Autophagy in Mammalian Systems, Part B

529 Pages·2009·11.037 MB·English

by  Daniel J. Klionsky (Eds.)

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
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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