltions,providedthatthelipiddistributionisasymmetric.Theseauthorspointedout that theobservedpotentialoriginatesfromadifferenceinthedipolemomentsofthetwoleafletsoftheasymmetricmembraneandisnotrelated to the transmembrane potential arising fromconcentration differences of ionic substances acrossthemembrane.
Lipid polymorphism not only induces lipidasymmetry between the two leaflets of membranes,but it is also responsible for phase separationwithinone monolayer leaflet. For example, it is assumed
that lipidpolymorphism is involved in the formationoflipidrafts,whichareenrichedinsphingolipidsandcholesterol (Simons et al., 1997). Due to its conicalshape, cholesterol may play the role of molecularspacer to fulfil the free space between sphingolipidmolecules,whichexhibitaninvertedconicalshape.
3. MoLecuLar DynaMIc oF MeMbranes
Biologicalmembranes are highly dynamic structures(Figure 3). Both the position (i.e. lateral order) andtheorientation(i.e. rotationalorder)ofa lipidwithinthe membrane bilayers are continuously changingwith time. Moreover, conformational changes (suchastrans-gaucheisomerisation)withinthehydrocarbonlipid chains may also occur (over time scales of afewpicoseconds)andaffecttheconformationalorderof lipid molecules. Different diffusion coefficientsareused tocharacterize the lipiddynamicwithin themembranes. The lateral diffusion coefficient (CD),rangingtypicallyfrom10-7to10-10cm2.s-1,determinestheabilityofalipidmoleculetolaterallyexchangewithone of its neighbours (this phenomenon occurs overtime scales less than a minute), while the rotationaldiffusion coefficient defines the angular rotation ofa lipidmolecule around its axis perpendicular to theplane of the bilayer (this motion takes place overtimesscaleofnanoseconds).Thetransferofonelipidmoleculefromoneleafletofthebilayertotheotheroneisaspecialcaseofmoleculardynamic.Suchprocess,called transversal diffusion or flip-flop, involves therotationofthelipidmoleculeintheplaneofthebilayerfollowedbyitstranslationperpendicularlytotheplaneofthebilayer.Thetransversaldiffusionisaveryslowprocess (of the order of hours, possibly days) and is
Figure 3.Schemeillustratingthedynamicsofmembranelipids—Schéma illustrant la dynamique des lipides membranaires.a:intramoleculardynamics—dynamique intramoléculaire;b:lipiddiffusioninbiologicalmembranes—diffusion lipidique au sein des membranes biologiques.
Trans-gauche isomerisation
Transversal diffusion
Lateral diffusion
Rotationaldiffusion
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HC1C1
a b
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Biologicalmembranesandbiomimeticmodels 723
energeticallyunfavorableasitforcesthepassageofthepolar lipidheadgroup through thehydrophobic coreof the lipid bilayer. However, some lipid moleculessuchascholesterolareabletoundergoafastflip-flop(<1.s-1) between the two leaflets of the lipid bilayer(Mulleretal.,2002;Stecketal.,2002).Suchpropertymostlikelyarisesfromtheverysmalleffectiveareaofthepolarheadgroupofcholesterol,whichislimitedtoonehydroxylgroup.
Proteins may also diffuse laterally within thebiological membranes, but their diffusion rate istypicallyahundredtimesslowerthanlipiddiffusion.However,proteinsarenotabletodiffusetransversallybetweenthetwoleafletsofthelipidbilayers.
4. therMotropIc phase behavIor oF MeMbranes
In aqueous medium, lipid bilayer constituting thebiologicalmembranes can exist in different physicalstates, which are characterized by the lateralorganization,themolecularorderaswellasthemobilityof the lipidmoleculeswithin the bilayer (Figure 4).
Consequently, physicochemical parameters such astemperature,pH,ionicstrengthandotherfactorssuchasthechemicalstructureofthelipidconstituentsandthe presence of cholesterol strongly influence thenatureofthelamellarphase.
The two extreme lipid phases that occur inbiologicalmembranes are the so-called gel andfluidphases (Figure 4). In the gel phase (Lβ’ or Lβ), alsocalledsolid-ordered(So)phase,thelipidsarearrangedonatwo-dimensionaltriangularlatticeintheplaneofthemembrane(Janiaketal.,1979).Thehydrocarbonlipid chains display an all-trans configuration andare elongated at the maximum, giving rise to anextremely compact lipid network. Consequently, thelateral diffusion of lipids is strongly reduced (CD ~10-11cm2.s-1).Notethat,asafunctionofthehydrationlevel,thehydrocarbonchainsoflipidsinthegelphasemaybetilted(Lβ’)ornottilted(Lβ)withrespecttothemembranenormal,theangleoftiltincreasingwiththeincreaseofwatercontent (Tardieuetal.,1973).Asaresult, the thicknessofa lipidbilayer in thegelstatedecreases as the amount of water increases. Otherparameterssuchasthenatureofthepolarheadgroupand the presence of counterions, which affect theheadgroup conformation,may also influence the tiltof the lipid alkyl chains in the gel phase (McIntosh,1980).Forexample,whilethehydrocarbonchainsofhydratedPCaretiltedwithrespecttothebilayers,thealkylchainsofhydratedPEareapproximatelynormalto theplaneof thebilayers.Suchadifference in thedegreeoftiltarisesfromthesmallerheadgroupofPEcompared to PC and from the fact that hydrated PEbilayersdonotcontainasmuchwaterashydratedPCbilayers(McIntosh,1980).
Inthefluidphase,alsocalledliquid-disordered(LαorLd)phase,trans-gaucheisomerisationoccursgivingrisetomuchlessextendedlipidchains.Moreover,thetwo-dimensional triangular lattice is completely lost.Asaresult,boththelateraldiffusion(CD~10
-8cm2.s-1)and the rotational diffusion of lipids are favored influidlipidbilayers.
The transition between the gel and fluid phasesoccurs at a specific temperature called thermotropicphasetransition(Tm).Thephasetransitiontemperatureofamembranelipid,i.e.thetemperaturethatisrequiredforinducingthelipidmeltingfromasolid-orderedtoa liquid-disorderedphase, isdependingonthenatureof itshydrophobicmoietyandcanbedeterminedbyusingthedifferentialscanningcalorimetrytechnique.
For some membrane lipids, such as phospha-tidylcholines, the lipid disordering occurs in twostepswhenincreasingtemperature.AfirsttransitionisobservedafewdegreesbelowthemaintransitionTm.Thispretransitionmaybeduetochangesinthevicinityofthepolarheadgroupsuchanincreaseoftheinteractionof the lipidheadgroupswith thesolvent (Heimburg,
Figure 4. Scheme illustrating the different physical statesadoptedbya lipidbilayer in aqueousmedium—Schéma illustrant les différents états physiques adoptés par une bicouche lipidique en phase aqueuse.
Tm:mainphasetransition—transition de phase principale.
2000). For example, phosphatidylethanolamines thatdiffer from phosphatidylcholines by the nature ofthe polar head group do not display a pretransition(McIntosh, 1980). According to Heimburg (2000;2007),pretransitionandmaintransitionarebothpartof thechainmelting transitionwith the splitting intotwotransitionsbeingtheconsequenceofsimultaneouschanges in the lipid order and membrane curvature.Consequently,forthelipidsthatexhibitapretransitiontemperature,anadditionallamellarphaseexists.Thisphase,calledtheripplephase(Pβ),ischaracterizedbyperiodicone-dimensionalundulationsonthesurfaceofthelipidbilayer(Janiaketal.,1979)(Figure 4).Asthisphaseappearspriortothemainchainmelting,itmustcorrespond to a partially disordered lipid phase. Forthisreason,ithasbeensupposedthattheundulationsobserved on the top of the lipid bilayers arise fromperiodicarrangementsoflinearorderedanddisorderedlipiddomains(Heimburg,2000;2007;deVriesetal.,2005).
of cholesterol into a solid-ordered lamellar phasedisturbsthelateraltriangularlatticeandconsequentlyreduces the ordering of the lipid chains. At theopposite, in a liquid-disordered lamellar phase, therigidhydrophobicmoietyofcholesterolisintercalatedbetween the lipid chains and favors a trans chainconformation(Sankarametal.,1990b).Consequently,theliquid-orderedphasedisplaysbothalateralandarotational diffusion that are close to the ones of theliquid-disorderedphase(Almeidaetal.,1993;Filippovetal.,2003),butaconformationalordersimilartotheoneofthesolid-orderedphase(Gallyetal.,1976).
As shown in figure 5, liquid-disordered andliquid-ordered phases as well as liquid-ordered andsolid-orderedphasescancoexistinasamelipidbilayer(Vist et al., 1990).For example, a phase-coexistencebetween a cholesterol-poor liquid-disordered phaseand a cholesterol-rich liquid-ordered phase has beenexperimentally observed for lipid bilayers composedof phosphatidylcholine/cholesterol (Sankaram et al.,1991) and sphingomyelin/cholesterol (Ahmed et al.,1997)mixtures.
The preferential partitioning of membrane lipidsinto a liquid-disordered or a liquid-ordered phaseis strongly depending on their chemical structure.Most of glycerophospholipids found in biologicalmembranesarecomposedofanunsaturatedfattyacid
chain inposition sn-2of theglycerol backbone.Thepresenceofdoublebondsinconfigurationcisinducesakink in thehydrocarbonchain andhampers averycompact assembling of the lipids. Consequently,this class of membrane lipids has very little affinityfor highly ordered lipid domains. At the opposite,sphingolipidsdisplay longsaturatedalkylchainsandsegregatetogetherviavanderWaalsandhydrophobicinteractions. Moreover, hydrogen bonds betweenthe hydroxyl groups of sphingomyelin polar heads(Ramstedtetal.,2002)orbetweentheoligosaccharidicheadgroupsofglycosphingolipids(Rocketal.,1990)may also accentuate the auto-assembling of theselipids.Therefore,sphingolipidshaveahightendencytoformorderedlipidphases(Wangetal.,2000).
Thelateralorganizationofmembranelipidsisalsoinfluenced by the nature of their polar head group.Membrane lipids displaying a relatively small polarheadgroupsuchasphosphatidylethanolaminesallowa more compact lipid assembling due to a reducedsteric hindrance (Brown et al., 2002; Rappolt et al.,2004). Furthermore, cholesterol differently interactswith glycerophospholipids as a function of theirpolarheadgroup.Forexample,cholesterolexhibitsahigher affinity for negatively charged phospholipidscompared to their zwitterionic analogues (Sankarametal.,1990a).
The existence of phase-separated zones in thelipid bilayers affects also the lateral organization ofmembraneproteins.Proteinscomprisingoneorseveralsaturatedaliphaticchainsdisplayahighertendencytosegregate into ordered lipid phases. It is notably thecaseforglycosyl-phosphatidylinositol(GPI)-anchoredproteins (Brownet al., 1992;Schroeder et al., 1994)and other acylated proteins such as the Src kinases(Shenoy-Scariaetal.,1994).
5. MeMbrane coMpLexIty at the nanoMetre scaLe
The assumption of Singer et al. (1972) that plasmamembranes and organelle membranes in eukaryotesare composed of a unique liquid-disordered lamellarphaseinwhichthelipidsarerandomlydistributedandallowthespanningofmembraneproteinsisnotfullycorrect.Experimentalandtheoreticaldataobtainedinthelasttenyearsinthefieldofmembranebiophysicsareallinfavoroftheexistenceofaphaseseparationintheplaneofthemembrane.
Attheendofthenineties,ithasbeenpostulatedthatmembranes are constituted of small, heterogeneous,and highly dynamic domains which are believed tobe enriched in sphingolipids and sterols and to beinvolved inmanybiologicalprocesses (Brownetal.,1997;Simonsetal.,1997;Rietveldetal.,1998).These
membranemicrodomains,betterknownaslipidrafts,exhibit thephysicalpropertiesofarelativelyorderedliquidcrystalline lamellarphaseandcoexistwithinaliquid-disordered environment.They are supposed tobe responsible for the lateral distribution of proteinsand the concentration of membrane constituents insmallcompartmentsfacilitatingtheirinteraction.
Theexistenceoflipidraftsinmembranesishoweverstill under debate. The raft hypothesis is originallybasedonthedetergentextractionofmembranelipids.As the lipids involved in putative membrane raftsformliquid-ordered(Lo)phases,theypresentalowersolubilityinnon-ionicdetergents(e.g.TritonX-100andBrij58)atlowtemperature(4°C)thanlipidsfromthesurroundingliquid-disorderedphase.Theuseofsuchanextractionprocedureforinvestigatingthepresenceof lipid rafts in membranes is today questionable.Indeed, the extraction of lipid constituents at verylow temperaturemay affect the lipid organization ofthenativemembraneandinducealateralaggregation,which would not occur in physiological conditions(deAlmeidaetal.,2003).Dependingonthenatureandtheconcentrationofthenon-ionicdetergentaswellason the extraction parameters (temperature, duration),changes in terms of lipid/protein composition anddistributionmayalsotakeplacewithinthetwoleafletsoflipidbilayers(Schucketal.,2003;Shogomorietal.,2003). In addition, TritonX-100 has been shown toinduce the formation of liquid-ordered domains inmodel membranes by decreasing the proportions ofsphingolipidsandcholesterol in theliquid-disorderedphase (Heerklotz, 2002). This detergent could bealso responsible for the fusionof rafts entities in themembrane and the formation of large interconnectedmembraneaggregates(Giocondietal.,2000;Simonsetal.,2004).Asaconsequence,itisveryunlikelythatmembranecompartmentsthatcannotbesolubilisedinnon-ionicdetergentreflectboththenativecompositionand organization of lipids within membrane rafts(Lichtenbergetal.,2005).
Thefactthatputativeraftsarethoughttobehighlydynamic structures makes also their characterizationextremelydifficult.Ithasbeenpostulatedthatlipidraftsexist in biologicalmembranes only if they are smallentitieswithashortlifetime(Subczynskietal.,2003).Itisgenerallyacceptedthattheselipidmicrodomainsdisplayasizedistribution(10-200nm)thatisinferiortotheresolutionoftheconventionalopticalmicroscopy(Simonsetal.,1997;Bagatollietal.,1999;Jacobsonetal.,1999;Simonsetal.,2000;Pike,2003).Thesizeoflipidraftsseemstobeinfluencedbythelocallipidcomposition, the incorporation of externalmoleculesthat can act as nucleation sites for the formation oflarger membrane domains (Brown et al., 1998b;Radhakrishnanetal.,2000;Andersonetal.,2002),aswellasbytheproteinconformation,whichcanperturb
thelipidassemblingwithinthesedomains(Heerklotz,2002).Lipid raftsmayalsoauto-associatewithin themembrane leaflets to form larger lipidplatforms thatbecomedetectablebyopticalmicroscopy(Subczynskiet al., 2003; Simons et al., 2004). The aggregationof these small entities may arise from protein-lipidinteraction,proteinoligomerisationorfromthebindingof proteins to specific antibodies at the cell surface(Friedrichsonetal.,1998;Harderetal.,1998).
Alternative theories are nowadays proposed forexplainingthesubmicronlateralheterogeneitiesincellplasmamembranes.Thepresenceofsubmicronlipid-richentitiesintheplaneofbiologicalmembranescouldarise from dynamic submicron critical fluctuations,inhomogeneous lipid mixing, 2-D microemulsions,orsmall-scalestructurewithinasinglegel(So)phase(Veatchet al., 2005;Honerkamp-Smithet al., 2009).Simple ternary lipid mixtures constituted of a steroland two other lipid components (one with a highchain melting temperature Tm and one with a lowchain melting temperature) are good model systemsforinvestigatingthelateralorganizationinbiologicalmembranesastheselipidmixturesphase-separateandform micron-scale liquid domains as a function oftemperature (Veatch et al., 2005). Bymeasuring themiscibilitytransitiontemperatureasafunctionofthelipidcomposition,thermodynamicphasediagramsthatarespecificoftheternarylipidmixturesofinterestcanbemapped(Goñietal.,2008).UsingthecombinationoffluorescencemicroscopyanddeuteriumNMR,ithasbeenobservedthatdynamicsubmicronliquiddomainsexhibiting a large distribution of sizes, compositionsandlifetimesarecreatedinthevicinityofmiscibilitycritical points (Veatch, 2007; Veatch etal., 2007).Critical fluctuations have been also found in giantplasma membrane vesicles, which are sphericalvesiclesisolateddirectlyfromtheplasmamembranesoflivingcells,neartheirtransitiontemperature(Veatchetal.,2008).Suchamanifestationofsubmicroncriticalfluctuations in model lipid systems could explainsomeofthenanometrescalemembraneheterogeneityattributedtoputativelipidraftsinbiologicalmembranes(Veatchetal.,2008).
The coexistence of liquid-ordered and liquid-disordered phases in the plane of themembrane andthe lateral distribution of proteins play certainly animportant role in many biological processes. It iswidelyacceptedthatsuchphasesegregationisinvolvedin the sorting and the transport of both membraneproteinsandlipidsduringendocytosisandexocytosisphenomena, incascade signallingaswell as inothercellularprocessessuchasapoptosis,membranefusion,cell adhesion and migration (Brown et al., 1998a;Simonsetal.,2000).
pathogensortoxins(vanderGootetal.,2001;Duncanet al., 2002;Manes et al., 2003).They could indeedconcentratecellularreceptorsthatarenecessaryforthebindingofpathogenstotheplasmamembraneoftargetcellsorfor theoligomerisationof toxinsfavoringbythiswaytheirentryinthecell.Orderedlipiddomainswithin the membrane may also provide preferentialplatforms for the assembling and the budding ofviral particles (such as Ebola, influenza, and humanimmunodeficiency-1 viruses) as well as for theformation of pathological forms of the prion proteinandoftheβ-amyloidpeptide,whichisassociatedwithAlzheimer’s disease (Campbell et al., 2001; Fantinietal.,2002).
Nowadays, while there is no doubt about thepresence of phase separation between liquid-orderedand liquid-disordered phases in the plane of themembrane(Swamyetal.,2006;Senguptaetal.,2007),additional research involving both cell membranesandbiomimeticmodelmembranesisstillrequiredtofurtherinvestigatethenanoscalelateralorganizationoflipidsinbothintracellularandextracellularmembraneleafletsaswellas tobetterunderstand thebiologicalfunctions associated to these phase-separated lipiddomains.
6. bIoMIMetIc MoDeL MeMbranes
Asbiologicalmembranesareverycomplexsystems,many model membranes have been developed overthe last century for studying membrane properties,structure and processes as well as for investigatingthemembraneactivityofdiversenaturalorsyntheticcompounds such as surfactants, peptides, and drugs.The most well-known and common biomimeticsystemsusedforsuchpurposesarelipidmonolayers,lipid vesicles and supported lipid bilayers. Whileeach of these systems exhibits advantages anddisadvantages,theyallmimicthelipidarrangementofnaturalcellmembranes.
6.1. Lipid monolayers
Lipid monolayers provide a simple model formimicking biologicalmembranes and for evaluatingmembrane insertion of amphipathic compounds(Brockman, 1999; Maget-Dana, 1999). Thesemonomolecular insoluble films, also referred to asLangmuir monolayers, are formed by spreadingamphiphilicmolecules at the surface of a liquid andcan be considered as half the bilayer of biologicalmembranes. These two-dimensional systems displaymany advantages compared to the other modelmembranes. Parameters such as the nature and thepackingof the spreadmolecules, the compositionof
Lipid monolayers are very useful to characterizedrug-lipid or lipid-lipid interactions at a molecularlevel. Such a characterization can be deduced fromcompression isotherms, which are obtained bymeasuring the surfacepressure (Π) of the interfacialfilm as a function of the mean molecular area (A)of the compounds spread at an air-water interface(Figure 6). Under compression, a two-dimensionalinsoluble monolayer adopts different physical states(typicallygaseous,liquidexpanded,liquidcondensedandsolid-likestates),whicharerelatedtothelevelofconformationalorderofthemoleculesattheinterfaceand to the presence of intermolecular interactionswithinthemonolayer.
During its compression, an insoluble monolayeris also characterized by changes in terms of two-dimensional compressibility (Cs). This parametercorrespondstotheslopeofthecompressionisothermandcanbedeterminedforeachpointoftheΠ-Acurveusingthefollowingequation(Equation1):
Cs =-1dA Equation1
A dΠ
Molecular interactions occurring at an air-waterinterface between molecules of different nature canbeevaluatedbyperforminga simple thermodynamicanalysis.FromtheΠ-Aisothermofpureandofmixedmonolayers, information about the mixing behaviorofspreadmoleculescanbeobtained.Whenthemeanmolecularareaofamixedmonolayer(Am)atadefinedsurfacepressurecorrespondstotherelativesumofthemolecularareasof theseparatedcomponents(A1and
A more detailed analysis of the thermodynamicsof the system, by calculating the excess free energyofmixing∆Gex(Equation3)developedbyGoodrich(1957), can provide further information about themiscibilityprocessandthepossiblespecificinteractionsbetweentheinterfacialcomponents.
ΔGex=∫ AmdΠ-X1∫A1dΠ-(1-X1)∫ A2 dΠ
Equation3
Positive values of ∆Gex signify that mutualinteractionsbetween the twocomponentsareweakerthan interactions between the pure compoundsthemselvesandsuggestphaseseparationbetweenthecomponentsat the interface.Negativevaluesof∆Gexindicate thepresenceofstrongmutual interactionsatthe interface and are in favor of complex formationbetween the monolayer constituents (Maget-Dana,1999).
Lipid monolayers are also excellent modelmembranesforevaluatingtheinsertionofamphipathiccompounds such as antimicrobial peptides,biosurfactantsanddrugs into themembraneof targetcells(Maget-Dana,1999).Forthisspecificpurpose,aninsolublemonolayermimickingthelipidcompositionofthebiologicalmembraneofinterestisformedattheair-waterinterfaceofaLangmuirtrough(Figure 7a).Afterstabilizationofthelipidmonolayeratadefinedinitial surface pressure (Πi), the active compound,solubilised in an appropriate solvent, is injected intothewater subphase.At thispoint, the increaseof thesurfacepressure resulting from the interactionof theactivecompoundwiththelipidmonolayerisrecorded(Figure 7b).
By plotting the maximum surface pressureincrease (ΔΠ) observed as a function of the initialsurfacepressureof the lipidmonolayer,anexclusionsurfacepressure(Πe)isdetermined(Figure 7c).Thisparameter corresponds to the initial surface pressureof the lipid monolayer above which no more activecompound can penetrate the lipid film and increasethe surface pressure. In other words, this parameterreflectsthepenetrationpoweroftheactivecompoundofinterestintoawell-definedtwo-dimensionalmodelmembrane.
Solid-like state
Liquid condensed state(LC)
Liquid expanded state(LE)
LC - LE coexistence
Gaseous state
Mean molecular area (Å.mol-1)
Sur
face
pre
ssur
e (m
N. m
-1)
Figure 6. Theoretical Π-A isotherm obtained bycompressing an insoluble lipid monolayer formed at anair-water interface— Isotherme de compression théorique obtenue en comprimant une monocouche lipidique insoluble étalée à une interface air-eau.
In order to visualize the interfacial organizationoflipidconstituentsofamonolayerorthechangesinthe interfacial behavior resulting from the insertionof a compound of interest into the monolayer, theLangmuir trough technique can be easily combinedwithfluorescenceorBrewsteranglemicroscopy.Thelattertechniquepresentstheadvantageofnotusingafluorescentprobethatmayresultindomaininstabilityfor highly compressed monolayers (McConlongueet al., 1997). Fluorescence and Brewster anglemicroscopyofferalateralresolutioninthemicrometrerange and are thus not suited to visualize the phasepropertiesoflipidmonolayersathighresolution.Forsuch purpose, atomic forcemicroscopy (AFM) is anexcellentalternativeprobingtechniquesinceitallowsthe visualizationof lipid domains in phase-separatedfilmswithananometrescaleresolution(Dufrêneetal.,1997; Reviakine et al., 2000; Milhiet et al., 2001).However, the use of AFM for imaging monolayersimpliesthetransferoftheinterfacialfilmontoasolidsupport.
The most common technique used to transfer amonolayer is the Langmuir-Blodgett (LB) technique(Motschmann et al., 2001). The solid support canbe either hydrophilic or hydrophobic.When using a
hydrophilic support, the lipid polar heads are facingtowards the support, whereas the transfer onto ahydrophobic support is obtained via hydrophobicinteractions with the lipid hydrocarbon chains. Thetransfer of an interfacial film onto a solid supportis performed at constant surface pressure and ismonitored via the so-called transfer ratio. In orderto maintain a constant surface pressure during thetransfer process, the interfacial film is continuouslycompressed resulting in adecreaseof themonolayersurfacearea.The transfer ratio is thusdefinedas theratioofthedecreaseofthemonolayersurfaceareatotheareaof thesolidsupportwhichhasbeencoveredby the constituents of the interfacial film.A transferratioclosetooneindicatesthatthedepositionprocesshas been successful, i.e. the supportedmonolayer isrepresentativeofthespreadmonolayerattheair-waterinterface. However, it has to be kept in mind that,dependingonboththenatureoftheconstituentsoftheinterfacialfilmand thesurfacepressureatwhich themonolayerhasbeentransferredontothesolidsupport,changes in themolecular organization at the supportsurfacemayoccur.
The Langmuir trough technique can also becombinedwith spectroscopic (e.g. polarized infrared
a
To Π-recording system
b
Drug injection
Exclusion pressure
SurfacepressureΠi
Surfacepressureincrease
ΔΠ
ΔΠ
c
Time
Sur
face
pre
ssur
e Π
i
Πe
Figure 7.a:SchematicrepresentationoftheLangmuirtroughtechniqueusedforevaluatingthepenetrationpowerofabioactivecompoundintoabiomimeticlipidmonolayer—Représentation schématique de la technique de la cuve de Langmuir utilisée pour évaluer la pénétration de molécules actives au sein d’une monocouche lipidique biomimétique;b:Penetrationkineticfollowingtheinjectionofthedrugintothesubphase—Cinétique de pénétration théorique obtenue suite à l’injection d’un composé amphiphile dans la phase aqueuse;c:Surfacepressureincreasevsinitialsurfacepressureplotusedfordeterminingtheexclusionpressureofthelipidmonolayer—Graphique de l’augmentation de la pression de surface vs la pression de surface initiale utilisé pour déterminer la pression d’exclusion d’une monocouche lipidique.
Biologicalmembranesandbiomimeticmodels 729
spectroscopy), reflection and scattering (e.g.ellipsometryandgrazing-incidenceX-ray)techniquesin order to obtain direct structural information (i.e.conformation and orientation of the monolayerconstituents) in phase-separated two-dimensionalsystems.
6.2. Lipid vesicles
Lipid vesicles or liposomes are versatile biomimeticmodel membranes commonly used for studyingmembrane phase behavior and membrane processessuch as membrane fusion, molecular recognition,cell adhesion, andmembrane trafficking.These lipidassemblies enclose a small aqueous compartmentand are produced from the aqueous dispersion ofmembrane lipids (single lipid component ormixtureofdifferenttypesoflipids).Whereaslipidmonolayersareconstitutedofonlyone lipid leafletand thereforedonotreflectthecomplexityofbiologicalmembranestructure, lipid vesicles are composed of two lipidleaflets,whicharearrangedinawaythatissimilartothatofbiologicalmembranes.
According to the method of preparation,different types of bilayer structures can be obtained(Gregoriadis,1991;Muietal.,2003;Lorinetal.,2004;Uhumwanghoetal.,2005).Whenadriedlipidfilmisvigorously hydrated at temperatures above the lipidphase transition, multilamellar lipid vesicles (MLV)areformed.Thesevesiclesdisplayasizerangeof0.5-10µmandarecharacterizedbyseveralconcentriclipidbilayers,whichareseparatedbywatermolecules.ThesizeoftheseMLVscanbereducedandhomogenizedbyperformingseveral freeze-thawcycles.Theextrusionof MLVs through a porous membrane gives rise tothe formation of large unilamellar vesicles (LUV)whilesmallunilamellarvesicles(SUV)areformedbysonicatingtheMLVsinaclassicalbath-typesonicatororusingaprobesonicator.LUVsandSUVsarebothcharacterizedbya single lipidbilayer.SUVsusuallyexhibit a mean diameter inferior to 50nm whereasthe size of LUVs varies from 100 to 500nm.Giantunilamellar vesicles (GUV) are liposomes havinga size range of 5-100µm. These model membranescan be obtained by hydrating a dried lipid film attemperaturesabovethelipidphasetransitioneitherforalongperiodoftime(upto36h)(i.e.gentlehydrationmethod)or inpresenceof anexternal electricalfield(i.e.electroformationmethod)(Bagatollietal.,2000;Rodriguezetal.,2005;Wesolowskaetal.,2009).Thesize of these giant vesicles allows their visualizationbyopticalmicroscopysuchasfluorescenceorconfocalmicroscopy, as well as the micromanipulation ofindividual vesicles. Although these techniques havea lower lateral resolution thanAFM, they allow theinvestigation of molecular interactions with lipid
The main disadvantages of using lipid vesiclesas biomimetic model membranes are that the lipidasymmetry found in native biological membranescannotbemimickedandthatthefinallipidcompositionof the vesicles may be relatively different from theinitial lipid mixture used for vesicle formation. Asdemonstrated by phase diagrams of complex lipidmixtures,smalldifferencesincompositionmaystronglyaffectthephasebehavioroflipidsystems(Goñietal.,2008).Consequently,anappropriatecontrolofthefinallipidcompositionneedstobeperformedbeforeusingthe model for studying membrane properties and/orprocesses.InthecaseofGUVs,ithasbeendemonstratedthat the lipidcompositionof thevesicles is closer totheoneoftheinitiallipidmixturewhenpreparingthevesiclesviathegentlehydrationmethodratherthanviatheelectroformationmethod(Rodriguezetal.,2005).However, the former technique is responsible for ahigherpercentageofdefectsinGUVs,whicharelipidstructures bound to the inner or outer lipid leaflet orencapsulatedinsidethelipidvesicles(Rodriguezetal.,2005).Consequently,dependingontheapplicationoftheGUVs,oneofthetwopreparationtechniqueswillbepreferred.
Itisworthtonotethat,aslipidvesiclesareusuallyformedfromdilutelamellardispersionswiththeinputofmechanical(e.g.sonicationorextrusion),chemical(e.g.changeofsolubilityconditions,incorporationofexternal compounds) or electrochemical (e.g. changeof pH, ionic strength) energy, they are metastablestructures offering poor long-term stability (Lasic,1990; Madani et al., 1990; Marques, 2000). Thismeans that, upon aging, vesicle dispersion mayaggregate(clusteringformation),fuseorevolvetothethermodynamicallystabletwo-phaseregion(consistingofalamellarphasedispersedinlargeexcessofsolvent)fromwhichtheywereformed.However,dependingonthecompositionandthesizeoflipidvesiclesaswellasontheenvironmentalparameters(temperature,pH,ionic strength, presence of external molecules andions),thesethermodynamicallynonstablesystemscanbestableforprolongedperiodsoftime(uptoseveralmonths) and are then suitablemodelmembranes forinvestigating membrane properties and biologicalprocesses.Inparticular,theyareveryinterestingmodelsystems for studying cell adhesion and membranefusionphenomenawhicharemediatedbynon-covalentprotein-protein and protein-carbohydrate interactions(Voskuhletal.,2009).
In addition to be relevant biomimetic modelmembranes for investigating membrane properties,structureandprocesses,lipidvesicleshavebeenprovedto be suitable transport vehicles for drugs, proteins,enzymes, or DNA. The applications of these lipid
systems are abundant in particular in pharmacologyand in dermato-cosmetology where these lipidassemblies are used as drug delivery systems andallowthepredictionofpharmacokineticpropertiesofdrugs such as their transport, their distribution, theiraccumulation,andhence theirefficacy(ElMaghrabyetal.,2008;Peetlaetal.,2009).
6.3. supported lipid bilayers
Supportedlipidbilayers(SLBs)arebiomimeticmodelmembranesconstitutedofaflatlipidbilayersupportedontoasolidsurfacesuchasmica,glassorsiliconoxidewafers.Insuchmodelsystem,thepolarheadgroupsofthefirstlipidmonolayerarefacingtowardsthesupportwhilethehydrocarbonchainsofthislipidmonolayerareincontactwiththelipidchainsofthesecondmonolayer.SLBsoffermanyadvantagesoverlipidvesicles(Looseetal.,2009).Thesemodelmembranescanbepreparedquite easily and are much more stable than lipidvesicles.Besides,boththeoverallcompositionandthelipidasymmetryofSLBscanbecontrolledwhileitisnot thecasewhenusingvesicularmodel systems. Inaddition,asthesemembraneassembliesareconfinedtothesurfaceofasolidsupport,theycanbecharacterizedmuch easier than free-floating vesicles using a largevariety of surface sensitive techniques such asAFM(Linetal.,2007;Mingeot-Leclercqetal.,2008;Goksuetal.,2009),secondaryionmassspectrometry(SIMS)(Chan et al., 2007), fluorescencemicroscopy (Craneet al., 2007), optical ellipsometry (Puu et al., 1997),quartz-crystalmicrobalance(Kelleretal.,1998),X-rayreflectivity(Milleretal.,2006)andneutronreflectivity(Vacklinetal.,2007).
DifferenttechniquesarecommonlyusedtoprepareSLBs. The first one is the LB technique. After thetransfer of a lipidmonolayer spread at the air-waterinterface of aLangmuir trough onto a solid support,thesamesupport is immersedasecond time throughtheinterfaceinordertoobtainasupportedlipidbilayer(Figure 8a).
AsecondmethodforpreparingSLBsisthefusionof lipidvesiclesontoasolidsupport.Thismethod isrelativelysimpleandcanbecompletedinfewhours.Thedetailedprotocoltoachievethefusionoflipidvesicleshasbeenrecentlyreviewedintheliterature(Mingeot-Leclercqetal.,2008).Briefly,thefusionisobtainedbyheatingaSUVsuspensionincontactwiththesupportat temperatures above the lipid phase transition.Thefusionprocessisnotyetfullyunderstoodbutinvolvesthe adsorption of the lipid vesicles on the surface,followedbytheirdeformation,theirflatteningandtheirrupture.Thefusionoftheedgesofthebilayerpatchesthrough hydrophobic interactions gives rise in finalto a continuous supported lipid bilayer (Figure 8b)(Jass et al., 2000: Reviakine et al., 2000; Richter et
al., 2005;Anderson et al., 2009).As SLBs preparedfromthefusionoflipidvesiclesrequirestemperaturesabove the lipidphase transition, this technique isnotappropriate when temperature-sensitive membranecomponents such as proteins has to be incorporatedinSLBs.However,itiscurrentlythemostfrequentlyusedmethodforpreparingSLBs.
Supportedmodelmembranescanbealsoobtainedfrom micellar solutions composed of a mixtureof surfactants and phospholipids (Tiberg et al.,2000;Vacklin et al., 2005; Lee et al., 2009). In thismethod, the surfactant (e.g. non-ionic β-D-dodecylmaltoside) is used as a lipid solubilising agent andactsasatransportertodrivethewater-insolublelipidto the surface. When mixed surfactant-phospholipidmicellesadsorbatthesolidsurface,theconcentrationofmixedmicelles at the vicinity of the surface (i.e.within the stagnant layer) is reduced compared totheir concentration in the bulk solution (Figure 8c).The formation of phospholipid-enriched supportedbilayersisfavoredbythesolubilitydifferencebetweenthe phospholipid and the surfactant, and is obtainedby repetitively rinsing the adsorbed layer with bulksolutionsofdecreasingmicelleconcentration.Indoingso,themoresolublesurfactantmonomersadsorbedtothe surface are progressively solubilised while boththesolidsupportandthemixedmicellesaregraduallyenriched in the less soluble component. After eachaddition of more diluted bulk solutions, a rinsingstepisusuallyperformedtoremoveanyexcessofthesolublesurfactant.Asphospholipidsexhibitverylowwater solubility, the progressive solubilisation of thesurfactantfromthesolidsupportisresponsiblefortheformationofapurephospholipidbilayer.
Since their development two decades ago asbiomimetic model membranes (Tamm et al., 1985),supported lipid bilayers have been largely used bythe biophysical community to predict the phasebehaviorandthemolecularorganizationofbiologicalmembranes. Moreover, over the past ten years, ithas been demonstrated that these supported lipidbilayers are also highly relevant model membranesfor investigating the molecular interactions of drugswithcellmembranes.Anoverviewoftheapplicationsof supported lipid bilayers as well as of otherbiomimetic model membranes for investigating thepharmacokineticpropertiesofdrugshasbeenrecentlyreportedbyPeetlaetal.(2009).
One of the main drawbacks of using classicalsupportedlipidbilayersisthattheproximitybetweenthe lipid bilayer and the solid substrate may affectthe membrane properties of the biomimetic system,suchasthemobilityofmembranecomponentsortheincorporationof transmembraneproteins. Inorder tosolvesuchproximityproblem, tethered lipidbilayersmadeupofalipidbilayerspacedfromthesolidsurface
Biologicalmembranesandbiomimeticmodels 731
a
c
b
Supportregion
BulkStagnant layer
Micelle-monomerexchange
Figure 8. Preparationmethodsofsupportedlipidbilayers—Méthodes de préparation des bicouches lipidiques supportées.
byspacermoleculesorlayershavebeendeveloped.Thedifferentstrategiescurrentlyavailableforseparatingalipidbilayerfromasolidsupporthavebeencriticallyreviewed by Rossi et al. (2007). The formation ofthese tethered bilayers is usually achieved via theaddition of a polymer film or the self-assembling ofchemicallymodifiedlipidsonthesolidsurface,orviathedirectfusionofspacerlipidscontainingvesiclesonfunctionalizedsurfaces,andinvolvestheLBtechnique,thefusionoflipidvesicles,orthecombinationofbothtechniques.
Newapplicationsofsuchtetheredsupportedlipidbilayersareconstantlydiscovered(Rossietal.,2007).Asthesebiomimeticsystemsallowproteinincorporationin non-denaturing conditions, they are very suitablemodelmembranesforinvestigatingmembrane-proteininteractionsinafunctionalmanner.Thereconstitutionof membrane receptors in such lipid bilayers opensalsodoorsforthedesignofspecificsensorsthatcouldbeusedinaverynearfutureforavarietyofmedicalapplicationsforexampleaspharmaceuticalscreening.
7. LIMItatIons oF bIoMIMetIc MoDeL MeMbranes
Over the last century, model membranes have beenproved to play a considerable role in the elucidationof the structure and the properties of biologicalmembranesaswellasintheunderstandingofbiologicalprocesses thatoccurat themembranesurfaceor thatareassociatedwithcellmembranes.
However,ithastobekeptinmindthatthesemodelsystemshavesomelimitationsastheydonotcapturethewholecomplexityofbiologicalmembranes.Whilethe simplification of themembrane system is crucialfor the analysis of specificmolecular interactions atthemembrane level, itcanalsobeanobstacle to theaccurateunderstandingofsomemembranefunctions.
Some of the main limitations associated withthe use of model membranes have been highlightedrecentlybyVestergaardetal.(2008)andarepresentedinthispaper.
Thenumberofcomponentsthatcanbeincorporatedin a model system is relatively limited mainly dueto experimental constraints and to the capabilities ofanalysis of the currently available technologies. Forexample,mostofbiophysicalstudiesbasedonmodelmembranesonlyinvolveuptothreeorfourdifferentlipid species, while biological membranes enclosemorethanthousanddifferentlipids(vanMeer,2005).
Anotherlimitationofthesemodelsystemsliesonthefactthatitisrelativelychallengingtoreconstituteproteins in model membranes (Chan et al., 2007).Consequently, proteins are much less considered inmembraneresearch,whilethesemembraneconstituents
affect also themembrane structure and contribute tomembranepropertiesandfunctions.
Other limitationsarealsodirectlyassociatedwiththe preparation technique of these model systems.While lipidmonolayers can be quite easily preparedfrom each type of membrane lipids, the preparationtechnique of lipid vesicles is more complex andselective. For example, it is very challenging toreconstitutemembranelipidswithahighchainmeltingtemperature (such as ceramides) into vesicles as thevesicle formation requires an aqueous dispersion oflipids at temperatures above their phase transitiontemperature.
8. concLuDIng reMarKs
Toconcludeitisworthtonotethatthisarticledoesnothave the pretention to present the last developmentsin membrane research resulting from the use ofbiomimeticmodelmembranes.
While also revisiting the fundamental propertiesof biological membranes in terms of membranecomposition, membrane dynamic and molecularorganization at the nanometre scale, this articleprovides a general and consistent description of themainmodelmembranesusedinmembraneresearch.
This article should be then considered as a firstreference document for scientists who desire to beinitiated into the fascinating world of biologicalmembranes.Afterwards,thereaderisreferredtomorespecialized articles and reviews for more in-depthinformationrelatedtothedifferenttopicsdiscussedinthepresentpaper.
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