METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N. ABELSON MELVIN I. SIMON AND Division of Biology California Institute of Technology Pasadena, California, USA Founding Editors SIDNEY P. COLOWICK NATHAN O. KAPLAN AND AcademicPressisanimprintofElsevier 525BStreet,Suite1900,SanDiego,California92101-4495,USA 30CorporateDrive,Suite400,Burlington,MA01803,USA 32JamestownRoad,LondonNW17BY,UK Copyright#2009,ElsevierInc.AllRightsReserved. Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans, electronic or mechanical, including photocopy, recording, or any information storage and retrievalsystem,withoutpermissioninwritingfromthePublisher. Theappearanceofthecodeatthebottomofthefirstpageofachapterinthisbookindicatesthe Publisher’sconsentthatcopiesofthechaptermaybemadeforpersonalorinternaluseofspecific clients.Thisconsentisgivenonthecondition,however,thatthecopierpaythestatedpercopyfee through the Copyright Clearance Center, Inc. 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You may also complete your request on-line via the Elsevier homepage (http://elsevier. com), by selecting ‘‘Support & Contact’’ then ‘‘Copyright and Permission’’ and then “Obtaining Permissions.” ForinformationonallElsevierAcademicPresspublications visit our Web site at elsevierdirect.com ISBN-13: 978-0-12-374547-7 PRINTEDINTHEUNITEDSTATESOFAMERICA 09 10 11 9 8 7 6 5 4 3 2 1 C ONTRIBUTORS ChantalBauvy INSERMU756,Universite´ Paris-Sud11,Chaˆtenay-Malabry,France EttoreBergamini CentrodiRicercaInterdipartimentalediBiologiaePatologiadell’Invecchiamento, Universita` diPisa,Pisa,Italy CherylL.Birmingham DepartmentofMolecularGeneticsandInstituteof MedicalScience,Universityof Toronto,Toronto,Ontario,Canada GeirBjørkøy Biochemistry Department, Institute of Medical Biology, University of Troms(cid:1), Norway AndreasBrech Department ofBiochemistry,Institute forCancer Research,Norwegian Radium Hospital,Oslo,Norway JohnH.Brumell DepartmentofMolecularGeneticsandInstituteofMedicalScience,Universityof Toronto, Toronto, Ontario, Canada, and Cell Biology Program, Hospital for SickChildren,Toronto,Ontario,Canada BrendaG.Byrne Department of Microbiology and Immunology, University of Michigan Medical School,AnnArbor,Michigan,USA GabriellaCavallini CentrodiRicercaInterdipartimentalediBiologiaePatologiadell’Invecchiamento, Universita` diPisa,Pisa,Italy TrinadChakraborty Institute of Medical Microbiology, Justus-Liebig University Giessen, Giessen, Germany PatriceCodogno INSERMU756,Universite´ Paris-Sud11,Chaˆtenay-Malabry,France xiii xiv Contributors Mar´ıaIsabelColombo LaboratoriodeBiolog´ıaCelularyMolecular,InstitutodeHistolog´ıayEmbriolog´ıa (IHEM),FacultaddeCienciasMe´dicas,UniversidadNacionaldeCuyo-CONICET, Mendoza,Argentina AnaCoto-Montes Departamento de Morfolog´ıa y Biolog´ıa Celular, Facultad de Medicina, UniversidaddeOviedo,Oviedo,Spain AnaMariaCuervo Department of Developmental and Molecular Biology, Marion Bessin Liver Research Center, Institute for Aging Research, Albert Einstein College of Medicine,Bronx,NewYork,USA JayantaDebnath DepartmentofPathology,UniversityofCalifornia,SanFrancisco,California,USA MonicaA.Delgado Departmentof MolecularGeneticsandMicrobiology,Universityof NewMexico, HealthSciencesCenter,Albuquerque,NewMexico,USA PatrickB.Dennis UniversityofCincinnati,GenomeResearchInstitute,DepartmentofCancerand CellBiology,Cincinnati,Ohio,USA VojoDeretic Departmentof MolecularGeneticsandMicrobiology,Universityof NewMexico, HealthSciencesCenter,Albuquerque,NewMexico,USA AlessioDonati CentrodiRicercaInterdipartimentalediBiologiaePatologiadell’Invecchiamento, Universita` diPisa,Pisa,Italy Jean-FrancoisDubuisson Department of Microbiology and Immunology, University of Michigan Medical School,AnnArbor,Michigan,USA ZvulunElazar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,Israel RashaA.Elmaoued Departmentof MolecularGeneticsandMicrobiology,Universityof NewMexico, HealthSciencesCenter,Albuquerque,NewMexico,USA Eeva-LiisaEskelinen DepartmentofBiologicalandEnvironmentalSciences,DivisionofBiochemistry, UniversityofHelsinki,Helsinki,Finland Contributors xv JunjiEzaki Department of Biochemistry, Juntendo University School of Medicine, Hongo, Tokyo,Japan NaonobuFujita Department of Cellular Regulation, Research Institute for Microbial Diseases, OsakaUniversity,Osaka,Japan MoniqueGannage´ ViralImmunobiology,InstituteofExperimentalImmunology,UniversityHospital ofZu¨rich,Zu¨rich,Switzerland DaleW.Hailey Section on Organelle Biology, Cell Biology and Metabolism Branch, NICHD, NationalInstitutesofHealth,Bethesda,Maryland,USA TorstenHain Institute of Medical Microbiology, Justus-Liebig University Giessen, Giessen, Germany SungkiHong LifeSciencesInstitute,UniversityofMichigan,AnnArbor,Michigan,USA TsuneoIkenoue LifeSciencesInstitute,UniversityofMichigan,AnnArbor,Michigan,USA KenInoki Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA, and Life Sciences Institute, UniversityofMichigan,AnnArbor,Michigan,USA TerjeJohansen Biochemistry Department, Institute of Medical Biology, University of Troms(cid:1), Norway EijaJokitalo Institute of Biotechnology, Electron Microscopy Unit, University of Helsinki, Helsinki,Finland MotoniKadowaki Department of Applied Biological Chemistry, Faculty of Agriculture, Graduate SchoolofScienceandTechnology,NiigataUniversity,Niigata,Japan Md.RazaulKarim Department of Applied Biological Chemistry, Faculty of Agriculture, Graduate SchoolofScienceandTechnology,NiigataUniversity,Niigata,Japan SusmitaKaushik Department of Developmental and Molecular Biology, Marion Bessin Liver xvi Contributors Research Center, Institute for Aging Research, Albert Einstein College of Medicine,Bronx,NewYork,USA ShunsukeKimura Department of Cellular Regulation, Research Institute for Microbial Diseases, OsakaUniversity,Osaka,Japan MasaakiKomatsu Department of Biochemistry, Juntendo University School of Medicine, Hongo, Tokyo,Japan EikiKominami Department of Biochemistry, Juntendo University School of Medicine, Hongo, Tokyo,Japan MondiraKundu Department of Pathology and Laboratory Medicine, Abramson Family Cancer ResearchInstitute,UniversityofPennsylvania,Philadelphia,Pennsylvania,USA TrondLamark Biochemistry Department, Institute of Medical Biology, University of Troms(cid:1), Norway JenniferLippincott-Schwartz Section on Organelle Biology, Cell Biology and Metabolism Branch, NICHD, NationalInstitutesofHealth,Bethesda,Maryland,USA AlfredJ.Meijer Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam,Amsterdam,TheNetherlands CarolA.Mercer UniversityofCincinnati,GenomeResearchInstitute,DepartmentofCancerand CellBiology,Cincinnati,Ohio,USA NoboruMizushima Department of Physiology and Cell Biology, Tokyo Medical and Dental University,Tokyo,Japan ChristianMu¨nz ViralImmunobiology,InstituteofExperimentalImmunology,UniversityHospital ofZu¨rich,Zu¨rich,Switzerland IchiroNakagawa DivisionofBacteriology,DepartmentofInfectiousDiseaseControl,International ResearchCenterforInfectiousDiseases,InstituteofMedicalScience,Universityof Tokyo,Tokyo,Japan Contributors xvii PaulA.Ney Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee,USA TakeshiNoda Department of Cellular Regulation, Research Institute for Microbial Diseases, OsakaUniversity,Osaka,Japan MichinagaOgawa Division of Bacterial Infection, Department of Microbiology and Immunology, InstituteofMedicalScience,UniversityofTokyo,Tokyo,Japan AndersØverbye Proteomics Section DNR, Department of Cell Biology, Institute for Cancer Research,TheNorwegianRadiumHospital,Oslo,Norway AudØvervatn Biochemistry Department, Institute of Medical Biology, University of Troms(cid:1), Norway SerhiyPankiv Biochemistry Department, Institute of Medical Biology, University of Troms(cid:1), Norway SimonG.Pfisterer Autophagy Laboratory, Department of Molecular Biology, Interfaculty Institute forCellBiology,UniversityofTu¨bingen,Tu¨bingen,Germany JamesM.Phang Metabolism and Cancer Susceptibility Section, Laboratory of Comparative Carcinogenesis,CenterforCancerResearch,NationalCancerInstitute,Frederick, Maryland,USA MarisaPonpuak Departmentof MolecularGeneticsandMicrobiology,Universityof NewMexico, HealthSciencesCenter,Albuquerque,NewMexico,USA TassulaProikas-Cezanne Autophagy Laboratory, Department of Molecular Biology, Interfaculty Institute forCellBiology,UniversityofTu¨bingen,Tu¨bingen,Germany MinooRazi LondonResearchInstitute,CancerResearchUK,London,UK FrankSætre Proteomics Section DNR, Department of Cell Biology, Institute for Cancer Research,TheNorwegianRadiumHospital,Oslo,Norway xviii Contributors ChihiroSasakawa Division of Bacterial Infection, Department of Microbiology and Immunology, InstituteofMedicalScience,UniversityofTokyo,Tokyo,Japan,andDepartment of Infectious Disease Control, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan, and CREST,JapanScienceandTechnologyAgency,Kawaguchi,Japan RuthScherz-Shouval Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,Israel PerO.Seglen Proteomics Section DNR, Department of Cell Biology, Institute for Cancer Research,TheNorwegianRadiumHospital,Oslo,Norway OleksandrSeleverstov Department of Animal Science, College of Agriculture, University of Wyoming, Laramie,Wyoming,USA ElenaShvets Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,Israel MicheleS.Swanson Department of Microbiology and Immunology, University of Michigan Medical School,AnnArbor,Michigan,USA SharonA.Tooze LondonResearchInstitute,CancerResearchUK,London,UK TakashiUeno Department of Biochemistry, Juntendo University School of Medicine, Hongo, Tokyo,Japan CristinaLourdesVa´zquez Laboratorio de Biolog´ıa Celular y Molecular, Instituto de Histolog´ıa y Embrio- log´ıa (IHEM), Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo-CONICET,Mendoza,Argentina IgnacioVega-Naredo Departamento de Morfolog´ıa y Biolog´ıa Celular, Facultad de Medicina, UniversidaddeOviedo,Oviedo,Spain HelenaVihinen Institute of Biotechnology, Electron Microscopy Unit, University of Helsinki, Helsinki,Finland Contributors xix Pa¨iviYla¨-Anttila DepartmentofBiologicalandEnvironmentalSciences,DivisionofBiochemistry, UniversityofHelsinki,Helsinki,Finland SadakiYokota Faculty of Parmaceutical Science, Nagasaki International University, Sasebo, Nagasaki,Japan YukoYoshikawa Division of Bacterial Infection, Department of Microbiology and Immunology, InstituteofMedicalScience,UniversityofTokyo,Tokyo,Japan TamotsuYoshimori Department of Cellular Regulation, Research Institute for Microbial Diseases, OsakaUniversity,Osaka,Japan OlgaZabirnyk Metabolism and Cancer Susceptibility Section, Laboratory of Comparative Carcinogenesis,Center for Cancer Research,NationalCancerInstitute,Frederick, Maryland,USA JiZhang Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, Tennessee,USA P REFACE Researchintothetopicofautophagystartedinthelate1950s.Atthattime, and for the following several decades, there were few methods available for studying this process. The initial methodology relied primarily on electron microscopy, sometimes coupled with subcellular fractionation, and electron microscopy remains one of the principal methods of analysis. Additionaltechniqueswereeventuallyadded,whichincludedsequestration andproteindegradationassays,andallofthesearedescribedinthisvolume of Methods in Enzymology. Overall, however, the methods for examining autophagy in mammalian cells have been relatively limited. In the 1990s, the autophagy-related (ATG) genes were identified in variousfungi,whichinpartopenedanewera,allowinganunderstandingof the molecular mechanism of autophagy. In addition, the identification of homologuestothefungalgenesinhighereukaryotesprovidedevidencefor theroleofautophagyinagrowingnumberofprocesses,inpathophysiology and also in development. Although there is tremendous conservation among the autophagy- relatedgenesacrossspecies,therelativedifficultyofusingmoleculargenetic approaches in mammalian cell culture and, in some cases, the need to monitor autophagy in intact organisms or tissue samples, has limited the development of techniques for assessing autophagy in mammalian systems. By far, the most versatile methodology among the molecular approaches reliesondetectionoftheAtg8homologuemicrotubule-associatedprotein1 lightchain3,orLC3.Theprimaryreasonsfortheutilityofthisproteinare that it is often upregulated following autophagy induction, it undergoes posttranslationalmodificationsthatcanbeusedtomonitorcertainaspectsof autophagy,anditispresentlytheonlyAtgproteinthatisreliablyassociated with the completed autophagosome membrane. The LC3 protein can be followed by western blot, fluorescence and immunoelectron microscopy. Accordingly,manyofthechaptersinthisvolumedescribedifferentwaysto use LC3 for following autophagy, and one of the main benefits of these chapters is that they describe specific variations that are applicable to particular systems and/or questions. DespitetheoverallrobustnessofLC3-dependentassays,theentranceof many researchers into this field has also led to the development of new techniques, aswell asmodifications ofearlier approaches.Accordingly,the chaptersinthisvolumedescribetheuseofdyesincludingDQ-BSAthatcan be used to monitor amphisome fusion with the lysosome, alternative xxi