Spatial Association of Signaling Proteins and F-Actin Effects on Cluster Assembly Analyzed via Photoactivation Localization Microscopy in T Cells 英文参考文献.docVIP

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Spatial Association of Signaling Proteins and F-Actin Effects on Cluster Assembly Analyzed via Photoactivation Localization Microscopy in T Cells 英文参考文献.doc

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Spatial Association of Signaling Proteins and F-Actin Effects on Cluster Assembly Analyzed via Photoactivation Localization Microscopy in T Cells 英文参考文献

SpatialAssociationofSignalingProteinsandF-Actin EffectsonClusterAssemblyAnalyzedvia PhotoactivationLocalizationMicroscopyinTCells Chih-JungHsu1,TobiasBaumgart1,2* 1DepartmentofChemistry,UniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesofAmerica,2DepartmentofChemicalandBiomolecularEngineering, UniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesofAmerica Abstract RecognitionofantigensbyTcellreceptors(TCRs)triggerscellularsignalingcascadesinitiatedbyrecruitmenttotheplasma membraneofnumerouseffectormoleculestoformsignalingmicroclusters(MCs).Hereweshowthatthemethodofhigh- resolutionphotoactivationlocalizationmicroscopy(PALM)imagingcanbeusedtoanalyzethespatialcorrelationbetween kinaseZAP70andadaptorSLP76MCsatthecellperipheryandtheeffectsofF-actinonMCassembly.Wefirstdetermined thephotophysicalrateconstantsofDronpaandtdEosfluorescenceprobes,whichallowedustooptimizeourdual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander’s colocalizationcoefficientsfromPALMimages,whichrevealedincreasingspatialsegregationofZAP70andSLP76MCsatthe cellperipheryafterinitiationofsignaling.Weshowedthatthisspatialsegregationatthecellperipheryoccurredinparallel withthereductionofMCphosphorylationlevels.Furthermore,weusedRipley’sKfunctiontoanalyzespatialrandomness, anddeterminedaverageradiiofclustersasafunctionof activationtime.The averageradiiofSLP76 andLATMCs were foundtodecrease,whereasZAP70MCradiiremainedrelativelyconstant.Finally,effectsofF-actindepolymerizationonMC morphologywerestudiedbydeterminingradialdistributionsofclustercircularity.OurdatasuggestthatMCmorphologyis affectedbyactinpolymerization.Thequantitativeanalysisofsub-diffractionPALMimagesmayprovideastartingpointfora molecularinterpretationofcluster-clusterinteractionsandoftheregulationofTcellsignalingMCsbythecytoskeleton. Citation:HsuC-J,BaumgartT(2011)SpatialAssociationofSignalingProteinsandF-ActinEffectsonClusterAssemblyAnalyzedviaPhoto