Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains Evidence for Phase Compartmentation of Natural Lipids 英文参考文献.docVIP

SegregationofFluorescentMembraneLipidsinto DistinctMicrometricDomains:EvidenceforPhase CompartmentationofNaturalLipids? LudovicD9auria1,PatrickVanDerSmissen1,Fre′de′ricBruyneel2,PierreJ.Courtoy1. ,Donatienne Tyteca1*. 1CELLUnit,deDuveInstituteandUniversite′ catholiquedeLouvain,Brussels,Belgium,2CHOMUnit,Universite′ catholiquedeLouvain,Louvain-la-Neuve,Belgium Abstract Background:Werecentlyreportedthatsphingomyelin(SM)analogssubstitutedonthealkylchainbyvariousfluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneouslyconcentratedintomicrometricdomains.Despitesharingthesamefluorescentceramidebackbone,BODIPY- SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenousSM. Methodology/Principal Findings: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or - glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol(GPI)-anchoredfluorescentproteinreporter,andlateraldiffusionbyFRAP,thusdemonstrating differentlipidphasesandboundaries.WhereasBODIPY-D-e-LacCerbehavedlikeBODIPY-GlcCer,itsartificialstereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporterandshowedmajorrestrictiontolateraldiffusionwhenphotobleachedinlargefields.Thisfunctionalcomparison supportedathree-phasemicrometriccompartmentation,ofdecreasingorder:BODIPY-GSLs.-SM.-PC(orartificialL-t- LacCer).Co-existenceofthreesegregatedcompartmentswasfurthersupportedbydoublelabellingexperimentsandwas confirmed by additive occupancy, upto ,70%cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulationofcorrespondingendogenoussphingolipidssuggestedthatdistinctf