There Is No Simple Model of the Plasma Membrane ... - Frontiers

The plasma membrane fully covers the cell surface. Its continuity is especially important for membrane receptors or effector molecules which ... ThisarticleispartoftheResearchTopic Molecularorganisationofmembranes:wherebiologymeetsbiophysics Viewall 12 Articles Articles ManuelPrieto InstitutoSuperiorTécnico,UniversidadedeLisboa,Portugal DylanM.Owen UniversityofBirmingham,UnitedKingdom RichardM.Epand McMasterUniversity,Canada Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract BasicStructureofCellMembranes IntrinsicPropertiesofCellMembranesEssentialforTheirFunction ExtrinsicFactorsInfluencingthePlasmaMembraneOrganisation PlasmaMembraneOrganisation–GeneralModelsandConcepts ThereisNoUniversalModelofthePlasmaMembraneLateralOrganisation AuthorContributions Funding 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.,29September2016 |https://doi.org/10.3389/fcell.2016.00106 ThereIsNoSimpleModelofthePlasmaMembraneOrganization JorgeBernardinodelaSerna1,GerhardJ.Schütz2,ChristianEggeling3andMarekCebecauer4* 1ScienceandTechnologyFacilitiesCouncil,RutherfordAppletonLaboratory,CentralLaserFacility,ResearchComplexatHarwell,Harwell,UK 2InstituteofAppliedPhysics,TechnischeUniversitätWien,Wien,Austria 3MRCHumanImmunologyUnit,WeatherallInstituteofMolecularMedicine,UniversityofOxford,HeadleyWay,UK 4DepartmentofBiophysicalChemistry,J.HeyrovskyInstituteofPhysicalChemistry,CzechAcademyofSciences,Prague,CzechRepublic Eversincetechnologiesenabledthecharacterizationofeukaryoticplasmamembranes,heterogeneitiesinthedistributionsofitsconstituentswereobserved.Overtheyearsthisledtotheproposalofvariousmodelsdescribingtheplasmamembraneorganizationsuchaslipidshells,picket-and-fences,lipidrafts,orproteinislands,asaddressedinnumerouspublicationsandreviews.Insteadofemphasizingononemodelweinthisreviewgiveabriefoverviewovercurrentmodelsandhighlighthowcurrentexperimentalworkinoneortheotherwaydonotsupporttheexistenceofasingleoverarchingmodel.Instead,wehighlightthevastvarietyofmembranepropertiesandcomponents,theirinfluencesandimpacts.Webelievethathighlightingsuchcontroversialdiscoverieswillstimulateunbiasedresearchonplasmamembraneorganizationandfunctionality,leadingtoabetterunderstandingofthisessentialcellularstructure. Membranesareoneofthekeystructuresincellbiology.Besidesbeinginstrumentalincompartmentalizingandprotectingcells,theirroleasorganizingcentersfortaskssuchasmetabolismorsignalingisincreasinglyrecognized.Infact,amajorityofcellularprocessesareassociatedwithmembranes(Stryer,1995).Membranesprovideusefuldocksforcorrectlocalisationofproteinswhichisessentialfortheirfunction(MiosgeandZamoyska,2007;Greccoetal.,2011;HungandLink,2011).Importantly,inhumans,mislocalizationofmembraneproteinsleadstotheloss-of-functionand,frequently,candevelopintodiseases(Edwardsetal.,2000;Matsudaetal.,2008;HungandLink,2011;Schaefferetal.,2014).Nevertheless,thepresenceofproteinsataparticularmembraneisusuallynotsufficientfortheirfunction.Often,thenanoscopiclocalization,oligomerisationand/orclusteringofmembraneproteinscanaffecttheefficiencyofcellularprocesses(Cebecaueretal.,2010;Matthews,2012;Nussinov,2013;Garcia-Parajoetal.,2014).Membranes,thelipidenvironmentandmembranepropertiesingeneral,influencenanoscaleorganizationandfunctionofthesemolecules.Itis,therefore,importanttounderstandmoleculardetailsofmembranestructureandmechanismsresponsibleforitsdynamicsorganization. Here,wereviewmembraneproperties,modelsofmembraneorganizationandusefultechniquesforstudiesofmembraneorganizationanddynamics,withaspecialfocusontheplasmamembraneofhighereukaryotes(mammals).Ourspecificaimistore-emphasizecurrentlyomittedorunderestimatedbiophysicalprinciplesanddiscusstheirroleindynamicmembraneorganization.Weattempttoprovideacomprehensivedescriptionofmembranecomplexityandsuggestionshowtoavoidinterpretationofmembrane-associatedphenomenawithinthebordersofasingletheory.Asareaderwillsee,webelievethatthereisnouniversalmodeloftheplasmamembranedynamiclateralorganization.Thesemoregeneralissueswillbediscussedinthelastsection.First,letusstartwiththeverybasicstructureofmembranes. BasicStructureofCellMembranes Alipidbilayerformsthebasisofallcellularmembranes.Itisalamellarstructurewithahydrophobiccoreandapolarheadgroupregiononbothsides(Figure1A).Incells,itiscomposedofhundreds,ifnotthousands,ofdifferentphospholipidspecies.Thesedifferintheirpolarheadgroupmoietybutmainlyinthelengthandsaturationofacylchainsformingahydrophobiccoreofalipidbilayer.Otherlipidandfattyacidspeciesaddtothiscomplexity.Ofthose,sterols(cholesterolinmammals)arethemostabundantintheplasmamembraneandcanrepresentupto40%oftotallipid(vanMeeranddeKroon,2011).Cholesterolhasaspecialstructure(Figure1A)enablingstrongimpactonbasicmembranepropertiessuchasviscosityorinterleafletcoupling,asdescribedmultipletimesincomprehensivearticles(Ipsenetal.,1987;MouritsenandZuckermann,2004;MaxfieldandvanMeer,2010). FIGURE1 Figure1.Schematicillustrationofthebasicstructureoflipidbilayerandproteo-lipidicmembranes.(A)Cellmembranesarelamellarstructureswithahydrophobiccoreandapolarheadgroupspace.Asexamples,phospholipidsandcholesterolareshownwithalmostatomisticdetail(redandgreenboxes).(B)Membraneproteinscanintegrateintomembranes(i),butcanuselipidanchors(iiandiii)orperipherallyassociatewithmembranesviaelectrostaticinteractions(iv).(C)Proteinscanfurtherassociatewithmembranesviaprotein-proteininteractionsonthecytosolicside(v)orattheinterfacebetweentheplasmamembraneandtheextracellularmatrix(vi).Outerleafletlipids(vii)andextracellulardomainsofproteins(viii)areoftenglycosylated. Proteinsconstituteapproximatelyhalfofthetotalplasmamembranemass(DupuyandEngelman,2008).Wedistinguishintegralandperipheralmembraneproteinsdependingontheiranchorageintoalipidbilayerviatransmembranedomain(s)oralipidmoiety,respectively(Figure1B).Inaddition,someproteinsmayassociatewiththemembraneviaelectrostaticinteractionswithlipidheadgroups(Figure1B;McLaughlinandMurray,2005)oravarietyofprotein-proteinorprotein-glycaninteractions(Figure1C;Stryer,1995).Suchproteinsarecommonlytermedas“membrane-associated.”Extracellularpartsoflipidsandproteinsarefrequentlyglycosylated(Figure1C).Indeed,glycansformadensestructureattheoutersurfaceoftheplasmamembrane(BerrierandYamada,2007).Thismolecularcomplexityofmembraneshasprobablyevolvedtoserveasaselectivebarrierandorganizingcenterwithahighfidelityandrobustness(Cebecaueretal.,2010).Butwhatarethoseuniquepropertieswhichwereselectedintheprocessofevolutiontocontrolcriticalcellularprocesseswithsuchefficiency? IntrinsicPropertiesofCellMembranesEssentialforTheirFunction Earlydefinitions,ofwhichthe“fluidmosaicmodel”ofSingerandNicolson(SNmodelSingerandNicolson,1971,1972)isthebestknown,highlightedfluidityasoneofthemostcriticalmembranefeatures.Indeed,fluidityofmembranesprovidesimportantadvantageoverothercellularstructuressuchasthecytoskeletonorribonucleoproteins.Itformsthebasisforthehighlydynamiccharacterofmembrane-associated(bio)chemicalreactionsandothercellularprocesses.Membranefluidityenablesthemajorityofmoleculestodiffusefreelyoverlongdistancesandrotateorre-orientatetoadoptoptimalconformation.Membranescanbeconsideredastwo-dimensionalsolutions.Thistwo-dimensionalcharacteralsodistinguishesmembranesfromotherthree-dimensionalcellularsolutes(e.g.,thecytosol).Thefundamentalimportanceoffluidityis,forexample,underlinedbythefactthatcellsmodifythesaturationoftheirlipidacyl-chainstokeeptheirmembranesfluidwhenadaptingtotheenvironment,e.g.,differenttemperatures(FraenkelandHopf,1940;Budaetal.,1994). Althoughmembranesarefluid,theyhavehigherviscosity(Box1)thanthecytosol(Luby-Phelpsetal.,1993).Thishasadirectimpactonthemobilityofmembranemolecules.Membraneviscositycanbemodifiedbylipidcompositionorotherfactors,suchasthepresenceofproteinsorpoorlymobilestructures,andwillthusvaryoverspaceandtime. Box1.MembraneFluidity,ViscosityandMobility Viscosityisamacroscopicparameterdescribingthebehaviorofalarge,rigidsphereinaNewtonianfluid.Itsuseformembranesisimperfectandshouldbetreatedwithcare(ValeurandBerberan-Santos,2012;Olšinováetal.,2014).Membranesarenanoscopicstructureswith2Dcharacterandhighlyheterogeneouscompositionintermsofsizeandchemistry.Duetoalackofabetterparameter,weusetheterm“viscosity”todescribemembranepropertiessuchasmembranelateralcompressibilityandacylchainordering,whichinfluencethemobilityofmembranecomponents.Otherterms,e.g.,“microviscosity”or“rigidity”werealsousedinliteraturetocoverthesepropertiesinoneword(ShinitzkyandInbar,1976;KowalskaandCierniewski,1983;Gutetal.,1985;Sherbet,1989). Theterm“fluidity”isfrequentlyusedtoreplace“viscosity”forbiologicalmembranesorotherhighlyheterogeneousmaterials(ValeurandBerberan-Santos,2012).Weuseterm“fluidity”inthisworktodistinguishmembranesfromothercellularstructureswhichexhibitmuchhigherstability(e.g.,nucleoproteins),therebylimitingrapid,long-rangemobilityofassociatedcompounds. Effortstomeasureviscosityofcellularmembranesareassociatedwithserioustechnicaldifficulties(ValeurandBerberan-Santos,2012;Olšinováetal.,2014).Instead,measurementsofrotationalorlateraldiffusionweresuccessfullyappliedtocharacterizemembraneviscosity.Incellularmembranes,lateraldiffusionisfrequentlysubstitutedwiththeterm“mobility.”Mobilityofmembranemoleculescanbeinfluencedbymanydifferentfactors,suchas(i)membraneorderingor,intheotherterms,howdenselylipidsandproteinsarepackedinthemembrane(Kahyaetal.,2003),(ii)lateralpressureofthemembranewhichispartiallylinkedtoorderingbutalsomembranehydration(polarity)anddirectlyinfluencesbilayercompressibilityandelasticity(Marsh,1996;Cantor,1999),and(iii)macromolecularcrowding(Saxton,1987;GuigasandWeiss,2015).Mobilityofmembranecomponentsisfurtherinfluencedbyotherintrinsicandextrinsicfactorsasdescribedinthemaintext. Toillustratedramaticdifferencesinthemobilityofmoleculesinsyntheticandcellularenvironments,weprovideafewvaluesofdiffusioncoefficientsinTable1.Theseshouldbeconsideredasasimpleguidelineduetodifferencesintheprecisionwithwhichthesevaluesweremeasured.Wealsoprovidethetimescaleamoleculerequirestotraversethedistanceof20μm(longitudalsizeofHeLacells)byrandom(Brownian)2Dmotion.Thisshouldunderlinedramaticdifferencesinthemobilityofmoleculesinrealspace. AnotherpropertyemphasizedintheSNmodeliscontinuityoftheplasmamembrane(SingerandNicolson,1972).Theplasmamembranefullycoversthecellsurface.Itscontinuityisespeciallyimportantformembranereceptorsoreffectormoleculeswhichneedrapidlytore-localize,e.g.,whenacellischangingitsdirectionofchemotacticmobility(JanetopoulosandFirtel,2008).Continuityalsosupportsintermolecularinteractionsortheformationofmulti-molecularassemblieswithinoratthesurfaceofmembranes.Insomecells,membranecontinuityislimitedtotheapicalorbasalsideduetothepresenceoftightjunctionseliminatingfreemobilityofmembranemolecules(BaldaandMatter,2008).Wewilldiscussviscosityandcontinuity,andtheirimpactontheorganizationofmembranesinmoredetailfurtherinthetext. Almostallmoleculescaninteractandinfluenceeachotherincellularmembranes.Asaconsequence,coexistenceofmoleculesinmembraneshascooperativecharacter.CooperativityofmoleculeswasalreadymentionedforfluidcellularmembranesintheSNmodel(SingerandNicolson,1972)butseemstoberecentlyoverseen.Thispropertyhasadramaticimpactonexperiments,inwhichsystemicperturbanceofmembranes(e.g.,bychemicalorgenetictreatment)wasemployedtosupportspecificmodelsofmembraneorganization. Lipidmembranesundergointerleafletcoupling,meaningthatacylchainsoflipidsinoneleafletinterdigitateintothespaceoftheotherleaflet(Figure2A;Nickelsetal.,2015).Theoreticalpredictionssuggestthatinterleafletcouplingcancoordinatetheorganizationofmoleculesbetweenthetwoleaflets(Schmidtetal.,1978;Duzgunesetal.,1988;Merkeletal.,1989;Kiesslingetal.,2006;Raghupathyetal.,2015;WilliamsonandOlmsted,2015).Yet,Whiteandco-workersrecentlyprovidedanalternativeview(Mihailescuetal.,2011;Capponietal.,2016).Theydonotnegatetheexistenceofstrongcouplingbetweenthetwoleafletsoflipidbilayerbutobservednodirectcomplementaritybetweentheoppositeacylchains(Capponietal.,2016).Cholesterol,whichwaspredictedtointensifyinterleafletcouplinginmembranelipiddomains,wasfoundtoreducethelevelofacyl-chaininterdigitation(Mihailescuetal.,2011).Theseworksindicatethatweneedmoreexperimentaldatainordertobetterunderstandtheeffectofinterleafletcouplinginlipidbilayers. FIGURE2 Figure2.Schematicillustrationsoftheselectedintrinsicmembraneproperties:(A)Interleafletcoupling[interdigitatinglipidacylchainsingreen-gray;zoom:interdigitatingethylgroupsofupper(green)andlower(red)leaflets];(B)Asymmetricdistributionoflipidsandions[righthand-side:color-codingoflipidspecies];(C)Negativelychargedlipids(yellow)oftheplasmamembraneinnerleaflet[fortheassociationofproteinswithbasic-richdomains(lightgreen)];(D)Lipidself-assemblies(pink);(E)Hydrophobicmismatch(purple);(F)Protein-lipidinteractions[*sphingolipid-and**cholesterol-bindingpockets]. Theplasmamembraneofeukaryoticcellsisasymmetric(Figure2B)intermsoflipidandsurfaceioncomposition,aswellasthepresenceofspecificproteins(RothmanandLenard,1977;vanMeeretal.,2008).Thelipidasymmetryismaintainedbyflippasesandotherlipidtranslocatingortransportproteins(Canagarajahetal.,2008;Devauxetal.,2008).Chemicalasymmetry,agradientofions,drivesanumberofvitalcellularprocesses(e.g.,generationofchemicalenergyandmetabolism).Ontheotherhand,lipidasymmetryfurtheraddstothediversityandcomplexityofcellularcompartments,therebyhelpingtooptimizecellularprocesses.Forexample,negativelychargedlipidheadgroupsintheinnerleafletprovidethebindingsurfaceforproteinswithspecificbindingdomains(Figure2C;McLaughlinandMurray,2005).Thiscancauseproteinrelocalisationoftenleadingtotheinitiationofsignalingevents(Yeungetal.,2008).Inaddition,chemicalasymmetryandthepresenceofionsinducesheterogeneousdistributionoflipids,atleastinsimulationsandinmodelsystems(Váchaetal.,2009;Jurkiewiczetal.,2012).Whetherthiseffectcontributestotheorganizationofplasmamembraneinlivingcellsisexperimentallydifficulttotest;anasymmetricmembraneisindispensableforcellviability.Atthesametime,theformationofasymmetricmodelmembranesinvitroisaratherdelicateprocessandwassuccessfullyperformedonlyinafewcasesinpast(Kiesslingetal.,2006;CollinsandKeller,2008;Chiantiaetal.,2011).Therefore,datademonstratinglateral(re)organizationduetomembraneasymmetryarestillrare. Eventhoughlipidsinteractonlyweakly,preferentialself-assembliesofcertainlipidspeciesorconformations(Figure2D)weredemonstratedinmodellipidmixtures(Björkbometal.,2010;Ivankinetal.,2010).Undercertaincircumstances,lipidself-assemblingmayextensivelyreducemiscibilityofitsmolecules,i.e.,generatephysico-chemicalheterogeneities.Awell-knownexampleoflipidself-assemblyandsegregationistheformationofseparatedlipidphasesinvesiclescomposedoftwoormorelipidspecieswithdifferentmeltingpoints(BagatolliandGratton,1999;Korlachetal.,1999;BernardinodelaSernaetal.,2004;VeatchandKeller,2005).Importantly,lipidsarepronetophaseseparationormiscibilitytransitionsalsoincellmembrane-derivedvesiclesandblebs,aswellasartificialvesiclesgeneratedfromlipidsextractsandfromnativemembranes(BernardinodelaSernaetal.,2004;Baumgartetal.,2007;Veatchetal.,2008).Alltheseobservationswereachievedusingequilibratedmembranes;however,cellsarenon-equilibriumsystems(Stryer,1995).Indeed,nomiscibilityphasetransitionswereobservedinlivingcellsoverawiderangeoftemperatures(Leeetal.,2015).Putativeimpactoflipidself-assemblyandorderedlipidmembranesoncellmembranesisdiscussedinthesection“Plasmamembraneorganization–generalmodelsandconcepts.” Hydrophobicthicknessofalipidbilayerisdefinedmainlybythelengthandsaturationofacylchainsandthepresenceofsterols.Bilayerlipidsinteractnon-specificallyandtransientlywithtransmembranedomainsofintegralproteins(Marsh,1993).ImparityofthehydrophobicthicknessofthebilayerandthehydrophobiclengthofTMD(s)iscalledhydrophobicmismatch(Figure2E).Hydrophobicmismatchwasproposedtoinducemolecularaggregation/segregationinlipidbilayers,asdescribedinthemattressmodel(MouritsenandBloom,1984).Forexample,lipidswithlongerandmoresaturatedacylchainswillpreferentiallyresideintheannulusofhelicalTMDwithlonghydrophobiclength.Moreaboutthemattressmodelisdiscussedinthesection“Plasmamembraneorganization–generalmodelsandconcepts.” Lipidscanalsointeractwithproteinsinamorespecificmanner(Haberkantetal.,2008;FantiniandBarrantes,2013;Yeagle,2014).Severalproteinscarrylipid-bindingdomains(Ernstetal.,2010;Contrerasetal.,2011;FantiniandBarrantes,2013)towhichlipidsbindwithahigheraffinitycomparedtothelipidsofthefirstshellinteractingwithtransmembranedomainsnon-specifically.Suchprotein-lipidinteractions(Figure2F)canbehighlyspecificinawaythatlipidheadgroup,acylchainlengthanditssaturationdeterminetheaffinityofsuchinteractions(Contrerasetal.,2012).Specificprotein-lipidinteractionshavebeenshowntomodulateproteinstabilityanditsfunction(UittenbogaardandSmart,2000;Hansonetal.,2008;Contrerasetal.,2012)oraredirectlyinvolvedintransportoflipidsbetweensubcellularcompartments(Kwonetal.,2009).Butwhatistheirimpactonthelateralorganizationofplasmamembraneistodateunclear. Theabovementionedintrinsicpropertiescanbeascribedtoanyproteo-lipidmembranes,independentofwhethertheseareartificialorcellularstructures.Butwhatissospecificaboutmembranesoflivingcells?Can“clever”useoftheseintrinsicproperties,theirlocalamplification,reductionand/orcombinationleadtosuchlimitlessconcertofeventssuchasmetabolismandsignaltransduction?Oristhereaneedforextrinsicfactorstosupportthosebasalmembraneproperties? ExtrinsicFactorsInfluencingthePlasmaMembraneOrganisation Theplasmamembraneisbuilttointeractwithsurroundingstructuressuchascorticalactin,theextracellularmatrixoravarietyofligandmolecules.Theseformthebasisofextrinsicfactorswhichcanshapetheplasmamembrane. Weassignedprotein-proteininteractionstothesectionofextrinsicfactors,giventhefactthatextra-membranous(extracellularandcytosolic)domainsarethepredominantstructuresinvolvedinpersistentassociationsofproteins.Further,sincetheseinteractionsofteninvolvenon-membranousproteinscaffolds,webelievethatprotein-proteininteractionshave,tosomeextent,extrinsiccharacter. Incontrasttolipids,proteinscaninteractwithhighaffinityandthusformrelativelystablestructures(Figure3A)withinaseaoflipidmolecules.Indeed,theinteractionofproteinsisacommonprocessassociated,forexample,withleukocytesignalingorcellularadhesion,bothtakingplaceatthesurfaceofcells(DouglassandVale,2005;Rossieretal.,2012).Supramolecularcomplexesofproteinscanberelativelylargeandcanfurtherinteractwithothercellularcomponentssuchasthecytoskeleton,therebyformingproteinnetworkswhichcanhavelocalorsystemicimpactonmembranes(seebelow). FIGURE3 Figure3.Schematicillustrationsoftheselectedextrinsicmembraneproperties.(A)Protein-proteininteractions.Putativemembraneproteins(greenandred)formingheterodimerscanassembleintolargerclustersandbestabilizedbyfurtherinteractionwithcytosolicproteins(darkyellow).(B)Membranecurvature.Certainproteins(blueandgreen-brown)mayprefercurvedmembranes.Curvedmembranescanbestabilizede.g.,byBARproteins(lightyellow).(C)Intracellularcorticalactinskeleton.Actin-bindingproteins(darkyellowandorange)canassociatewithintegralmembraneproteinsandformlargerassemblieswithreducedmobility.(D)Extracellularglycocalyx.Interactionofcertainmembraneproteins(darkblueanddarkgreen)withtheextracellularmatrixmayleadtotheformationoflargerassemblieswithreducedmobility.(E)Endo-/exocytosis.Membranelipidsandproteinsarerapidlyendocytosed(red)orexocytosed(green). Morerecently,aconceptofproteinislandswaspresentedbasedonthefactthatproteinsweredetectedindistinctdomainsinterspacedwithprotein-freeareas,whenmembranepatcheswereimagedbyelectronmicroscopy(Wilsonetal.,2000;Lillemeieretal.,2006).Heterogeneousdistributionofproteinsinentitiesreminiscentofsuch“proteinislands”wereoftenfoundbysuper-resolutionfluorescenceimagingofthecellularplasmamembrane(forexampleSieberetal.,2007;Lillemeieretal.,2010;Letschertetal.,2014;Sakaetal.,2014;seealsoFigure4).However,itisnotyetclearwhethersuchentitiesarecreatedandstabilizedbyprotein-proteininteractionsorothermechanismsareinvolved.Theimpactoftheunderlyingactincytoskeletononproteinislandswasreportedinthepast(Wilsonetal.,2001;Lillemeieretal.,2006). FIGURE4 Figure4.Examplesofheterogenousdistributionofproteinsintheplasmamembraneofmammaliancells.(A)HeterogenousdistributionofMHCmoleculesasobservedonmurinelymphoidcellsin1967byCerottiniandBrunnerusingepifluorescencemicroscopy(AdaptedbypermissionfromJohnWileyandSonsLtd;(CerottiniandBrunner,1967).(B)MHCclusteringonmurineredbloodcellsasdetectedin1971byNicolsonandcolleaguesbyelectronmicroscopy(EM;scalebar:200nm;AdaptedbypermissionfromRUPress:©1971Nicolsonetal.,1971).(C)DistributionofindividualTCRmoleculesonactivatedprimaryhumanTcellsanalyzedbydSTORM(AdaptedbypermissionfromMacmillanPublishersLtd:NatureMethods(Rubin-Delanchyetal.,2015),copyright2015).Showing3×3μmarea.(D)DistributionofproteinsinmembranesheetsderivedfromaneuroendocrinecelllineasrevealedbySTEDmicroscopy[AdaptedbypermissionfromMacmillanPublishersLtd:NatureCommunications(Sakaetal.,2014),copyright2015].Scalebar:500nm. Certaincytosolicproteinscaninteractwithheadgroupsofselectedlipidspecies(e.g.,negativelychargedphosphatidylserinesandphosphoinositols)viaelectrostaticinteractions(Figure2C;McLaughlinandMurray,2005).Incells,bindingofproteinstochargedheadgroupsofinnerleafletlipidsiswell-documentedtocontrolimportantcellularprocesses,e.g.,phagocytosis(Botelhoetal.,2000).Inaddition,peripheralproteininteractionsattheinnerleafletoftheplasmamembranemodulatelocalizationandmobilityofchargedlipids,aswellassomeother,probablyassociated,molecules(Golebiewskaetal.,2008,2011;Yeungetal.,2008).Butwhethersuchperipheralinteractionscanmodulatethemobilityofothermembranecomponents(e.g.,attheouterleaflet)andhaveamoregeneralimpactontheplasmamembraneorganizationawaitsitsdirectproof. Duetomembraneplasticityandflexibity,certainlipidswithnon-conicalshape,e.g.,lysophospholipidsorphosphatidylethanolaminesfoundalsoincellmembranes,candeformtheplanarstructureoflipidbilayersbybending(Figure3B),therebychangingitscurvature(Šachletal.,2011).Similartoprotein-proteininteractions,curvatureisnotatypicalextrinsicfactor.Butincells,highlycurvedmembranesareprevalentlygeneratedbycurvature-formingproteins(e.g.,BAR-domaincontainingproteins)orcytoskeleton-inducedmechanicalforces(McMahonandGallop,2005;MattilaandLappalainen,2008).Boththeseprocessesareexternallyregulatedandrequireenergyand/orcofactors(Mimaetal.,2008;Frolovetal.,2010).Forexample,processesofendo/exocytosisareinitiatedbyprotein-inducedmembranebendingandATP/GTPhydrolysis(Vilmart-Seuwenetal.,1986;HansenandNichols,2009;Stachowiaketal.,2013).Theplasmamembranehasthecapacitytoformspecializedextensionswithhighcurvaturetoaccomplishsomeofitsspecificfunctions,e.g.,theformationofmicrovilliinpolarizedcellsfortheefficientuptakeofnutrients(Crawleyetal.,2014),orofmembranenanotubesfortheinter-cellularcommunication(Onfeltetal.,2004).Intheory,curvaturecanmodulatethedistributionofmembranemolecules(Bozicetal.,2006;WuandLiang,2014).Indeed,someproteinsaccumulateincurvedorfilamentousmembranesincells,butthemechanismsresponsibleforsuchdiversityareprobablybasedontargeteddeliveryofmoleculestothesespecificstructuresandpartialimpermeabilityofthebasalregionofsuchmembraneextensions,e.g.,ofcilia(TrimbleandGrinstein,2015).Inmodelmembranes,specificproteinsundergocurvature-drivensortingwhileothersdonot(Hatzakisetal.,2009;Aimonetal.,2014;Quemeneuretal.,2014).Specificintermolecularinteractionsbetweenlipidsandproteinsweresuggestedtoberesponsibleforsuchselectivity(Callan-Jonesetal.,2011),nonethelessitisstillunclearwhethercurvature-basedproteinandlipidsortingcanoccurinhighlydynamicmembranesofcells. Thecorticalactin(CA)skeleton(Figure3C)helpstokeepandmodulatetheshapeoflivingcells(Muraseetal.,2004).Inaddition,itisinvolvedintheregulationofmembranetraffickingandsignaling(forexampleSuzukietal.,2007;Jaqamanetal.,2011;Gowrishankaretal.,2012;Johnsonetal.,2012).TheimpactoftheCAontheorganizationoftheplasmamembraneiswelldescribedandformsthebasisofakeymodeldiscussedinthefollowingsection. Similarly,theglycocalyx(Figure3D),apartoftheextracellularmatrix(ECM)invertebrates,formsadensestructureatthesurfaceofeukaryoticcells(Stryer,1995).Glycosaminoglycansandassociatedglycoproteinsandproteoglycansoftheglycocalyxwereshowntomodulatesignalingbydirectassociationwithsurfacereceptors(Bassetal.,2007;Morganetal.,2007)orbybindingofligands(Hynes,2009).Inthisway,theglycocalyxandtheextracellularmatrixregulatetheshapeofmulticellularorganisms.Theglycocalyxwasalsopredictedtoinfluencethegeneralorganizationoftheplasmamembrane(Jacobsonetal.,1987).Indeed,glycosylatedextracellulardomainswereshowntomodulatetheorganization(AndersonandFambrough,1983)andmobility(WierandEdidin,1986;Zhangetal.,1991;Harteletal.,2015)ofmembraneproteins.Themolecularmechanismisstillunknownand,toourknowledge,hasnotbeenstudiedindetail. Cellskeeptheirmembranes“healthy”byarapidturnoverofitscomponents.Thisisachievedmainlybyvesiculartransport—endo-/exocytosis(Figure3E)—butalsobyalesswellunderstoodprotein-mediatedlipidtransportmechanism(s)(Lev,2012).Eachexo-/endocyticeventdeliversorremovesamaterialequivalenttoasurfaceareaof≈30.000nm2(estimatedfortheaveragediameterofexo-/endocyticvesiclestobe~100nm).Therefore,everysucheventcantransiently,butdramaticallychangethelocalmembranecompositionand,thereby,organization.Whereasnopreferredsitesofexo-/endocytosiswerereportedunderrestingconditions(Schmoranzeretal.,2000),stimulationofcellscanresultinmorelocalizedvesiculartransportandfusion/fissionhotspots(Stinchcombeetal.,2001;Gaffieldetal.,2009).Thiscanfurtheracceleratechangesintheplasmamembrane. Alternatively,membranecomponents(specificallylipids)canbedeliveredtotheplasmamembranebyspecificlipidtransporters(Raychaudhurietal.,2006;Voelker,2009;Tarlingetal.,2013).Thesemaytravelthroughthecytosolbydiffusionor,moreprobably,sucheventscantakeplaceatmembranecontactsitesbetweentheendoplasmicreticulum(ER)andplasmamembrane(seeFigure5inFernández-Busnadiegoetal.,2015).Thesesitesareresponsibleforthesynthesis,transportoflipidsbetweentheERandplasmamembrane(e.g.,byOshsteroltransportersRaychaudhurietal.,2006)andregulationoflipidmetabolismintheplasmamembrane(Stefanetal.,2011). Vesicularandprotein-mediatedtransportarethetwomainmechanismsresponsibleforarapidturnoverofmembranemolecules(estimatedtoexchangealmostallofitscomponentswithin1h),butotherroutessuchasfreediffusionofsmallmolecules(e.g.,glucose,ionsCortizoetal.,1990)orinfectionofcellsbyvirusesandotherpathogens(MazzonandMercer,2014)canfurthermodulatemembranecompositionandorganization. Activetransportofprotonsandions,togetherwithchemicalasymmetry,generatesanelectrostaticpotentialacrosstheplasmamembraneoflivingcells(HodgkinandHuxley,1952).Inadditiontothefunctionofthemembranepotentialinmetabolismandtransportofessentialmoleculesinandoutofcells,ithasanimpactonpropertiesofmodelandcellmembranes(O'Sheaetal.,1984;Grossmannetal.,2007;Hermanetal.,2015).Evenintheabsenceofions,asymmetricdistributionoflipidsinthebilayercangenerateatransmembranepotential(GurtovenkoandVattulainen,2007).Asaconsequence,itistechnicallychallengingtouncouplemembranepotentialandasymmetry.Ofnote,theavailabletheoreticalandexperimentalevidencerelatedtotheelectrostaticpotentialandtheorganizationofcellmembraneswasrecentlyreviewed(Malinskyetal.,2016). Ontheirown,extrinsicfactorsdonothavethecapabilitytocontrolallplasmamembraneprocesses.Hence,moreholistichypothesescombiningintrinsicandextrinsicfactorsareneeded.Inthefollowingsection,wewillbrieflydescribeamoregeneralconceptandfivemostpopularmodels.Areaderwillfindmoredetaileddescriptionsofthesemodelsandsomealternativeviewsinrecentlypublishedreviews(e.g.,LingwoodandSimons,2010;Owenetal.,2010;KlammtandLillemeier,2012;KlotzschandSchütz,2013;Nicolson,2014;RaoandMayor,2014;MouritsenandBagatolli,2015;SevcsikandSchütz,2016). PlasmaMembraneOrganisation–GeneralModelsandConcepts Letusbeginthissectionwithabriefinspectionofthemobilityofmembranecomponents.Thiswillindicatehowsimpleconceptshighlightingintrinsicproperties,namelyviscosityandcontinuitycan,tosomeextent,explaincertainpuzzlesrelatedtotheplasmamembraneorganizationandfunction.Measurementsoflateraldiffusionofmembranecomponentsoverthelastfewdecadesuncoveredmuchslowermolecularmobilityofmoleculesincellmembranescomparedtotheirmodelcounterparts(WierandEdidin,1986;Jacobsonetal.,1987;Lippincott-Schwartzetal.,2001).Onaverage,lipidtracers(e.g.,DiIorBODIPY-DPPE)diffuseaboutfourtimesfasterinmodelmembranesthanintheplasmamembraneoflivingcells(Box1;Table1).Thisdifferencecanbeexplainedbythecompositionalcomplexityoftheplasmamembrane.Thelargeproportionoflipidswithlongandsaturatedacylchainsandcholesterol(vanMeeretal.,2008)causeahigherrigidity(Sezginetal.,2015)and,thereby,viscosityofmembranes(Kuciketal.,1999).Inaddition,thepresenceofintegralmembraneproteinsfurtherincreasesthelocalviscosityintheirimmediateenvironment,whichreducesthemobilityofmembraneconstituentsingeneral(PetersandCherry,1982;ChazotteandHackenbrock,1988;Fricketal.,2007;Saxton,2008;Niemeläetal.,2010).Aplethoraoflipid-lipidandlipid-proteininteractions,heterogeneitiesingeneral,canfurthercontributetothisreductioninmobility. TABLE1 Table1.Examplesofdiffusioncoefficientsandtheirtranslationtothetimesneededtotraverseadistanceof20μm(e.g.,HeLacell). Therefore,intrinsicproperties,particularlyviscosity,canberesponsibleforthereducedlong-rangediffusionratesmeasuredforlipidsincellmembranes.Sincemembranesarecontinuous,allofitslipidcomponentsshouldbeinfluencedsimilarlyandequallythroughoutthewholearea.Forlipidswhichdonotcomplywiththisstatement,localizationandmobilityisregulatedbyotherfactorssuchasproteinsinteractingwithchargedlipidheadgroups,endocytosis,…etc.Thissimpleconceptworksforlipids.Buttheextremelyslowmobilityofmanyplasmamembraneproteins—one-to-twoordersofmagnitudelowercomparedtomodelmembranes–callsforamoreelaborateexplanation. FluidMosaicModel(SNModel;Figure5A).TheSNmodelinalargedetailsummarizestheunderstandingoftheplasmamembranecomposition,structureandthermodynamics45yearsago(SingerandNicolson,1972).Theemphasisisplacedonthefluidityofthemembraneandcoexistenceoflipidsandproteinsinthisessentialcellularstructures.Wehavealreadydescribedcrucialissuesofthismodelintheprevioussections.Here,wewouldliketounderlinethattheword“mosaic”intheSNmodelwasprimarilyusedtoaccentamixedcharacterofcellmembraneswherediverselipidsandproteinscoexistinasinglelamellarstructure.Later,thiswasfrequentlymisinterpretedashomogeneousorrandomdistributionofmolecules.Butheterogeneityofcellmembraneswasobservedandreportedasearlyasin1960s(Figures4A,B;CerottiniandBrunner,1967;Aokietal.,1969;Kourilskyetal.,1971;Nicolsonetal.,1971).Indeed,Nicolsondescribedputativemechanismsresponsibleforclusteringofproteins(orformationofdomains)inhispillarworkalreadyin1979(Nicolson,1979andFigure4therein).Theseassumptionsarestillvalidalmost40yearslater(Nicolson,2014). HydrodynamicModel(Figure5B).Themobilityoftransmembraneproteinsandtheiraggregatesincellmembranescanbedefinedbythehydrodynamicmodel(SaffmanandDelbrück,1975).Thismodelhypothesizesthatmoleculardiffusionratesdependmainlyonmembraneviscosityandthickness,andonlyweaklyonthesizeofproteinsandaggregates.Thismodelwaslaterupdatedmanytimes(e.g.,forarbitraryviscosityofmembranesandsolutes(Hughesetal.,1981)orasymmetricmembranesEvansandSackmann,1988),andexperimentallyconfirmedinmodelmembranes(e.g.,Ramaduraietal.,2009).However,itappliesonlyforfreelymovingmoleculesabsentofinteractionswithobjectswhichdonotco-diffuseasasingleentity.Themodelisfurtherlimitedbythedensityofobjectsinthemembraneandtheirlipidenvironment.First,thepresenceofslowlymovingobstaclesandmolecularcrowdingcanstronglyinfluencethemobilityofmembranecomponents(Saxton,2008;GuigasandWeiss,2015).Second,lipidsinthevicinityofTMDsofintegralmembraneproteins(annularlipidsorlipidshells)exhibitreducedlateraldiffusion(Meieretal.,1987;AndersonandJacobson,2002).ThisisprobablycausedbythefactthatTMDsformrelativelylargeandrigidstructureinthebilayer(Meieretal.,1987;Niemeläetal.,2010)butalsoduetotheroughsurfaceofTMDs.Therefore,thecomplexityofcellmembranesevidentlydoesnotallowtheapplicationofhydrodynamicmodeloritsvariantsasageneralmodeloftheplasmamembraneorganization.Nevertheless,itcanprovideausefulalternativetomorecomplexmodelsforlocalchanges(nanoscale;seebelow). Self-AssembliesofLipidsandOrderedLipidDomains(Figure5C).Observationofproteinclusters(DePierreandKarnovsky,1973),lipidsegregation(ShimshickandMcConnell,1973a,b;Klausneretal.,1980)andheterogeneousdistributionofcertainlipidsandproteinsbetweenapicalandbasalmembranesofpolarizedcells(vanMeerandSimons,1982)ledtothesuggestionsthatlipidsandtheirself-assembliescandeterminethefateofnewlysynthesizedorrecycledmembranemolecules(Karnovskyetal.,1982;SimonsandvanMeer,1988).ThisconceptwasmodifiedbySimonsandIkonen(SimonsandIkonen,1997)whoproposed“lipidrafts”astheplasmamembraneplatformsofhighmolecularorderenrichedincholesterolandsphingolipids,inwhichproteinsinvolvedinsignalingcanselectivelyinteractwitheffectormolecules.Inparallel,biochemicalanalysesrevealedinefficientsolubilisationofsome,butnotall,membraneproteinsandlipidsinmilddetergents,formingthebasisofdetergentresistantmembranes(DRMs).Throughouttheyears,theorderedlipidcharacterof“modellipidrafts”wasemphasizedandsuggestedtocorrespondtodomainspresentintheplasmamembraneofcells.Alltheseterms,lipidrafts,DRMsandorderedlipiddomains,wereusedinconsistentlyandfrequentlyledtomisinterpretationswhichwerehighlightedinrecentreviews(Cebecaueretal.,2009;Owenetal.,2010;Kraft,2013;SevcsikandSchütz,2016).Inaddition,thedatasupportingspontaneousformationoflipiddomainsinlivingcellsarerathercontroversialandinconclusive(e.g.,Eggelingetal.,2009;Brameshuberetal.,2010;Owenetal.,2012;Honigmannetal.,2014;Sevcsiketal.,2015).Ontheotherhand,anundisputablecapacityofcertainlipids(e.g.,gangliosides)toself-aggregate(Fujitaetal.,2007;Chenetal.,2008),anomalousdiffusionand/ordistributionoflipidsinhighlycomplexmixtures(Kusumietal.,2005;Eggelingetal.,2009;HeandMarguet,2011;Jeonetal.,2012)andspontaneousformationoffluidnanoclusters(vanZantenetal.,2010;Amaroetal.,2016)weredemonstratedinsilico,inmodelmembranesaswellasinlivingcells.Suchfluctuationscanpotentiallycontributetotheoverallheterogeneityoftheplasmamembraneandthepeculiarmobilityofcertainlipidsandproteinstherein.Yet,thedirectobservationofsuchanomalyremainschallengingduetherequiredspatialandtemporalresolutiontodisclosemolecular-scaleobjectsatsub-millisecondrates,albeitrecentadvancesinsuper-resolutionopticalmicroscopyandultrafastsingle-moleculetrackingindicateremediestothislimitation. MattressModel(Figure5D).Asmentionedabove,lipidsinthevicinityofTMDsexhibitabnormalbehavior(Lee,2004;Niemeläetal.,2010),particularlyincellmembraneswithalargevarietyoflipidspeciesandTMDs.TheaveragemembranehydrophobicthicknessincreasesbetweentheER,Golgiapparatusandplasmamembrane(Mitraetal.,2004).Duringproteintranslation,proteinswithlongTMDsareincorporatedintotherelativelythinmembraneoftheER,causinghydrophobicmismatch.LipidswithlongerandsaturatedacylchainscanformmetastableshellssurroundingsuchTMDs,therebygeneratingheterogeneityinthemembraneoftheER.Atalargerscale,hydrophobicmismatchwasproposedtoinducetheformationoflipid/proteindomainsalsointheplasmamembrane(MouritsenandBloom,1993;AndersonandJacobson,2002;Kaiseretal.,2011).Significantimpactofhydrophobicmismatchiswell-documentedforthesortingofproteinsincellmembranes(Munro,1995;Sharpeetal.,2010;Chumetal.,2016).Butwhethersimilar“sorting”oflipidsandproteinsduetohydrophobicmismatchcontributestothenanoscaleorganizationoftheplasmamembraneinlivingcellshassofarnotbeenexperimentallyproven,mainlyduetoaforementionedlimitationsonspatialandtemporalresolutionofpotentialdirectobservationmethods. CorticalActinSkeleton(Figure5E).Membrane-proximalpositioningoftheCAskeletonanditsdirectassociationwiththeplasmamembraneviaactin-bindingproteinsorcomplexesmakesitthefirst-handstructuretoinfluencethemobilityofplasmamembranemoleculesandtheirlateralorganization.Indeed,theactinskeletonwasdemonstratedtoaffectmembranemoleculesinnumerousworksemployingavarietyofexperimentalapproaches(e.g.,GolanandVeatch,1980;Sheetzetal.,1980;Tanketal.,1982;Fujiwaraetal.,2002;Ritchieetal.,2003;Muraseetal.,2004;Muelleretal.,2011;Andradeetal.,2015).TheeffectoftheCAskeletonistodatethemostacceptedmodelformembraneorganization,independentofwhetherwespeakaboutindirectstericalhindrance(picket-and-fencemodel;(Koppeletal.,1981;Jacobsonetal.,1984;SakoandKusumi,1995;Machtaetal.,2011)ordirectinteractionsofproteinswiththeCAskeleton(Saxton,1990;Sheetzetal.,2006;Muelleretal.,2011;RaoandMayor,2014).Itsundisputableimpactwasdescribedinmoredetailincurrentreviews(Kusumietal.,2010;RaoandMayor,2014).Ontheotherhand,theCAskeletonprovidesagoodexplanationformany,butprobablynotallmembrane-associatedphenomena(seebelow). FIGURE5 Figure5.Schematicillustrationsoftheplasmamembraneorganizationmodels.(A)Fluidmosaicmodel.Themembranesurfacewasartisticallydecoratedtoindicatenon-homogenousdistributionofmolecules.Coloredobjectsrepresentvariousspeciesofmembraneproteins,stringsofcoloredhexagonsillustrateglycosylationofproteinsandlipids.(B)Hydrodynamicmodel.Similarmobilityoflipidsandproteinsareindicatedbyorangeandpinktrajectories.Largeassemblies(redcircle)withsignificantlylargerradiuscanexhibitslowerdiffusion(dashedredtrajectory).(C)Lipidmembranedomains.Darkmembranepatchesindicatelipidself-assembliesanddifferentlipid(andprotein)composition.(D)Mattressmodel.DarkmembranepatchesindicateaccumulationoflipidspeciesduetoincreasedhydrophobiclengthofproteinTMDs.(E)Picket-and-fencemodel.AccumulationofproteinsaroundtheunderlyingCAskeletonandformationoffences(dashedblackline)whichmayrestrict“free”diffusionofnon-associatedproteins(red)toalimitedarea(redtrajectory).Forlong-distancemobility,proteinshaveto“hop”overthefence(yellowarrow-line)whichlimitstheirlong-rangediffusioncoefficient. ThereisNoUniversalModelofthePlasmaMembraneLateralOrganisation Modelslistedintheprevioussections,betterorworse,contributetotheoverallunderstandinghowcellspotentiallyorganizemoleculesintheirplasmamembrane.Someofthesemodelspassedthroughtheirgloriousperiods,inwhichalmostanyarticleassumedtheapplicabilityofthisoneparticularmechanismforthefunctionand/ororganizationofthestudiedmembranemolecule(s).Ahandfulofrecentexperimentalwork(e.g.,Kenworthyetal.,2004;Friszetal.,2013;Honigmannetal.,2014;Letschertetal.,2014;Sevcsiketal.,2015;Wilsonetal.,2015)andreviews(Kraft,2013;SevcsikandSchütz,2016)argueagainsttheseuniversaltheories.Improvementsintechnologyforobservingmembranestudieshavemoreandmorereducedtheaffectionforsuchasingle,universaltheory.Adynamicandcomplexplasmamembraneistheenvironmentwhereallmoleculesplayinconcerttoachievetheoptimalphysiologicaloutput. Asametaphor,onecanthinkofhumansociety.Similartocellmembranes,itishighlycomplexanddynamic,withactivitiesdifficulttoinvestigate.Asanexampleonecanconsiderclustering.“Clustering”occursinhumansocietyatthenanoscale(e.g.,families),mesoscale(e.g.,clubs,classesorothersmallinterestgroups),ormacroscale(e.g.,villages,cities,states).Theformationofsuch“clusters”dependsonintrinsicpropertieslikeaffectionoranimosity,thelocalorglobaleconomicsituation,butalsothehealthandmobilityoftheindividuals.Asananalogyforextrinsicparameterswemayconsidertheenvironmentalsituation(sunshine/rain,drought/flooding),localfactors(alpinelandscapevs.influenceofthesea),butalsotheinteractionwithother“clusters.”Asweknowfromexperience,socialsystemsmaydevelopratherstablephenotypesatthemacroscale(e.g.,thecurrentwesternsociety),whicharestillcharacterizedbyhighdynamicsatthenano—ormesoscale.Onthecontrary,thereareperiodsinhistory,inwhichnostablesituationwasreachedformanyyears.Ourpointis,thatitisvirtuallyimpossibletopredictthebehaviorofalargesocietyfromsimplemodels,eveniftheintrinsicandextrinsicparametersarewell-knownathighdetail.Or,ifwereturntothetopicofcellmembranes:currently,itseemsimpossibletoexplaintheplasmamembraneorganizationbasedonindividualmodelsdescribedintheprevioussection.Hence,futurechallengeswillincludetheclevercombinationofthisprinciplemodelsintomoreholisticmeta-modelstoincreasetheirpredictivepower.Or,intheotherwords,webelievethereisnosimple,universalmechanismunderlyingtheorganizationoftheplasmamembraneofmammaliancells. Whywebelievethisisso?Andwhataretheconsequences? Startingwiththefirstquestion,onehastolookatthesectionswiththelistsofintrinsicandextrinsicfactorsinfluencingthebehaviorofmoleculesintheplasmamembrane.Both,intrinsicandextrinsicfactorsarehighlyinterconnectedandcanoccuratthesametimeor,moreprobably,inrapid,sequentialevents.Ifintrinsicpropertiesshouldbeconsideredasrathergeneralfactors,towhichallmoleculesmustadapt,extrinsicfactorsmayhavemorespecificeffects.Tuningofintrinsicproperties(e.g.,fluidityorviscosity)requiressignificantchangesinmolecularcomposition.Thiscanrapidlyoccurlocally(atthenanoscale)andtransiently(sub-second),butwouldrequiresubstantialcostsofenergytoinducelarge-scaleandmorestablechanges.Onthecontrary,extrinsicfactors(e.g.,theCAskeletonorglycocalyx)canaffectlargersurfaceareasforlongerperiodsoftimewithhigherefficiency.Itis,therefore,probablyacombinationofthesefactorswhichregulatesbehaviorofmoleculesintheplasmamembraneatafullspectrumofspatialandtemporalscales. Thisbringsustothesecondquestionabouttheconsequencesofthenon-existenceofomnipotent,universalmodelapplicabletoallplasmamembranecomponentsandevents.First,wheninterpretingdataacquiredduringtheanalysisofcellmembranes,oneshouldnotignoreintrinsicmembraneproperties.Eventhoughlessvisible(detectable),theseformthebasisofmembraneorganizationandfunction.Extrinsicfactorsareimportantbutmaybeconsequential.Inordertofullyunderstandmembrane-associatedprocessesandavoidundesirablebordersofasingletheory,acarefulanalysisofsequentialevents,whichmayleadtotheobservedeffect,needstobeperformed(Box2). Box2.SequentialEventsInfluencingthePlasmaMembraneOrganisation Imagingtechniquesareexcellenttoolstomonitorchangesoftheplasmamembraneorganization.Highdetailscanbeexploredusingcurrentadvancedtechniques(Eggeling,2015).Butallmethodssufferfromthefactthatthepreeminentfeature(e.g.,CAskeletonreorganization)canhideoneormorelesswelldetectableevents(e.g.,changesinlocalviscosity)accompanyinganobservedprocess.Insomecases,theseundetectablefluctuationsmaybethedeterminingfactorsortriggersofatransformationprocess. Toillustratetheconsequencesoftheabovementionedlimitation(s),let'simagineaputativemembrane-associatedprocess:Aligandbindstoitsreceptorwhichisfollowedbyreceptoroligomerisationornanoscaleclustering.Suchincreasedproteindensitycausesincreasedmembraneviscositywhich,inturn,reducesmobilityofmoleculesinthevicinityofacluster(PetersandCherry,1982;Niemeläetal.,2010).Asaconsequence,anactin-bindingproteincancollidewithareceptorcluster,enhancelow-affinityinteractionsbycrosslinkingclustercomponentsandtriggerCAskeletonreorganization.Itisthelasteventwhichwillstabilizetheoverallstructureand,atthesametime,itisthebestdetectablefeatureofthisimaginaryprocess.ButtheinterpretationthattheCAskeletonisresponsiblefortheobservedchangesisonlypartofthestory.Inthiscase,theabilitytodetectsmall-scaleviscosityfluctuationswouldhelptobetterunderstandsuchprocess.Unfortunately,atpresent,suchtoolsarenotavailableforlivingcells. Anotherconcernwiththeinterpretationofmembrane-focuseddataisthesystemicuseofchemicalandgenetictoolsasaproofofoneortheothermodeloftheplasmamembraneorganization.Specificside-effectsofsomeofthesetreatments(e.g.,detergents,methyl-β-cyclodextrin,cytochalasinDortemperaturechanges)havebeendescribedinpast(AilenbergandSilverman,2003;Lichtenbergetal.,2005;Mageeetal.,2005;Shvartsmanetal.,2006;ZidovetzkiandLevitan,2007).Duetothefluidityandcooperativity,systemictreatment(both,chemicalandgenetic)willofteninfluencethebehaviorofmany(ifnotall)moleculespresentinorassociatedwiththemembrane,insteadofonlyspecificones.Inaddition,theprocedureofobservingthesystemmaypotentiallyintroduceartifacts,forexamplelabelsorintenselightsourcesemployedinfluorescencemicroscopy(Sezginetal.,2012;MagidsonandKhodjakov,2013).Therefore,employmentoftreatmentsorobservationtechniquesrequirescautiousinterpretationandexperimentsperformedwithextensivenumberofcontrols.Leavingspaceforalternativeinterpretationsandemphasisonpossibleside-effectsshouldbeagoodpracticeinthiskindofworks. Insummary,weprovidehereacomprehensivelistofmembranefeaturesandperipheralstructureswhichwerepreviouslydemonstratedorproposedtocontrollateralmobilityandorganizationoftheplasmamembraneinmammaliancells.Wealsoofferalternativeviewshowtointerpretresultsmeasuredontheplasmamembraneoflivingcells.Were-emphasizetheimpactoftheintrinsicmembranepropertieswhichwerediscoveredandcharacterizedmorethan20yearsagobutweresometimesoverlookedinmorerecentworks.Wefinishwiththehopethatdevelopmentofnovelimprovedobservationtechniquessuchasfastsingle-moleculetracking(Ritchieetal.,2005;Ortega-ArroyoandKukura,2012),TOCSSL(Brameshuberetal.,2010),STED-FCS(Eggelingetal.,2009;Muelleretal.,2013;Eggeling,2015),iMSDorrelatedimagecorrelationtechniques(Hebertetal.,2005;Digmanetal.,2009;DiRienzoetal.,2013),willberewardedwithamorepreciseinformationaboutplayersresponsiblefortheuniquenessoftheplasmamembrane.Incasetheimprovementswillbestillinsufficient,weshouldprobablyoverpassthebarrier(obstacle)betweenresearchersstudyingmammaliancellsandthosefocusedonyeastsandplants.Theseorganismsownmembraneswhichbehavemuchfriendlierontemporalscalecomparedtotheplasmamembraneofmammaliancells.Suchmembranesarehighlyheterogeneousandcanbeimagedwiththeuseofexistingmethods(Malínskáetal.,2003;Spiraetal.,2012).CellcycleregulationandRNAinterferencewerealsodiscoveredinyeastandplants. AuthorContributions JBdlS,GS,CE,andMCdefinedthetopicandwrotethemanuscript. Funding ThisworkwasfundedbyCzechScienceFoundation(15-06989S;MC),theMedicalResearchCouncil(MRC,grantnumberMC_UU_12010/unitprogrammesG0902418andMC_UU_12025;CE),MarieCurieCareerIntegrationGrant(JBdlS),andtheAustrianScienceFund(FWFprojectsP26337-B21,P25730-B21). ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WewouldliketothankMarieOlsinova,DanielaGlatzova,TonyMagee,MartinHof,LukaszCwiklik,PiotrJurkiewicz,andTomasChumforcriticaldiscussionswhichledtowritingofthisarticle. 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Editedby:ManuelJosePrieto,InstitutoSuperiorTécnico,Portugal Reviewedby:DylanMyersOwen,UniversityofNewSouthWales,AustraliaRichardM.Epand,McMasterUniversity,Canada Copyright©2016BernardinodelaSerna,Schütz,EggelingandCebecauer.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:MarekCebecauer,[email protected] COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>