清華物理學(xué)課件-生物物理

俺怎么越看越象乒乒乓乓，不要扔西紅柿～～～-_-b俺們的進(jìn)山全家福，hohoCOOHNH24.細(xì)胞膜中的脂筏及膜穴系統(tǒng)

很早以前，人們就發(fā)現(xiàn)許多真核生物的細(xì)胞中都可以分離得到抗去垢劑的膜微疇結(jié)構(gòu)，英文簡(jiǎn)稱為DRMs（detergent-resistantmembranedomains），但直到近年來(lái)DRMs才引起人們的廣泛關(guān)注。這是因?yàn)镈RMs在細(xì)胞內(nèi)的分選和細(xì)胞表面信號(hào)傳導(dǎo)過(guò)程中都表現(xiàn)出其特有的重要性。這些在4℃去垢劑不溶的膜區(qū)域被認(rèn)為是由鞘脂類和膽固醇的動(dòng)態(tài)聚集而形成，它們組成了相對(duì)穩(wěn)定的具有一定功能的疇結(jié)構(gòu)漂浮于二維流動(dòng)的細(xì)胞膜中，人們形象地稱之為“脂筏”（Lipidrafts）。

FunctionalraftsincellmembranesNature387(1997)571Anewaspectofcellmembranestructureispresented,basedonthedynamicclusteringofsphingolipidsandcholesteroltoformraftsthatmovewithinthefluidbilayer.

------Simons&Ikonen,Nature387(1997)569-572.

在胞吞、脂類運(yùn)輸和信號(hào)傳導(dǎo)過(guò)程中，質(zhì)膜的表面會(huì)出現(xiàn)一種無(wú)籠形蛋白覆蓋的穴樣凹陷，這些穴樣凹陷呈現(xiàn)4℃去垢劑不溶性，人們把這種DRMs稱作“膜穴”（caveolae）。目前發(fā)現(xiàn)膜穴與功能筏在分子水平上有著類似的組成和結(jié)構(gòu)，因此可以說(shuō)膜穴是功能筏的一種特殊表現(xiàn)形式。

Caveolae:lipidraftsincellsurfaceinvaginationscontainingcaveolinNature387(1997)571CaveolaeinendocytictrafficCaveolaearenon-clathrincoatedinvaginations(50-100nm)intheplasmamembraneinmanycelltypes.Caveolaeareformedbyself-associatingcaveolinmolecules(makingahairpinloop)inthemembraneinteractingwithraftlipids.Toformsmallsignallingcompartments:anumberofsignallingproteinsareanchored,suchashetrotrimericGproteins,Src-familykinases,H-Ras.Beinvolvedinendocytosisandtranscytosis.LipidcompositionofraftsSphingolipid(glycosphingolipids,sphingomyelin)andcholsterol

sphingomyelin-andcholsterol-richDIGscanalsobeisolatedfromcells.glycosphingolipidsarenotabsolutelyrequired!DIGsintrans-Golgi-network:richincholesterolandanchoredbyGPIprotein!以甘油為骨架的磷脂即甘油分子中三個(gè)羥基有兩個(gè)與高級(jí)脂肪酸形成酯，另一個(gè)與磷酸衍生物形成酯：其中R1、R2為脂肪酸碳?xì)滏湣８鶕?jù)X的成分不同，可以形成不同的磷脂。以神經(jīng)鞘氨醇為骨架的鞘脂類(sphingolipid)神經(jīng)鞘氨醇(sphinogsine)的C－2上的氨基(-NH2)與脂肪酸(R)縮合生成神經(jīng)鞘脂類(sphingolipid),C－l上的羥基與磷酸衍生物(X)縮合即生成磷酸神經(jīng)鞘脂類(phosphasphingolipid):若:X＝磷脂膽堿(PC)，則生成神經(jīng)鞘磷脂(sphingomyelin,SM).

若:X＝－H,則生成神經(jīng)酸胺（ceramide）。Sphingolipids:longandsaturatedfattyacylchianshigherTm:Glycosphingolipid60-70oC.Sphingomyelin37-41

oC.Detergent-insolubleglycolipid-enrichedcomplexes(DIGs)arisefromlipid-lipidinteractionsdetergentinsolubilitywasobservedevenintheabsenceofprotein.saturated-chain,high-TmDPPCisTritoninsolubleinDIGs-containingliposomes,whilethelowTm,unsaturated-chainDOPCismuchTritonsoluble.correlationofacylchainstructure,Tm,anddetergentinsolubilitynotfromlipidhead-groupinteractions.Phaseseparationexistsinmodelmembranesco-existinggelandfluidphasesphaseseparationoftwoliquidphasesliquid-cryst.andliquid-orderedphasesproteininducesmicrodomainformationDoesphaseseparationoccurinbiologicalmembrane?ProteinsinDIGsGPI-anchoredproteins----thefirstproteinstobeidentifiedinDIGs.Byacyltailstheproteinsbindtothecytoplasmicleaflet----suchastheSrc-familykinases.Proteinsassociatingthroughtheirtransmembranedomains----suchasinfluenzavirushaemagglutinin(HA).ThefunctionoflipidraftsMembranesortingandtraffickingSignaltransductionTheintracellulartransportofsphingolipid-cholesterolraftsshowsaapicalroute.

ApicalsortingsignalGPIanchorsspecificmembrane-spanningregionsN-glycansbasolateralsortingsignals

tyrosineordileucinecontainingmotifsofthecytoplasmicdomainsofbasolaterallytargetedproteinsPNAS95(1998)6460PNAS95(1998)3966FunctionalraftsinneuralpolarityEMBOJ.16(1997)4932EMBOJ.15(1996)5218SignallingoccursinaraftFollowingdimerization(oroligomerization)theproteinesphosphorylated(bluecircle)inrafts.Signallingoccursbyalteringproteinpartitioninaraft

Followingdimerization(oroligomerization)theproteinesphosphorylated(bluecircle)inrafts.ClusteringofraftstriggerssignallingThereareseveralraftsinthemembrane,whichdifferinproteincomposition.Clusteringwouldcoalescerafts(red),sothattheywouldnowcontainanewmixtureofmolecules,suchascrosslinkersandenzymes.Clusteringcouldoccureitherextracellularly,withinthemembrane,orinthecytosol(a–c).RaftclusteringcouldalsooccurthroughGPIanchoredproteins.Rafts的確定方法TechniquestoidentifyraftsTheExistenceofraftsincellmembranes

BiochemicalcrosslinkingofGPI-anchoredproteinswhentheyareinproximityinrafts.VisualizationofraftsandclusteredraftsinIgEsignallingbyelectronmicroscopyAntibodycrosslinkingofraftproteinsintopatchessegregatingfromnon-raft.

----Thefirstdemonstrationthatclustersofraftssegregateawayfromnon-raftproteins.

Bulkseparationofmembranephasescausedbyclusteringofmembranecomponents.(A)Microdomainswithmembraneproteinsinthesedomainsaredispersedintheplasmamembrane.(B)Cross-linkinggenerateslargeandstabilizedmembranedomainsthatcoalescetoformpatches.Iftwomembranecomponentsshareapreferenceforalipidenvironmentsuchasraftmicrodomainsthemarkerswillcopatchintotightlyassociateddomains.Iftwomarkerspartitionintodifferentmembraneenvironmentssuchasraftandnon-raftmarkersthepatcheswillbeseparated.TheExistenceofraftsincellmembranes

BiochemicalcrosslinkingofGPI-anchoredproteinswhentheyareinproximityinrafts.

VisualizationofraftsandclusteredraftsinIgEsignallingbyelectronmicroscopyClearvisualizationofraftclusteringbyimmuno-electronmicroscopy

LynassociateswithFceRIinrestingmastcells.MembranesheetswerepreparedfromuntreatedRBL-2H3cellsandlabeledfromtheinsidewith5-nmgoldparticlesspecificforLynandwitheither3-(A)or10-nm(B)goldparticlesspecificforFceRIb.Inbothmicrographs,asubstantialportionof5-nmgoldparticlesmarkingLynarecolocalizedwithFceRIb(circles).(C)Demonstratestheabsenceofbackgroundbindingwhenbothsizesofgoldparticlesareincubatedwithmembranesheetsintheabsenceofspecificantibodies.TheExistenceofraftsincellmembranes

(Raftsinlivingcells)

Fluorescenceresonanceenergytransfermeasurementsusingfluorescentfolatetoshowinteractionsoffolatereceptorswhentheyareinproximityinraftsinlivingcells.

Photonicforcemicroscopymeasurements

ofthesizeofraftsinlivingcells.CFP:EX436;EM476.YFP:EX516;EM529TheExistenceofraftsincellmembranes

(Raftsinlivingcells)

Fluorescenceresonanceenergytransfermeasurements

usingfluorescentfolatetoshowinteractionsoffolatereceptorswhentheyareinproximityinraftsinlivingcells.

Photonicforcemicroscopymeasurementsofthesizeofraftsinlivingcells.Figure1.Scaledmodeloftheexperimentalsituation:asphere(r5108nm)boundviaanadsorbedantibodytoaGPI-anchoredproteinthatispartofaraftdomain.Thelipidbilayerissymbolizedbythedoublerowofgraydotswithblacksectionssymbolizingraftdomains.Theextentofthethermalpositionfluctuationsobservedintheexperiments(660nm)ismarked.Itismuchsmallerthanthesmallestestimatesofthespacingofimmobilecytoskeleton-anchoredobstaclestofreediffusionof300–500nm(SakoandKusumi,1995).The