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首页 细胞生物学实验:方法精要 Cell Biology Assays:Essentialmetho…

细胞生物学实验:方法精要 Cell Biology Assays:Essentialmethods.PDF

细胞生物学实验:方法精要 Cell Biology Assay…

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简介:本文档为《细胞生物学实验:方法精要 Cell Biology Assays:Essentialmethodspdf》,可适用于自然科学领域,主题内容包含CELLBIOLOGYASSAYSESSENTIALMETHODSThispageintentionallyleftblankEditedbyGER符等。

CELLBIOLOGYASSAYSESSENTIALMETHODSThispageintentionallyleftblankEditedbyGERIKREITZERWeillMedicalCollegeofCornellUniversityFANNYJAULINWeillMedicalCollegeofCornellUniversityCEDRICESPENELWeillMedicalCollegeofCornellUniversityAMSTERDAM•BOSTON•HEIDELBERG•LONDON•NEWYORK•OXFORDPARIS•SANDIEGO•SANFRANCISCO•SINGAPORE•SYDNEY•TOKYOAcademicPressisanimprintofElsevierCELLBIOLOGYASSAYSESSENTIALMETHODSAcademicPressisanimprintofElsevierJamestownRoad,LondonNWBY,UKCorporateDrive,Suite,Burlington,MA,USABStreet,Suite,SanDiego,CA,USAFirsteditionCopyrightElsevierIncAllrightsreservedNopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinanyformorbyanymeanselectronic,mechanical,photocopying,recordingorotherwisewithoutthepriorwrittenpermissionofthepublisherPermissionsmaybesoughtdirectlyfromElsevier’sScienceTechnologyRightsDepartmentinOxford,UK:phone()()fax()()email:permissionselseviercomAlternatively,visittheScienceandTechnologyBookswebsiteatwwwelsevierdirectcomrightsforfurtherinformationNoticeNoresponsibilityisassumedbythepublisherforanyinjuryandordamagetopersonsorpropertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseoroperationofanymethods,products,instructionsorideascontainedinthematerialhereinBecauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationofdiagnosesanddrugdosagesshouldbemadeMaterialinthisworkoriginallyappearedinCellBiology,ThirdEdition,editedbyJulioECelis,(Elsevier,Inc)BritishLibraryCataloguinginPublicationDataAcataloguerecordforthisbookisavailablefromtheBritishLibraryLibraryofCongressCataloginginPublicationDataAcatalogrecordforthisbookisavailablefromtheLibraryofCongressISBN:ForinformationonallAcademicPresspublicationsvisitourwebsiteatelsevierdirectcomTypesetbyMacmillanPublishingSolutionswwwmacmillansolutionscomPrintedandboundinUnitedStatesofAmericavPrefaceviiListofContributorsixIMEMBRANEPROTEINTRANSLOCATIONASSAYS:EXOCYTOSIS,ENDOCYTOSIS,ERGOLGI,TRANSLOCATIONTHROUGHTHEERPermeabilizedEpithelialCellstoStudyExocyticMembraneTransportStudyingExitandSurfaceDeliveryofPostGolgiTransportIntermediatesUsingInVitroandLiveCellMicroscopyBasedApproachesUseofPermeabilizedMastCellstoAnalyzeRegulatedExocytosisCellSurfaceBiotinylationandOtherTechniquesforDeterminationofSurfacePolarityofEpithelialMonolayersAssaysMeasuringMembraneTransportintheEndocyticPathwayMicrosomeBasedAssayforAnalysisofEndoplasmicReticulumtoGolgiTransportinMammalianCellsCotranslationalTranslocationofProteinsintoMicrosomesDerivedfromtheRoughContentsEndoplasmicReticulumofMammalianCellsIILOADINGPROTEININTOCELLSTOASSESSPMFUNCTIONSyringeLoading:AMethodForAssessingPlasmaMembraneFunctionasaReflectionofMechanicallyInducedCellLoadingIIIPROTEINTRANSLOCATIONBETWEENCYTOPLASMANDCOMPARTMENTSOTHERTHANPLASMAMEMBRANEProteinTranslocationintoMitochondriaPolarographicAssaysofMitochondrialFunctionsAnalysisofNuclearProteinImportandExportinDigitoninPermeabilizedCellsHeterokaryons:AnAssayforNucleocytoplasmicShuttlingIVMEASURINGINTRACELLULARSIGNALINGEVENTSUSINGFLUORESCENCEMICROSCOPYCaasaSecondMessenger:NewReportersforCalcium(CameleonsandCamgaroos)RatiometricPericamVINSITUCELLELECTROPORATIONTOSTUDYOFSIGNALINGPATHWAYSDissectingPathwaysInSituElectroporationfortheStudyofSignalTransductionandGapJunctionalCommunicationInSituElectroporationofRadioactiveNucleotides:AssessmentofRasActivityorPLabelingofCellularProteinsTrackingIndividualChromosomeswithIntegratedArraysoflacopSitesandGFPlaciRepressor:AnalyzingPositionandDynamicsofChromosomalLociinSaccharomycescerevisiaeVICYTOSKELETONASSAYSMicrotubuleMotilityAssaysInVitroAssaysforMitoticSpindleAssemblyandFunctionInVitroMotilityAssayswithActinUseofBrainCytosolicExtractsforStudyingActinBasedMotilityofListeriamonocytogenesPedestalFormationbyPathogenicEscherichiacoli:AModelSystemForStudyingSignalTransductiontotheActinCytoskeletonListeriamonocytogenes:TechniquestoAnalyzeBacterialInfectionInVitroMeasurementofCellularContractileForcesUsingPatternedElastomerIndexviCONTENTSviiPrefaceOneoftheprimaryaimsofbiologyistounderstandhowexpression,activationandinactivationofgenesandproteinsisorchestratedtogeneratethewidevarietyofcells,organsandtissuesthatcomprisealivingorganismUltimately,elucidationofregulatorynetworkscontrollingtheseprocesseswillprovidekeymechanisticandmolecularinsightintonormaldevelopmentandhavethepotentialtoleadtothediscoveryofcellularfactorsthatcanbetargetedinattemptstocombatnumerousdiseasesthataffectcellandtissuearchitectureandfunctionRegardlessofwheretheworkstarts,atsomepointtheneedtostudyspecificgenesandproteinswithinthecellularmilieu,becomescentraltowardachievingthisgoalThechaptersincludedinthisvolume,takenfromCellBiology:ALaboratoryHandbook,rdedition,describeanarrayofcellbiologicalassaysthatcanbeusedtodefineandcharacterizeexperimentallythemachinerythatregulateseventscriticaltothefunctionofdiversecelltypesBroadlydefined,theseencompassmethodstoanalyzetranslocationofproteinsbetweensubcellularcompartments,intracellularsignalingandcytoskeletalorganizationEachoftheseprocessesiskeytothedefinitionofcellshape,functionandresponsivenesstobothintracellularandextracellularcuesThemethodsdescribedinthesechapterscanbeseparatedintothreegeneralcategoriesThefirstgroupofassayslaysouttherationaleandtechniquestomeasureproteintransportbetweensubcellularcompartmentsortheplasmamembraneinvitro,insemiintactcellsandinintactcellsEachoftheseassayshavebeenusedtogainfundamentalinsightintothemechanismsregulatingexocytosisandendocytosis,nuclearcytoplasmicshuttlingandtranslocationofproteins(includingcotranslationaltransportintotheendoplasmicreticulum)betweenintracellularcompartmentsIncombination,thesetechniquesprovideresearcherswithafullarsenalofapproacheswithwhichtoidentify,characterizeanddeterminetheminimalessentialmachineryforproteintransportincellsThesecondgroupofassaysprovidesindepthmethodstostudyintracellularsignalingandlocalizedproteinactivitywithinsingle,livingcellsTheseassaystakeadvantageofvitalfluorescentsensorstomeasurelocalchangesinintracellularcalcium,proteinconformation,proteinphosphorylationandnucleotidetriphosphateoccupancy,allreadoutsofproteinactivityInaddition,theseassaysincorporatemethodstocontrolandanalyzesignalingeventswithtremendousspatialandtemporalresolutionincellsThethirdgroupofmethodsdetailinvitroandcellbasedassaystoevaluatetheregulationandorganizationofactinandmicrotubulenetworksinresponsetoindividualproteins,definedcytosol,signalingcascadesandenvironmentalinsultbybacteriaTheseassaysalsoprovideresearcherswiththeabilitytoidentifymoleculareffectorsofcytoskeletaldynamicsandorganizationthatplaykeyrolesinhowcellsrespondphysicallytoavarietyofphysiologicalstimuliResultsobtainedinanyonegroupsofassayscanbefruitfullyincorporatedintoexperimentsusingtheotherstogainmorecompleteviiiPREFACEinsightintoregulationindividualcellularprocessesIndeed,invitroexperimentsshowingthatmotorproteinsmovealongactinandmicrotubuleshavebeencombinedsuccessfullywithproteintransportandsignalingassaystoidentifycytoskeletalmotorsresponsiblefornumerousproteinandorganelletransporteventsincellsUsingdetailedexperimentalexamples,thechaptersinthisvolumeexamineawiderangeoftechniquesandassaysthatcanbeappliedinmostlaboratoriesWhileseveralofthesemethodsaretechnicallydemanding,mostarestraightforward,utilizeequipment,toolsandsuppliescommonlyavailableatresearchinstitutions,andcanbeadaptedtosuitspecificquestionsofindividualscientistsInthiseraofcellbiology,theabilitytoapplydiversetechnicalapproachesenablesresearcherstoaddress,atamolecularlevel,themanyquestionsassociatedwithcomplexbiologicaleventsTogether,thestepbystepinstructionswithdetaileddiscussionofeachtechniquemakethislaboratoryhandbookanessentialresourceixListofContributorsCeliaAntonioDepartmentofBiochemistryMolecularBiophysics,CollegeofPhysiciansSurgeons,ColumbiaUniversity,WSTHHSC,NewYork,NYNathalieQBalabanDepartmentofPhysics,TheHebrewUniversityGivatRam,RacahInstitute,Jerusalem,,ISRAELWilliamEBalchDepartmentofCellandMolecularBiology,TheScrippsResearchInstitute,NorthTorreyPinesRoad,LaJolla,CAStefanieBeneschDepartmentofCellBiology,GesellschaftfurBiotechnoogischeForschung,MascheroderWeg,Braunschweig,D,GERMANYHeatherLBrownellOfficeofTechnologyLicensingandIndustrySponsoredResearch,HarvardMedicalSchool,ShattuckStreet,GordonHallofMedicine,Room,Boston,MANigelPCarterTheWellcomeTrust,SangerInstitute,TheWellcomeTrust,GenomeCampus,Hinxton,Cambridge,CBSA,UNITEDKINGDOMMariaCarmoFonsecaInstituteofMolecularMedicine,FacultyofMedicine,UniversityofLisbon,AvProfEgasMoniz,Lisbon,,PORTUGALSamitChatterjeeMargaretMDysonVisionResearchInstitute,DepartmentofOphthalmology,WeillMedicalCollegeofCornellUniversity,YorkAvenue,NewYork,NYMarkSFClarkeDepartmentofHealthandHumanPerformance,UniversityofHouston,HolmanStreet,GarrisonRmD,Houston,TXPascaleCossartUnitedesInteractionsBacteriesCellulesUnitéINSERM,InstitutPasteur,,rueduDocteurRoux,ParisCedex,F,FRANCERobertACrossMolecularMotorsGroup,MarieCurieResearchInstitute,TheChart,Oxted,Surrey,RHTE,UNITEDKINGDOMAmiDeoraMargaretMDysonVisionResearchInstitute,DepartmentofOphthalmology,WeillMedicalCollegeofCornellUniversity,YorkAvenue,NewYork,NYBernhardDobbersteinZentrumfurMolekulareBiologie,UniversitatHeidelberg,ImNeuenheimerFeld,Heidelberg,D,GERMANYDanielLFeebackSpaceandLifeSciencesDirectorate,NASAJohnsonSpaceCenter,BayAreaBlvd,Houston,TXKevinLFirthASKScienceProductsInc,VictoriaSt,Kingston,Ontario,KLZ,CANADAMargaridaGamaCarvalhoFacultyofMedicine,InstituteofMolecularMedicine,UniversityofLisbon,AVProfEgasMoniz,Lisbon,,PORTUGALSusanMGasserFriedrichMiescherInstitutefrBiomedicalResearch,Maulbeerstrasse,Basel,CH,SWITZERLANDBenjaminGeigerDepartmentofMolecularCellBiology,WeizmanInstituteofScience,WolfsonBuilding,Rm,Rehovot,,ISRAELCemalGurkanDepartmentofElectronMicroscopyTheCyprusInstituteofNeurologyandGenetics,Nicosia,CyprusJeanGruenbergDepartmentofBiochemistry,UniversityofGeneva,,quaiErnestAnsermet,Geneva,CH,SWITZERLANDGeraldHammondMolecularNeuropathobiologyLaboratory,CancerReasearchUKLondonResearchInstitute,Lincoln’sInnFields,London,WCAPX,UNITEDKINGDOMRebeccaHealdMolecularandCellBiologyDepartment,UniversityofCalifornia,Berkeley,Berkeley,CALedaHelenRaptisDepartmentofMicrobiologyandImmunology,Queen’sUniversity,RoomBotterellHall,Kingston,Ontario,KLN,CANADAxLISTOFCONTRIBUTORSFlorenceHedigerDepartmentofMolecularBiology,UniversityofGeneva,,QuaiErnestAnsermet,Geneva,CH,SWITZERLANDKlausPHoeflichDivisionofMolecularandStructuralBiology,OntarioCancerInstitute,DepartmentofMedicalBiophysics,UniversityofToronto,UniversityAvenue,A,Toronto,Ontario,MGM,CANADAElinaIkonenTheLIPIDCellBiologyGroup,DepartmentofBiochemistry,TheFinnishNationalPublicHealthInstitute,Mannerheimintie,Helsinki,FIN,FINLANDMitsuhikoIkuraDivisionofMolecularandStructuralBiology,OntarioCancerInstitute,DepartmentofMedicalBiophysics,UniversityofToronto,UniversityAvenueA,Toronto,Ontario,MGM,CANADAJeffAJonesSpaceandLifeSciencesDirectorate,NASAJohnsonSpaceCenter,TXRalphHKehlenbachHygieneInstitutAbteilungVirologie,UniversitatHeidelberg,ImNeuenheimerFeld,Heidelberg,D,GERAMNYGeriEKreitzerCellandDevelopmentalBiology,WeillMedicalCollegeofCornellUniversity,LC,NewYork,NYAnnaKofferPhysiologyDepartment,UniversityCollegeLondon,UniversityStreet,London,WCEJJ,UNITEDKINGDOMFrankLafontDepartmentofBiochemistry,UniversityofGeneva,,quaiErnestAnsermet,Geneva,CH,SWITZERLANDPaulLaPointeDepartmentofCellandMolecularBiology,TheScrippsResearchInstitute,NorthTorreyPinesRoad,LaJolla,CAAndreLeBivicGroupeMorphogeneseetCompartimentationMembranaire,UMR,IBDM,FacultedesSciencesdeLuminy,case,Marseillecedex,F,FRANCEChuanPULeeTheDepartmentofBiochemistryandMolecularBiology,WayneStateUniversitySchoolofMedicine,ScottHall,ECanfield,Detroit,MISilviaLommelDepartmentofCellBiology,GermanResearchCenterforBiotechnology(GBF),MascheroderWeg,Braunschweig,D,GERMANYPattiLynnPetersonDepartmentofNeurology,WayneStateUniversitySchoolofMedicine,LDetroitReceivingHospital,DetroitMedicalCenter,Detroit,MIAnneMueschDevelopmentalandMolecularBiology,AlbertEinsteinCollegeofMedicine,Bronx,NYAlanDMarmorsteinColeEyeInstitute,WeillMedicalCollegeofCornellClevelandClinic,EuclidAvenue,i,Cleveland,OHBrunoMartoglioInstituteofBiochemistry,ETHZentrum,BuildingCHN,RoomL,Zurich,CH,SWITZERLANDAtsushiMiyawakiLaboratoryforCellFunctionandDynamics,AdvancedTechnologyCenter,BrainScienceInstitute,InstituteofPhysicalandChemicalResearch(RIKEN),Horosawa,Wako,Saitama,,JAPANFrankRNeumannDepartmentofMolecularBiology,UniversityofGeneva,,QuaiErnestAnsermet,Geneva,CH,SWITZERLANDHendrikOttoInstitutfürBiochemieundMolekularbiologie,UniversitätFreiburg,HermannHerderStr,Freiburg,D,GERMANYBryceMPaschalCenterforCellSignaling,UniversityofVirginia,JeffersonParkAvenue,WestComplexRoom,Charlottesville,VAJavierPizarroCerdáUnitedesInteractionsBacteriesCellulesUnitéINSERM,InstitutPasteur,,rueduDocteurRoux,ParisCedex,F,FRANCEHelenPlutnerDepartmentofCellandMolecularBiology,TheScrippsResearchInstitute,NorthTorreyPinesRoad,LaJolla,CALindaJRobinsonEnriqueRodriguezBoulanMargaretMDysonVisionResearchInstitute,DepartmentofOpthalmology,WeillMedicalCollegeofCornellUniversity,NewYork,NYManfredRohdeDepartmentofMicrobialPathogenicity,GesellschaftfurBiotechnoogischeForschung,MascheroderWeg,Braunschweig,D,GERMANYSabineRospertInstitutfürBiochemieundMolekularbiologie,UniversitätFreiburg,HermannHerderStr,Freiburg,D,GERMANYUlrichSSchwarzTheoryDivision,MaxPlanckInstituteofColloidsandInterfaces,Potsdam,,GERMANYLISTOFCONTRIBUTORSxiAntonioSSechiInstituteforBiomedicalTechnologyCellBiology,UniversitaetsklinikumAachen,RWTH,Pauwelsstrasse,Aachen,D,GERMANYJamesRSellersCellularandMotilitySection,LaboratoryofMolecularCardiology,NationalHeart,LungandBloodInstitute(NHLBI),NationalInstitutesofHealth,CenterDrive,MSC,Bethesda,MDKaiSimonsMaxPlanckInstituteofMolecularCellBiologyandGenetics,Pfotenhauerstrasse,Dresden,D,GERMANYEviTomaiDepartmentofMicrobiologyandImmunology,Queen’sUniversity,RoomBotterellHall,Kingston,Ontario,KLN,CANADAAngelaTaddeiDepartmentofMolecularBiology,UniversityofGeneva,,QuaiErnestAnsermet,Geneva,CH,SWITZERLANDKevinTruongDivisionofMolecularandStructuralBiology,OntarioCancerInstitute,DepartmentofMedicalBiophysics,UniversityofToronto,UniversityAvenue,A,Toronto,Ontario,MGM,CANADAIsabelleVernosCellBiologyandCellBiophysicsProgramme,EuropeanMolecularBiologyLaboratory,Meyerhofstrasse,Heidelberg,D,GERMANYAdinaVulturDepartmentofMicrobiologyandImmunology,Queen’sUniversity,RoomBotterellHall,Kingston,Ontario,KLN,CANADAXiaodongWangSouthwesternMedicalCenter,DepartmentofBiochemistry,Dallas,TXJürgenWehlandDepartmentofCellBiology,GesellschaftfurBiotechnoogischeForschung,MascheroderWeg,Braunschweig,D,GERMANYYeXiongTheDepartmentofBiochemistryandMolecularBiology,WayneStateUniversitySchoolofMedicine,ScottHall,ECanfield,Detroit,MICharlesYeamanDepartmentofCellandDevelopmentalBiology,WeillMedicalCollegeofCornellUniversity,NewYork,NYChiaraZurzoloDepartmentofCellBiologyandInfection,PasteurInstitute,,rueduDocteurRoux,Paris,,FRANCEThispageintentionallyleftblankMEMBRANEPROTEINTRANSLOCATIONASSAYS:EXOCYTOSIS,ENDOCYTOSIS,ERGOLGI,TRANSLOCATIONTHROUGHTHEERSECTIONIThispageintentionallyleftblankCopyright,ElsevierScience(USA)CellBiologyAssays–EssentialMethodsAllrightsreservedPermeabilizedEpithelialCellstoStudyExocyticMembraneTransportFrankLafont,ElinaIkonen,andKaiSimonsCHAPTERIINTRODUCTIONPolarizedexocytictransportinepithelialcellscanbemeasuredbyperforminginvitroassaysonfiltergrownMadin–Darbycaninekidneycells(MDCKstrainIIcells)Techniqueshavebeendevelopedtoestablishconditionswherecytosoldependenttransferofaviralmarkerproteinis

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