Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major ...

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities ... ThisarticleispartoftheResearchTopic Molecularorganisationofmembranes:wherebiologymeetsbiophysics Viewall 12 Articles Articles MarekCebecauer J.HeyrovskyInstituteofPhysicalChemistry(ASCR),Czechia ErdincSezgin KarolinskaInstitutet(KI),Sweden HectorMartinez-Seara InstituteofOrganicChemistryandBiochemistry,AcademyofSciencesoftheCzechRepublic(ASCR),Czechia Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract MembraneAsymmetry LipidAsymmetryinthePlasmaMembrane TheRegistrationofMembraneNanodomainsintheTwoLeaflets SizeofDomains CrossingtheMid-Line—APrivilegeofAsymmetricLipidswithOneLongAcylchain ImperfectioninLipidAsymmetryandDomainRegistration Pinning Outlook AuthorContributions ConflictofInterestStatement Acknowledgments References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData REVIEWarticle Front.CellDev.Biol.,10January2017 |https://doi.org/10.3389/fcell.2016.00155 InterleafletCoupling,Pinning,andLeafletAsymmetry—MajorPlayersinPlasmaMembraneNanodomainFormation ToyoshiFujimoto1andIngelaParmryd2* 1DepartmentofAnatomyandMolecularCellBiology,NagoyaUniversityGraduateSchoolofMedicine,Nagoya,Japan 2ScienceforLifeLaboratory,MedicalCellBiology,UppsalaUniversity,Uppsala,Sweden Theplasmamembranehasahighlyasymmetricdistributionoflipidsandcontainsdynamicnanodomainsmanyofwhichareliquidentitiessurroundedbyasecond,slightlydifferent,liquidenvironment.Contributingtothedynamicsisacontinuousrepartitioningofcomponentsbetweenthetwotypesofliquidsandtransientlinksbetweenlipidsandproteins,bothtoextracellularmatrixandcytoplasmiccomponents,thattemporarilypinmembraneconstituents.Thismakeplasmamembranenanodomainsexceptionallychallengingtostudyandmuchofwhatisknownaboutmembranedomainshasbeendeducedfromstudiesonmodelmembranesatequilibrium.However,livingcellsarebydefinitionnotatequilibriumandlipidsaredistributedasymmetricallywithinositolphospholipids,phosphatidylethanolaminesandphosphatidylserinesconfinedmostlytotheinnerleafletandglyco-andsphingolipidstotheouterleaflet.Moreover,eachphospholipidgroupencompassesawealthofspecieswithdifferentacylchaincombinationswhoselateraldistributionisheterogeneous.Itisbecomingincreasinglyclearthatasymmetryandpinningplayimportantrolesinplasmamembranenanodomainformationandcouplingbetweenthetwolipidmonolayers.Howasymmetry,pinning,andinterdigitationcontributetotheplasmamembraneorganizationisonlybeginningtobeunraveledandherewediscusstheirrolesandinterdependence. MembraneAsymmetry Alreadyintheearly1970'sitwasknownthatthehumanerythrocytemembranedisplaysleafletasymmetryinthephospholipidcomposition(Bretscher,1972;Verkleijetal.,1973)withmostphosphatidylcholine(PC)andsphingomyelin(SM)presentintheouterleaflet,whereasphosphatidylserine(PS),phosphatidylethanolamine(PE),andphosphatidylinositol(PI)areintheinnerleaflet(Figure1).Thisarchitecturehasoftenbeenregardedasaprototypeoftheplasmamembraneofmammaliancells. FIGURE1 Figure1.Phospholipidasymmetryintheerythrocytemembrane.Thecolorsindicatetheasymmetricdistributionofphospholipids.Therangeinpercentagesindicatestheamountspresentinthepreferredleaflet[allbuttheGSLs(Lingwood,2011)fromTable1fromZachowski(1993)].Notethatmostphospholipidsarealsolikelytobepresentinthelessfavoredleaflet,albeitinasmallamounts.Cholesterolisalsoamajorcomponentofthemembranebutisnotshown. Inrecentyearsmethodstoproduceasymmetricphospholipidmembraneshavebeendevelopedandtogetherwithsophisticatedmolecularsimulationtechniques,theyhaveprovidedcompellingevidencethatlipidsinoneleafletofthemembranecaninfluencemoleculardiffusionanddomainformationoflipidsintheotherleafletwithoutproteinintervention,i.e.,interleafletcoupling.Inthesestudies,modelmembraneswithperfectphospholipiddistributionasymmetryareoftenused,forexamplewithPCand/orSMinoneoftheleafletsandPS,PE,and/orPIconfinedtotheoppositeleaflet.However,innaturalmembranes,nophospholipidspeciesislikelytodistributeexclusivelytoonlyoneleafletandalsosmall“imperfections”inasymmetrymighthaveasignificantimpactinsomemembranepropertiesaswediscussbelow. Tobetterunderstandthepropertiesofnaturalmembranes,itisimportanttoestablishthedegreeofasymmetricphospholipiddistribution.Wewillthereforesummarizewhatisknownaboutthedistributionofindividuallipidsintheplasmamembraneleafletsofmammaliancells.Herewehavetoemphasizetwopoints.Thefirstisthedivergencebetweencelltypes:themostwell-studiedhumanerythrocytemembraneandtheplasmamembraneofothercells.Thesecondisthedegreeofasymmetry:anasymmetricdistributionofaparticularlipidmeansthatthedistributionbetweenthetwoleafletsisnot50:50.Itcouldbe60:40,butitcouldalsobenearly100:0,andthedistributionoranychangethereofislikelytoinfluencemembraneproperties.Usingthelipiddistributiondatafromnaturalmembranes,weshouldbeabletogeneraterealisticmodelstoexplaintheroleoftheasymmetriclipiddistributionincellularprocessestakingplaceinmembranes. LipidAsymmetryinthePlasmaMembrane Phosphatidylcholine PCinthehumanerythrocytemembraneispredominantlyfoundintheouterleaflet,andtheproportionofPCintheouterleafletwasestimatedtobe76–78%(Verkleijetal.,1973;vanMeeretal.,1981).Thislevelofasymmetrydoesnotappeartoexistinothercelltypesoreveninerythrocytesofotherspecies.Forexample,theproportionofPCintheouterleafletofmouse,rat,andmonkeyerythrocytemembraneswasreportedtobe50%(Rawyleretal.,1985),62–63%(Renooijetal.,1976;CrainandZilversmit,1980),and67%(VanderSchaftetal.,1987).ThePCdistributionwasexaminedbybiochemicalmethods,utilizingcovalentbindingofmembrane-impermeablereagents(GordeskyandMarinetti,1973;WhiteleyandBerg,1974),enzymaticdigestion(Verkleijetal.,1973),oruseofphospholipidexchangeproteins(Barsukovetal.,1976),butthesemethodsarenotappropriateforaccuratemeasurementofasymmetry(OpdenKamp,1979;Etemadi,1980;Zachowski,1993).Nevertheless,thedivergentresultsfromerythrocytesofdifferentspecieswereobtainedbysimilarmethods,suggestingthatthePCdistributioninnon-humanerythrocytesmaynotshowsuchextremeasymmetryacrosstheplasmamembraneleafletasthatfoundinhumanerythrocytes. Morerecently,freeze-fracturereplicalabelingEMutilizingmetaboliclabelingwithaclickablecholineanalogindicatedthatPCexistsinequivalentamountsinbothleafletsoftheplasmamembraneincellsotherthanerythrocytes(Iyoshietal.,2014).Incontrast,studiesapplyingananti-PCantibodytofreeze-fracturereplicasshowedpredominantlabelingintheouterleaflet,butthecaptureratiowasextremelylow(Fujimotoetal.,1996;Murateetal.,2015),atleastpartiallybecausetheantibodypreferentiallydetectedonlysomePCsubpopulations(Nametal.,1990). AlthoughtheexactratioofPCinthetwoleafletsisnotclear,thebulkofthedatasuggeststhatmorethan20%ofPCcouldexistintheinnerleafletoftheerythrocytemembrane.Intheplasmamembraneofothercelltypes,theproportionofPCintheinnerleafletmaybeevenhigher. Aminophospholipids BothPSandPEwereinitiallythoughttobeconfinedtotheinnerleafletofthehumanerythrocytemembrane,andasimilarasymmetrywasassumedtobeageneralpropertyofallplasmamembranes.LackofcellsurfacebindingofPS-specificannexinV(Koopmanetal.,1994)andPE-specificRo09-0198(Emotoetal.,1996)innormalinterphasecellsfurtherstrengthenedtheassumption.However,theresultwithannexinVandRo09-0198onlysuggestedthatthelevelofPSandPEintheouterleafletisbelowathresholdlevel,andcannotnecessarilybeequatedwiththeircompleteabsence.Actually,biochemicalstudiesindicatedthatasizablefractionofPSandPEexistsintheouterleaflet,andmoreover,thattheproportionintheouterleafletishighlyvariableamongdifferentcelltypes,0–44%(PS)and0–73%(PE)(itisgenerallyhigherforPEthanPS;seetablesinDevaux,1991;Zachowski,1993forsummaryofresults).Althoughthemethodsusedinthosestudiesmaynotbeaccurateorquantitative(OpdenKamp,1979;Etemadi,1980;Zachowski,1993),itisdifficulttoexplainthedivergentresultsbythemethodologicalinsufficiencyalone.Wepresumethatnon-negligibleamountsofPSandPEdistributetotheouterleafletoftheplasmamembraneinmostcells. PhosphatidylinositolandPhosphoinositides PIisalsoassumedtoexistlargelyintheinnerleafletoftheplasmamembrane,butbiochemicalstudiesindicatedthatPImayalsobepresentintheouterleafletinthehumanerythrocytemembraneaswellasintheouterplasmamembraneleafletofseveralnucleatedcelltypes(Rawyleretal.,1985;Bütikoferetal.,1990;Gascardetal.,1991).ThelackofaPIspecificprobemakesitdifficulttoconfirmtheaboveresultmicroscopically,butseveralphosphoinositidescouldbelabeledatthecellsurfacebyapplyingmembrane-impermeableprobes(Gascardetal.,1991;Kaleetal.,2010).Thissuggeststhatinositolphospholipidsarepresentintheouterleafletoftheplasmamembraneasphysiologicalconstituents. Sphingomyelin TheproportionofSMintheouterleafletofthehumanerythrocytemembranewasreportedtobe80–85%(Verkleijetal.,1973)and79%(vanMeeretal.,1981).IncontrasttoPC,erythrocytesofotherspeciesalsoshowedasimilarorevenhigherfractionofSMintheouterleaflet(Renooijetal.,1976;CrainandZilversmit,1980;Rawyleretal.,1985;VanderSchaftetal.,1987).Alsoinothercelltypes,theproportionofSMintheouterleafletwasgenerallyshowntobehigherthanthatofPC(seetablesinDevaux,1991;Zachowski,1993forsummaryofresults).Nevertheless,itisnotablethatasignificantproportionofSM,e.g.,10–20%,isfoundintheinnerleafletwhenbiochemicalmethodsareused.Inaddition,freeze-fracturereplicalabelingEMalsoshowedlabelingoftheinnerleafletwithanSM-bindingtoxin,lysenin(Murateetal.,2015).Quantitativityandspecificityofthelyseninlabelinginfreeze-fracturereplicaneedstoberigorouslytested,buttheresultisconsistentwiththepresenceofSMintheinnerleafletoftheplasmamembrane. Glycosphingolipids Glycosphingolipids(GSLs)aregenerallybelievedtoexistonlyintheouterleaflet.GlucosylceramideandgalactosylceramidestarttheirsynthesisatthecytoplasmicsideoftheERandtheGolgi,andarethenflippedtotheluminalsideforfurtherglycosylations.ComplexGSLslikegangliosidesarenotlikelytoflipbacktothecytoplasmicleafletbecauseoftheirbulkyhydrophilicheadgroup,butshortcarbohydratechainGSLscantranslocateacrossthemembrane(Butonetal.,2002).Therefore,thepresenceofasmallamountofGSLintheinnerleafletoftheplasmamembranecannotbeexcluded. Cholesterol Despitethedevelopmentofnewmethodologies(Friszetal.,2013;Solankoetal.,2015),neitherthelateraldistributionofcholesterolnoritsdistributionacrossthetwoplasmamembraneleafletsiswell-characterized(Marquardtetal.,2015).Apolyeneantibiotic,filipin,hasbeenusedfrequentlytovisualizeendogenouscholesteroldistributionbothbyfluorescencemicroscopyandEM.FilipinhasbeenreportedtoprobealsoGM1(Arthuretal.,2011),butgiventhat5–7mol%cholesterolrequiredforfilipinvisualizationinmembranes(Behnkeetal.,1984)andthescarcityofGM1thisshouldnotbeaproblemincellstaining.However,membranedeformationcausedbyfilipin-cholesterolcomplexformationdoesnotindicateinwhichleafletofthemembranecholesterolexists.Asanalternative,analogueswithvariousfluorescenttagshavebeenused,butnoneofthemgivesatisfactoryresults,becausethepresenceoflargetagssignificantlychangesthemolecularpropertyofcholesterol,inparticularthequickflip-flopbetweenthetwoleaflets(KlymchenkoandKreder,2014;Sezginetal.,2016). Dehydroergosterol,thatisverysimilartocholesterolinthemolecularstructure,isconsideredtobethebestfluorescentanalog.Itsopticalpropertiesarenotfavorableforimaging,butastudyusingdehydroergosterolshoweditsenrichmentintheinnerleaflet(Mondaletal.,2009),whichisinaccordancewiththeoreticalconsiderations(GiangandSchick,2014;Falkovichetal.,2016).Incontrast,preferentialdistributionofcholesteroltotheouterleafletofthehumanerythrocytemembranewasshownbybiochemicalanalysisofaphospholipidmonolayerobtainedbyfreeze-fracture(Fisher,1976).Itisnotclearwhetherthedisparitybetweenthetwostudiesisderivedfromdifferencebetweenthecelltypesexamined,methodology,orboth. AcylChains Theacylchaincompositionofphospholipidsisthoughttobeacriticalfactortodeterminepropertiesofindividualmembraneleaflets,butourknowledgeofthisaspectisevenlesscompletethanthatoftheheadgroups.Classicalworkusingthinlayerchromatographyandphospholipasehydrolysissuggestedthattheproportionofsaturatedandunsaturatedacylchainsishighlyvariablebetweendifferentphospholipidspeciesandalsobetweendifferenttissues(YabuuchiandO'Brien,1968;WoodandHarlow,1969).Tandemmassspectrometricanalysisconfirmedthetissue-to-tissuevariation,butitalsoindicatedthatPS,PE,andPItendtohaveahigherproportionofpolyunsaturatedacylchainsthanPC,whichisenrichedwithmono-anddi-unsaturatedacylchains(Hicksetal.,2006). Itislargelyunknownhowphospholipidswithdifferentacylchaincompositionsdistributetwo-dimensionallyintheplasmamembrane.Notably,PCofparticularacylchaincompositionswereshowntohaveheterogeneousdistributionsalongtheneuronalaxonbothwhenassessedbyimagingmassspectroscopy(Yangetal.,2012)andbyuseofauniquemonoclonalantibody(Kugeetal.,2014).Butwiththesemethods,itisnoteasytodistinguishacylchainsinoneleafletofamembranefromanother.Freeze-fracturemaybeoneofafewpossiblewaystoseparatethetwoleaflets,buthasnotyetbeenusedforanalysisoftheacylchaincomposition. OrderedMembraneNanodomainsintheCytoplasmicPlasmaMembraneLeaflet Currentlytheco-existenceoftwoliquidphases,liquidordered(lo)-andliquiddisordered(ld),isthebestexplanationfordomainsofdifferentiallipidpackingobservedintheplasmamembranealthoughtheperfectprooftocharacterizethemasphasesisalmostaschallengingtoobtainastheabsoluteevidencefortheirdismissal.Thisneithermeansthatproteinsorlipidscouldnotalsoshowlocalenrichmentunrelatedtodifferentiallipidpacking,thatplasmamembranedomainsneedtobeinthemicronrangenorthatcellularmembranesareatequilibrium(AckermanandFeigenson,2015;MouritsenandBagatolli,2015). Anoutstandingquestioninthemembranenanodomainfieldiswhetherlo-domainscanforminthelipidmixespresentintheinnerleafletoftheplasmamembrane.Frommodelmembranestudiesitisclearthatthelipidmixesmimickingtheouterplasmamembraneleaflet(PC,SL,andcholesterol),canformco-existingld-andlo-domains(Ahmedetal.,1997;deAlmeidaetal.,2003;VeatchandKeller,2005).Themainphospholipidsintheinnerleaflet(PE,PS,andPC)canonlyformld-phaseeveninthepresenceofcholesterol(WangandSilvius,2001;Kiesslingetal.,2006).Infact,whentheacylchainsofPE,PS,andPCaremostlyunsaturated,astheyareinvivo,lipidmixeswithahighPEcontentdonotevenformabilayerbutformahexagonalorcubicphase(BoniandHui,1983)anditwasrecentlyproposedthatthehighbendingfreeenergyofPEiswhatattractscholesteroltotheinnerleaflet(GiangandSchick,2014).DespitethefailureofsymmetricPE/PC/PSlipidmixestosometimesformbilayers,theydomakeuponehalfofthecellularplasmamembranehighlightingthateffectsofasymmetriclipidcompositionscannotbepredictedfromstudiesofsymmetricalmodelmembranes.Moreover,thedifferenceinthemembraneorderoflo-andld-domainsinmostmodelmembranestudiesisfargreaterthanthatpossibleintheplasmamembraneconsideringitslipidcomposition,makingpredictionsofprobe,lipid,andproteinpartitioningintheplasmamembranefromsuchstudiesoflimiteduse. Inmodelmembraneslo-domainscanbecharacterizedbiochemicallybytheirinsolubilityinnon-ionicdetergents,TritonX-100(TX)beingthemostwidelyused,thatwhenusedat4°Cproducesdetergentresistantmembranes(DRMs)thatfloatinsucrosedensitygradients.TheamountofTX-DRMsreflectsthefractionoflo-domainsinthemembranes(Ahmedetal.,1997)andthelipidsretrievedinTX-DRMscanformlo-phase(Schroederetal.,1994).Incells,therelationshipbetweenDRMsandlo-domainsismoretenuous(Lichtenbergetal.,2005;AshrafzadehandParmryd,2015;SevcsikandSchütz,2016).TX-DRMsfromTcellsaregiganticratherthannanosize(MageeandParmryd,2003)andproteinsnormallyresidentinorganellesotherthantheplasmamembraneappearinTcellTX-DRMs(vonHalleretal.,2001).However,TX-DRMsdosuggestthatlo-domainsinthetwoplasmamembraneleafletsarecoupledsincetheyareenrichedinbothsphingomyelinlikelytooriginatefromtheouterleafletandsaturatedglycerophospholipidsprobablyoriginatingfromtheinnerleafletrelativetobothtotalcellandplasmamembranelipids(Fridrikssonetal.,1999).Moreover,TX-DRMscontainacylatedproteinsthatinintactcellsareanchoredtotheinnerplasmamembraneleafletindicativeof,butnotevidencefor,innerleafletlo-domains(Melkonianetal.,1999). Severalotherlinesofinvestigationindicatethatlo-domainscouldexistintheinnerplasmamembraneleafletandhighlightthatleafletasymmetrychangesthebehaviorofmonolayers.Inmoleculardynamics(MD)simulationsofasymmetricbilayers,lo-domainsinoneleafletcaninduceregisteredordereddomainsinanopposingleafletwithacompositionthatonitsownwouldnotallowlo-domainformation(PerlmutterandSachs,2011;Polleyetal.,2014).MDsimulationsandtheoreticalconsiderationshavealsosuggestedtheoppositepossibilitythatmembranefluidisationinoneleafletmaycauseadecreaseintheorderoforevenpreventphaseseparationintheopposingleaflet(Wagneretal.,2007;Sunetal.,2015).Inasymmetricsupportedbilayers,lo-domainsonthesupportedsidecaninducelo-domainsinthefreesidewithinnerleafletlipidmixesthatonlyformld-phaseontheirown(Kiesslingetal.,2006),aprocessthatwaslatershowntorequireacylchaindiversityamongtheinnerleafletlipids(Wanetal.,2008).Inblackmembranesithasalsobeendemonstratedthatldandlophaseseparationinoneleafletcaninducephaseseparationinanopposingld-phaselipidmixandmoreoverthatldandlophaseseparationcanbesuppressedbyld-phaselipidmixes(CollinsandKeller,2008).Alsoinasymmetricvesicles,producedbylipidexchange,registeredlo-domainscanbeinducedinaldlipidmixbyanopposingldandlophaseseparatedbilayer(LinandLondon,2015)and,conversely,agelphasebecomeslessorderedthanthatinsymmetricbilayerswhenfacingld-phase(Heberleetal.,2016).However,directevidenceoftheexistenceofinnerplasmamembraneleafletlo-domainsisstillmissing. TheRegistrationofMembraneNanodomainsintheTwoLeaflets Theaggregationofregistereddomainscontainingnon-transmembranesignalingmoleculescouldfacilitatesignaltransductionacrosstheplasmamembranebyseparatingdeactivatingmoleculesfromtheirsubstrates(SimonsandToomre,2000;Mageeetal.,2005;Mongrandetal.,2010),butwhetherdomainsincellplasmamembranesareregisteredislargelyunknown.Whenld-andlo-domainsco-existinbothleafletsofabilayer,theycanadopttwostrategiestominimizetheenergycostofthedomainthicknessmismatch.Domainscouldeitherberegisteredtominimizethecontactareabetweenthetwophases,oranti-registeredtominimizethedifferenceinthicknessoverthebilayer(Figure2).Notethatcompleteanti-registrationisonlypossiblewheneachdomaintypeoccupies50%ofthetotalmembranedistributedatanyfractionaldivisionbetweenthetwoleaflets.Sincetheproportionofplasmamembranelo-andld-domainsinlivecellsisnotfixed(Mahammadetal.,2010;Owenetal.,2012;Dinicetal.,2013;Golfettoetal.,2015),domainregistrationisthemorelikelyscenario.Moreover,thelikelysimilarityinlipidcompositionofplasmamembraneld-andlo-phasesfavorstheirregistration(Fowleretal.,2016). FIGURE2 Figure2.Domainregistrationacrossthebilayer.Interleafletinteractionsfavordomainsinthetwoleafletstoberegistered(A),whereashydrophobicmismatchfavorsdomainantiregistration(B),whichservestoeventhemembranethickness. Contributingfactorstodomainregistrationcouldbetransmembraneproteins,lipidinterdigitation,membranecurvature,linetension,cholesterolflip-flop,andelectrostaticinteractions(Nickelsetal.,2015)ofwhichchaininterdigitationhasbeeninvokedasthemajorcontributingfactor(May,2009)butalsohasbeentotallydismissed(Collins,2008).Arecentexperimentaldeterminationofthestrengthofthecouplingparameterthatcausesdomainregistrationmatchesatheoreticalpredictionswithvaluesaround0.01kBT/nm2(Putzeletal.,2011;Blosseretal.,2015),butconsiderablyhighervalueshavealsobeensuggestedfromtheoreticalconsiderations(May,2009).Intriguingly,MDsimulationsofpolymershaveshownthatforcedsplittingofregisteredlo-domainsmakethemmovetowardoneanotherlongbeforetheyareincontact,i.e.,theysenseeachotherspresencethroughtheld-phase(Pantanoetal.,2011).Thissuggeststhatcouplingbetweenlipidsissubstantialandinsymmetricbilayerstheenergyinvolvedinthecouplinghasbeenestimatedtobearound100cal/molofphospholipid(Zhangetal.,2007). Recently,ithasbeenarguedboththatlinetensionalone(Galimzyanovetal.,2015)orthecompetitionbetweenlinetensionandleafletcouplingdeterminesdomainregistration(WilliamsonandOlmsted,2015).However,linetensionintheplasmamembraneislikelytobesmallduetothewiderangeoflipidspeciesavailabletosmoothenanyhydrophobicmismatch—adiversitythatwouldalsoresultinahighsimilarityinthecompositionofco-existingld-andlo-domains.Thusthelipiddiversityandaswellastheasymmetriclipiddistributionneedtobeincorporatedintoboththeoreticalandmodelmembranestudies.Intheirabsencetheresultsarelessphysiologicallyrelevant. Registrationoflo-domainsinasymmetricbilayershasbeendescribedinMDsimulations,supportedbilayers,blackmembranesandvesicles(CollinsandKeller,2008;Wanetal.,2008;ChengandLondon,2011;Chiantiaetal.,2011;PerlmutterandSachs,2011;Polleyetal.,2014),indicatingthatregistrationisthefavoredorganizationalsoincells.Ofnoteisthatdomaincouplinginsupportedbilayersisdependentondistancingthemembranefromthesupport(Gargetal.,2007). Wepioneeredcellstudiesontheregistrationofplasmamembranenanodomainsbetweenleafletsbyusingaprobethatexclusivelyreportonthemembraneorderoftheouterplasmamembraneleaflet.Wefoundthatdecreasingthelevelofactinfilamentsattachedtoinnerplasmaleafletlipidsresultedinalowerfractionoflo-domainsintheouterleaflet(Dinicetal.,2013).Theimplicationofourresultsisthatadirecteffectononeplasmamembraneleafletiscommunicatedtotheotherwithouttheinvolvementoftransmembraneproteins. SizeofDomains Wheninterleafletcouplingisstudiedinliposomesandsupportedlipidbilayers,domainsof1μmorlargerareusuallyexamined,butsuchlargedomainsarenotlikelytoexistintheplasmamembraneofmostmammaliancells(LingwoodandSimons,2010;AshrafzadehandParmryd,2015).Actually,studiesusingsingleparticletrackingindicatedthatatransientdomainassmallas10nmcantransmitsignalstothecytoplasmtoinducephysiologicalreactions(Suzukietal.,2007a,b).Therefore,inordertostudyphysiologicalimportanceofdomainregistrationintheplasmamembrane,domainsinthesmallsizerangeneedtobeexamined. Thisisachallengingtask,however,consideringthemethodscurrentlyavailable.Forexample,GFP-taggedlipid-bindingproteinsarecommonlyusedasprobestodefinethelipiddistributionbyfluorescencemicroscopy.Butwhenoneprobemoleculebindstotheheadgroupofatargetlipid,whichshouldbeintherangeof~1nmindiameter,bindingofanotherprobemoleculetoadjacenttargetlipidsisprecludedduetosterichindrance.Thislimitationisnotderivedfromthelimitationinspatialresolutionofmicroscopes,butfromthelargesizeofprobescomparedtolipidheadgroups,sothatuseofsuper-resolutionmicroscopesisnothelpful(forotherpotentialproblemsofthemethod,seeTakatorietal.,2014).Interferometricdetectionofscatteringcircumventsseveraloftheproblemsofparticletrackingandhasalateralresolutioninthelownmscale(Lindforsetal.,2004),buthasnotyetbeenappliedtocells.Importantlytrackingmethodscan,ifcelltopographyisaccountedfor(Adleretal.,2010),detectanomalousdiffusioncausedbyplasmamembranedomainco-existencebutdonotrevealwhatiscausingdomainformation;forexampledifferentiallipidpacking,proteinconcentrationorchargeaggregation. Inelectronmicroscopy,colloidalgoldparticlesoflargerthan5–10nmindiameterareoftenusedasmarkersandonesuchgoldparticlemayoverlaymanylipidmolecules(Fujitaetal.,2007;Zhouetal.,2014).Therefore,aclusterofcolloidalgoldlabelsindicatesenrichmentofthetargetlipidinthearea,butdoesnotnecessarilyindicateclusteringoflipidmolecules(althoughclusteringoflipidsislikelytooccurmorefrequentlywhentheirlocaldensityishigher). Itisevenmoredifficultistoexaminewhetherdomainsinthetwoleafletsareregistered.Useofcomplementaryfreeze-fracturereplicashasenabledtheexaminationofmatchingareasinthetwoleaflets(Hagiwaraetal.,2005),butevenwiththismethod,theregistrationofsmalldomainsisdifficulttostudywithoutatechnicalbreakthrough. CrossingtheMid-Line—APrivilegeofAsymmetricLipidswithOneLongAcylchain Thelipidrafthypothesisstatesthatsmalltightlypackedlipiddomainsfloatinaseaoflesstightlypackedlipidsandproposesthatlipidraftsinthetwoleafletsarecoupledbytheinterdigitationoflongsaturatedacylchainsonglycosphingolipidsintheouterleafletextendingintotheinnerleaflet(SimonsandIkonen,1997).Theproposedtypeofinterdigitationwasdifferentfromtheinitialuseofthewordforlongacylchainsspanningtheentirehydrophobiccoreofthebilayermadeupoflipidswithconsiderablyshorteracylgroups(BoggsandKoshy,1994;SchramandThompson,1995).Insteadthenowacceptedmeaningofinterdigitation,longacylchainscrossingthemidplaneofthebilayerandpenetratingasmalldistanceintotheopposingleaflet(Figures1,3),washighlighted.Thistypeofinterdigitationisforinstanceobservedforlongacylchain(C24)glycosphingolipidswhentheyareminorcomponentsinabiologicallyrelevant(C16-C18)matrix(Mehlhornetal.,1988;Morrowetal.,1995).Althoughtheideasbroughttogetherinthelipidrafthypothesiswerenotnew,ithadanenormousimpactinpromotingmembraneresearch.Muchofthepolarizeddiscussioninthefieldhasbeencausedbytheuseofquestionablemethodology(KlotzschandSchütz,2013;AshrafzadehandParmryd,2015),buttheexistenceofplasmamembranenanodomainswithdifferentlipidpackingwassuggestedfromstudiesthatpredatethelipidrafthypothesisbydecades(Morrisettetal.,1975;Karnovskyetal.,1982).Althoughfrequentlymisinterpreted,the1972fluidmosaicmodeldoesnotruleout,butratherinsists,thatshort-rangeorderintheplasmamembraneexists(SingerandNicolson,1972),animportantfeaturethatwasrecentlyclarifiedbyoneofthemodelfounders(Nicolson,2014). FIGURE3 Figure3.Pinningasamechanismtonucleatemembranenanodomains.Immobilisingplasmamembranecomponentscaninducelo-domainformation.Thishasbeendemonstratedbothforinnerleafletphoshoinositideslinkedtoactinfilaments(A)andouterleafletcomponentslikeGPI-anchoredproteins(B)(Dinicetal.,2013).Thecreationofnewlo-domainsislikelytoplayanimportantroleinthemolecularsortingrequiredforcellularprocessessuchassignalingandmembranetrafficking. Sphingolipids(SLs)canhaveabigdifferenceinchainlengthbetweenthesphingosinechain,thattendstobeC18,andtheamide-linkedacylchains,thatdifferfromC16toC24innaturallyoccurringSLs,creatingmanyasymmetricSLspecies.Unsurprisingly,MDsimulationsofsymmetricbilayershaveshownthatthegreaterthelengthmismatchbetweenthematrixlipidacylchainsandthegreaterthelengthmismatchbetweentheacylandthesphingosinechains,themoreinterdigitationisobserved(Niemeläetal.,2006).BendingoflongacylchainstowardtheirownleafletisalsoapossibilityandhasrecentlybeenobservedinbothMDsimulationsofSLsandmodelmembranesstudiesoffreefattyacidsinglycerophospholipidbilayers(PazRamosetal.,2016;Rógetal.,2016).However,bendingtowardthewaterinterfacecanbegreatlyreducedbycholesterolrevealedbyamodelmembranestudyonmethyldistributions(Mihailescuetal.,2011).Interestingly,simulationsshowthatcouplingisstrongerifthereismorecholesterolintheouterthanintheinnerleaflet(Rógetal.,2016). ArecentsimulationstudyonsymmetricalmembranesmadeupofasymmetricPCswithdifferencesintheiracylchainlengthssuggestedthatlipidcomplementarityinchainlengthcouldacttoassureuniformacylchainpacking(Capponietal.,2016).Thisisalongthelinesofpreviouslipidcomplementaritystudiesindicatingthat,whenpresentatmatchingconcentrations,shortacylchainsarefoundoppositelongonestocreateasmoothmembrane(Zhangetal.,2004,2005;Stevens,2005).Thatsuchanorganizationshouldoccurinbiologicalmembranes,withdiverseandasymmetriclipidcompositions,islessclear,especiallysincedomainsofdifferentorderandthuspresumablythicknessarefoundintheplasmamembrane(Gausetal.,2003;Dinicetal.,2013). ThatasymmetriclipidsfrombothleafletscanaffectcouplinginasymmetricmembraneswasfirstdemonstratedinGUVswhereSMC24:0wasshowntoreducethediffusionofPOPCfarmorethanthatofDOPC(ChiantiaandLondon,2012).ArecentMDsimulationstudy,withasymmetricmembraneswithcompositionscloselymimickingcellplasmamembranes,supportedthisfindingwithanotherlipidclassandfoundastrongercouplingfirstlyinasymmetricvs.symmetricmembranesandsecondlythestrongestcouplingwhentheinnerleafletcontainedPS18:0/18:1andtheouterleafletcontainedSM24:0(Rógetal.,2016).ThatPS18:0/18:1playsanimportantbiologicalrolewasimpliedinarecentstudyrevealingthatPSisrequiredforretainingcholesterolintheinnerleafletincellsandthatPS18:0/18:1uniquelyhasthecapacitytoprotectcholesterolinmodelsymmetricalmembranesfromoxidation(MaekawaandFairn,2015).Intriguingly,GPI-anchorproteinclusteringwasalsoshowntobedependentoninnerleafletPSbutwithlongacylchains,ratherthanaspecificacylchaincombination,beingtherequirement(Raghupathyetal.,2015).ThiswasinterpretedasenablinginteractionwiththelongacylchainsrequiredfortheGPI-anchoredproteinstoclusterallowingcross-talkofspecificlipidsacrossthebilayermidplane.Inlightofthestudydiscussedabove(MaekawaandFairn,2015),wespeculatethatthePSmayhavebeenrequiredtomaintainthecholesterollevelintheinnerleafletatalevelsufficientfortheformationoflo-domainssinceclusteredGPI-anchoredproteinsarefoundinsuch(Dinicetal.,2013)andcholesterolpreviouslyhasbeenidentifiedasimportantfortheirclustering(Sharmaetal.,2004). ImperfectioninLipidAsymmetryandDomainRegistration Asdiscussedabove,lipidshaveasymmetricdistributionsintheplasmamembrane,buttheasymmetryisnotcompleteandnon-negligibleamountsofmostlipidsarelikelytoexistintheless-favoredleaflet.Suchimperfectioninasymmetry,especiallythatofSM,couldbeofconsiderableimportancefordomainregistration.Inmodelmembranesinterleafletcouplingcanoccurwithno(oraminimalamountof)SMintheinnerleaflet(Wanetal.,2008;Viscoetal.,2014;LinandLondon,2015).However,ifasmallbutsignificantamountofSMexiststheinnerleaflet,assuggestedbybiochemicalandhistochemicalstudies(Devaux,1991;Zachowski,1993;Murateetal.,2015),thatSMcouldpotentiallycontributetolo-domainformation,whichcouldmakecouplingwiththeouterleafletlo-domainsveryefficient(Hallingetal.,2008;Lönnforsetal.,2011).NaturallythisSM-SMinteractionmechanismdoesnotexcludeparticipationofotherphospholipidsinlo-domainformationandinterleafletcoupling.Rather,theSM-basedcouplingmayfunctionasaninitialseedtoinducefurtherchangesbylateralinteractionwithotherphospholipids,suchasPSorinositolphospholipids. Pinning IthasbeenshownrepeatedlythatthebehaviorofouterleafletlipidsandGPI-anchoredproteinsisinfluencedbyactindynamics(Fujiwaraetal.,2002;Kwiketal.,2003;Goswamietal.,2008;Fujitaetal.,2009;Muelleretal.,2011),buthowactininthecytoplasmcanexerteffectsontheouterleafletoftheplasmamembranehasbeenalongstandingenigma.Celltopographyandtheproblematicinterpretationofthreedimensionaldatain2Dcouldexplainatleastsomeofthisbehavior(Adleretal.,2010),butpinningisalsoimportantforthecommunicationbetweenactinfilamentsandtheouterleaflet. Pinning,i.e.,whenthemobilityofmembranecomponentsiseitherreducedorabsent,altersthemixingentropy,whichcanalterthephasebehaviorand/ormembraneorganization(PutzelandSchick,2009;ArumugamandBassereau,2015).Proteinpinningcantransmittherearrangementofproteinsfromoneleaflettotheother.Thiswasfirststudiedinerythrocyteghostswhereitwasshownthattheenclosureofantibodiestospectrin,aproteinassociatedwiththeinnerleaflet,causedtherearrangementofglycolipidsandglycoproteinsintheouterleaflet(NicolsonandPainter,1973).Analogously,bindingoflectinstotheouterleafletcausedtherearrangementofspectrin(JiandNicolson,1974).Lectinsarealsoknowntoaffectthephysicalpropertiesoftheplasmamembrane(EvansandLeung,1984).Forlipids,pinningcannucleateeitheranld-phaseasshowninphosphatidylinositol4,5-bisphosphate[PI(4,5)P2]-containinggiantunilamellarvesicles(GUVs)whereactinpolymerisationinducedphase-separation(LiuandFletcher,2006),anlo-phaseasshowninGUVsbyGM1cross-linking(Hammondetal.,2005)oruponadhesion(Gordonetal.,2008). Simulationsoncriticalpointfluctuationshaveindicatedthatpinningoflipidstoactinfilamentscouldpreventlarge-scalephaseseparation(Ehrigetal.,2011;Machtaetal.,2011)andearliersimulationssuggestedthatmembraneproteinobstaclesorpinningcouldhavethesameeffect(YethirajandWeisshaar,2007;Fanetal.,2010;Gómezetal.,2010),phenomenathatwererecentlyobservedinmodelmembranes(Honigmannetal.,2014;Arumugametal.,2015).Criticalpointfluctuationsmayhoweverbesuperfluousforphaseseparationsinceitcanbeachievedbycouplingofmechanicalforceslikeactinpolymerisationtomembranecomposition(SensandTurner,2011).Thatpinningdoespreventlarge-scalephaseseparationintheplasmamembraneissupportedbytheobservationthatvisiblelarge-scalephaseseparationcanbeseeningiantplasmamembranevesicles(GPMVs)thatdonotcontaincorticalactin(Senguptaetal.,2008;Leventaletal.,2011).Furthermore,membraneblebsinlivecells,wherethemembraneconnectiontoactinfilamentshasbeenlost(Charrasetal.,2005),arestructuresintheμmrangewithalowerlaurdangeneralizedpolarization-valuethanthatfoundinthebulkplasmamembraneofthesamecells,indicativeofdisorderedlipidarrangement(Dinicetal.,2013).Sincelo-domainsformwhereactinfilamentsarepinnedtotheplasmamembrane,blebsmaycontainld-phaseonlyandrepresentanexampleofmicroscopicallyvisibledomainsinlivecellsalongsidetheremainingintermittentlyactinfilamentpinned,andfromalipidpackingperspective,moreheterogeneousplasmamembraneoflivecells. Inasymmetricmodelmembranestudiesthegeneralassumptionisthatanouter-side-likelipidmixcouldinducedomainformationinacytoplasmic-side-likelipidmixthatcannotphaseseparateonitsown(Kiesslingetal.,2006;CollinsandKeller,2008;LinandLondon,2015)buttheoppositeislessfrequentlyconsidered.However,incellsphosphoinositidesintheinnerplasmamembraneleafletcanbepinnedtointracellularactinfilamentsandcausetheformationoflo-domainsintheouterplasmamembraneleaflet(Figure3A),demonstratingthatthereisaninsideoutcommunicationforthelipidpackingofthetwoleaflets(Dinicetal.,2013).Lo-domainscanalsoformwhenouterleafletcomponentslikeGM1andGPI-anchoredproteinsarepatched(Figure3B),demonstratingthatthephysicalstateoftheplasmamembraneisalsoinfluencedbythecell'sextracellularenvironment(Dinicetal.,2013).Thatimmobilizationbypinningcancausetheformationoflo-domainsinlivecellswasthusdemonstratedin2013(Dinicetal.,2013).DespitethisalaterprominentstudyusingsimulationsandGPMVsconcludedbyspeculatingthatpinninginducedlo-domainformationmightbepossibleinlivecells,claimingitasanovelandoriginalidea,reversingtheusualprocessofobservationfollowingspeculation(Raghupathyetal.,2015). AlinkbetweenGM1,pinningandinterdigitationhasrecentlybeenfoundinbothsimulationsandmodelmembranestudies(Spillaneetal.,2014;Sunetal.,2015).Itseemslikelythatthelo-domainformationobservedintheplasmamembraneaftercross-linkingouterleafletcomponents,describedabove,causesthepinnedmoleculestostretchandhencemakecontactwiththeinnerleafletlipids,bothnucleatingandstabilizingthedomains.Pinningmayalsoresultinmembranecurvaturethataffectsbothleaflets(Deveralletal.,2008).Althoughwearewell-awareofboththeimportanceandprevalenceofmembranecurvatureincells(Adleretal.,2010;ParmrydandOnfelt,2013),wehavechosentolimitthediscussioninthisreviewtoothermechanismsofimportanceformembranenanodomainformation. PI(4,5)P2isknowntointeractwithmanyactin-bindingproteins,regulatingtheiractivityandlocalization(Saarikangasetal.,2010)andmaybethephosphoinositidethatlinkschangesinactindynamicstotheproportionofplasmamembranelo-domains(Dinicetal.,2013).Interestingly,althoughPI(4,5)P2oftencontainsapolyunsaturatedacylchain(McLaughlinetal.,2002),whichmightnotbeexpectedtobefoundinlo-domains,PI(4,5)P2isneverthelessenrichedinplasmamembranelo-domains(Parmrydetal.,2003).Alikelyexplanationforthisapparentparadoxisthattheexpectationsarisefromthebehavioroflipidsinwell-separatedld-andlo-domainsinmodelmembranesthathaveanorderdifferenceimpossibletoachieveintheplasmamembrane. PSisalsoacandidatetomediatetheactin-outerleafletlipidcoupling,asitisknowntobindnumerouscytoplasmicproteins(forareview,seeStaceandKtistakis,2006),includingactin-bindingproteins(Cohenetal.,1986;Mugurumaetal.,1995;Makuchetal.,1997).Actually,themobilityofPSintheplasmamembranehasbeenshowntoincreaseuponactindepolymerization(Kayetal.,2012;Zhouetal.,2014),indicatingthatPSmaybeconstitutivelyboundtotheactinmeshworkoritsmovementsconfinedbytopographicalfeaturesofthemembrane(Adleretal.,2010).BindingbetweenPSandactin-linkingproteinsisthoughttooccurelectrostaticallytothePSheadgroupandthusshouldbeindependentoftheacylchaincomposition(WoodandHarlow,1969;Hicksetal.,2006),butitislikelytobeenhancedifPSisclustered. Interestingly,theacylchaincompositionofthedifferentphospholipidspecies,especiallyPS,variesbetweentissues(Hicksetal.,2006).Boththeefficiencyofdomainregistrationandinterleafletcouplingmaydifferwithacylchaincomposition:withastrongercoupling,theinnerleafletshouldbecomemoreorderedandhencesimilartotheouterleaflet.Howtheacylchaincompositionisregulatedisnotclear,butLand'scycle,remodelinginnerleafletphospholipids(Hishikawaetal.,2014),maybeinvolved.Theregulatorymechanismandthepossiblecorrelationbetweentheacylchaincompositionandinterleafletcouplingwarrantfurtherstudies. Outlook Comparedwithaminoacidsandnitrogenousbasesthelipiddiversityisenormousandintriguing.Similarly,comparedtoproteinsandnucleicacids,ourknowledgeofmembranesislimited.Toolsarenowbeingdevelopedtoforinstanceunraveltherolesofindividuallipidspeciesincellularprocesses,tostudytheeffectofleafletasymmetryandtoperformdetailedexaminationofthelipiddistributionwithinleaflets—alloutstandingquestionswhichrequireclosecollaborationsofscientistsfromphysical,chemical,andbiologicaldisciplines.Wepredictthatwithinthenextdecadeexcitinginterdisciplinarybreakthroughsinthemembranebiologyfieldwillunravelmechanismsoffundamentalbiologicalprocessesandthatwewillapproachaconsensusregardingthenatureofplasmamembranenanodomains. AuthorContributions IPconceivedtheidea.IPandTFwrotethereview. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WethankJeremyAdlerassistanceinpreparingfiguresandcriticalreadingofthemanuscript.ThisworkwassupportedbygrantstoTFfromGrants-in-AidforScientificResearchfromtheMinistryofEducation,Culture,Sports,Science,andTechnologyoftheGovernmentofJapantoTF(15H02500,15H05902)andtoIPfromtheSwedishResearchCouncil(2015-04764)andO.E.andEdlaJohansson'sFoundation. 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Editedby:MarekCebecauer,J.HeyrovskyInstituteofPhysicalChemistry(ASCR),Czechia Reviewedby:ErdincSezgin,UniversityofOxford,UKHectorMartinez-Seara,InstituteofOrganicChemistryandBiochemistry(IOCB)oftheCAS,Czechia Copyright©2017FujimotoandParmryd.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:IngelaParmryd,[email protected] COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>