Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.
Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
Klionsky
Daniel J
DJ
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA. klionsky@umich.edu
Abeliovich
Hagai
H
Agostinis
Patrizia
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Agrawal
Devendra K
DK
Aliev
Gjumrakch
G
Askew
David S
DS
Baba
Misuzu
M
Baehrecke
Eric H
EH
Bahr
Ben A
BA
Ballabio
Andrea
A
Bamber
Bruce A
BA
Bassham
Diane C
DC
Bergamini
Ettore
E
Bi
Xiaoning
X
Biard-Piechaczyk
Martine
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Janice S
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Dale E
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Brodsky
Jeffrey L
JL
Brumell
John H
JH
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Ulf T
UT
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Wilfried
W
Camougrand
Nadine
N
Cebollero
Eduardo
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Cecconi
Francesco
F
Chen
Yingyu
Y
Chin
Lih-Shen
LS
Choi
Augustine
A
Chu
Charleen T
CT
Chung
Jongkyeong
J
Clarke
Peter G H
PG
Clark
Robert S B
RS
Clarke
Steven G
SG
Clavé
Corinne
C
Cleveland
John L
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Codogno
Patrice
P
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María I
MI
Coto-Montes
Ana
A
Cregg
James M
JM
Cuervo
Ana Maria
AM
Debnath
Jayanta
J
Demarchi
Francesca
F
Dennis
Patrick B
PB
Dennis
Phillip A
PA
Deretic
Vojo
V
Devenish
Rodney J
RJ
Di Sano
Federica
F
Dice
J Fred
JF
Difiglia
Marian
M
Dinesh-Kumar
Savithramma
S
Distelhorst
Clark W
CW
Djavaheri-Mergny
Mojgan
M
Dorsey
Frank C
FC
Dröge
Wulf
W
Dron
Michel
M
Dunn
William A
WA
Duszenko
Michael
M
Eissa
N Tony
NT
Elazar
Zvulun
Z
Esclatine
Audrey
A
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Eeva-Liisa
EL
Fésüs
László
L
Finley
Kim D
KD
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JM
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Juan
J
Fujisaki
Kozo
K
Galliot
Brigitte
B
Gao
Fen-Biao
FB
Gewirtz
David A
DA
Gibson
Spencer B
SB
Gohla
Antje
A
Goldberg
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AL
Gonzalez
Ramon
R
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Cristina
C
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Sharon
S
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RA
Häussinger
Dieter
D
He
You-Wen
YW
Heidenreich
Kim
K
Hill
Joseph A
JA
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Maria
M
Hu
Xun
X
Huang
Wei-Pang
WP
Iwasaki
Akiko
A
Jäättelä
Marja
M
Jackson
William T
WT
Jiang
Xuejun
X
Jin
Shengkan
S
Johansen
Terje
T
Jung
Jae U
JU
Kadowaki
Motoni
M
Kang
Chanhee
C
Kelekar
Ameeta
A
Kessel
David H
DH
Kiel
Jan A K W
JA
Kim
Hong Pyo
HP
Kimchi
Adi
A
Kinsella
Timothy J
TJ
Kiselyov
Kirill
K
Kitamoto
Katsuhiko
K
Knecht
Erwin
E
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Masaaki
M
Kominami
Eiki
E
Kondo
Seiji
S
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Attila L
AL
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Guido
G
Kuan
Chia-Yi
CY
Kumar
Rakesh
R
Kundu
Mondira
M
Landry
Jacques
J
Laporte
Marianne
M
Le
Weidong
W
Lei
Huan-Yao
HY
Lenardo
Michael J
MJ
Levine
Beth
B
Lieberman
Andrew
A
Lim
Kah-Leong
KL
Lin
Fu-Cheng
FC
Liou
Willisa
W
Liu
Leroy F
LF
Lopez-Berestein
Gabriel
G
López-Otín
Carlos
C
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Bo
B
Macleod
Kay F
KF
Malorni
Walter
W
Martinet
Wim
W
Matsuoka
Ken
K
Mautner
Josef
J
Meijer
Alfred J
AJ
Meléndez
Alicia
A
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Paul
P
Miotto
Giovanni
G
Mistiaen
Wilhelm P
WP
Mizushima
Noboru
N
Mograbi
Baharia
B
Monastyrska
Iryna
I
Moore
Michael N
MN
Moreira
Paula I
PI
Moriyasu
Yuji
Y
Motyl
Tomasz
T
Münz
Christian
C
Murphy
Leon O
LO
Naqvi
Naweed I
NI
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Thomas P
TP
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Ichizo
I
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Ralph A
RA
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T
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Bernd
B
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M
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Nancy L
NL
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LJ
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Bulent
B
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Shoshana
S
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Glen E
GE
Papassideri
Issidora
I
Parkes
Miles
M
Perlmutter
David H
DH
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George
G
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Mauro
M
Pinkas-Kramarski
Ronit
R
Prescott
Mark
M
Proikas-Cezanne
Tassula
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KM
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Marco
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JJ
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IM
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A
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EC
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Hans-Uwe
HU
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A
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K
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Vickram
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M
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H
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PE
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CS
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Seiichiro
S
Sulzer
David
D
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Toshihiko
T
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Michele S
MS
Tabas
Ira
I
Takeshita
Fumihiko
F
Talbot
Nicholas J
NJ
Tallóczy
Zsolt
Z
Tanaka
Keiji
K
Tanaka
Kozo
K
Tanida
Isei
I
Taylor
Graham S
GS
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J Paul
JP
Terman
Alexei
A
Tettamanti
Gianluca
G
Thompson
Craig B
CB
Thumm
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M
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Aviva M
AM
Tooze
Sharon A
SA
Truant
Ray
R
Tumanovska
Lesya V
LV
Uchiyama
Yasuo
Y
Ueno
Takashi
T
Uzcátegui
Néstor L
NL
van der Klei
Ida
I
Vaquero
Eva C
EC
Vellai
Tibor
T
Vogel
Michael W
MW
Wang
Hong-Gang
HG
Webster
Paul
P
Wiley
John W
JW
Xi
Zhijun
Z
Xiao
Gutian
G
Yahalom
Joachim
J
Yang
Jin-Ming
JM
Yap
George
G
Yin
Xiao-Ming
XM
Yoshimori
Tamotsu
T
Yu
Li
L
Yue
Zhenyu
Z
Yuzaki
Michisuke
M
Zabirnyk
Olga
O
Zheng
Xiaoxiang
X
Zhu
Xiongwei
X
Deter
Russell L
RL
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071190:Autophagy-Related Protein 8 Family; D019411:Clinical Laboratory Techniques; D003627:Data Interpretation, Statistical; D005057:Eukaryotic Cells / Q000502:physiology*; D017408:Guidelines as Topic; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D010588:Phagosomes / Q000378:metabolism / Q000502:physiology; D010944:Plants / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins