清華物理學課件-生物物理

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

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

FunctionalraftsincellmembranesNature387(1997)571Anewaspectofcellmembranestructureispresented,basedonthedynamicclusteringofsphingolipidsandcholesteroltoformraftsthatmovewithinthefluidbilayer.

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

在胞吞、脂類運輸和信號傳導過程中，質(zhì)膜的表面會出現(xiàn)一種無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羥基有兩個與高級脂肪酸形成酯，另一個與磷酸衍生物形成酯：其中R1、R2為脂肪酸碳氫鏈。根據(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