A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis 英文参考文献.docVIP

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A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis 英文参考文献.doc

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A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis 英文参考文献

AHighPrecisionSurveyoftheMolecularDynamicsof MammalianClathrin-MediatedEndocytosis MarcusJ.Taylor1,DavidPerrais2,3,ChristienJ.Merrifield1* 1Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, 2Universite′ de Bordeaux, Interdisciplinary Institute for Neuroscience,UMR 5297,Bordeaux,France,3CNRS,InterdisciplinaryInstituteforNeuroscience,UMR5297,Bordeaux,France Abstract Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein–tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ,2s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappearfollowingscission.Wemeasuredtherecruitmentdynamicsof34typesofendocyticproteintoscissionevents: Abp1,ACK1,amphiphysin1,APPL1,Arp3,BIN1,CALM,CIP4,clathrinlightchain(Clc),cofilin,coronin1B,cortactin,dynamin1/ 2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2b1, and syndapin2. For each protein we aligned ,1,000 recruitment profiles to their respective scission events and constructed characteristic ‘‘recruitment signatures’’ that were grouped,asforyeast,torevealthemodularorganizationofmammalianCME.Adetailedanalysisrevealedtheunanticipated recruitmentdynamicsofSNX9,FBP17,andCIP4andshowedthatthesamesetofproteinswasrecruited,inthesameorder, toscissioneventsatCCSsofdifferentsizesandlifetimes.Collectivelythesedatarevealthefine-grainedtemporalstructure ofCMEandsugges