Abstract
Research in autophagy continues to accelerate,1and 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.2,3There 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 151-175 |
| Number of pages | 25 |
| Journal | Autophagy |
| Volume | 4 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 16 2008 |
Keywords
- Autolysosome
- Autophagosome
- Flux
- Lysosome
- Phagophore
- Stress
- Vacuole
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
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In: Autophagy, Vol. 4, No. 2, 16.02.2008, p. 151-175.
Research output: Contribution to journal › Review article › peer-review
}
TY - JOUR
T1 - Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
AU - Klionsky, Daniel J.
AU - Abeliovich, Hagai
AU - Agostinis, Patrizia
AU - Agrawal, Devendra K.
AU - Aliev, Gjumrakch
AU - Askew, David S.
AU - Baba, Misuzu
AU - Baehrecke, Eric H.
AU - Bahr, Ben A.
AU - Ballabio, Andrea
AU - Bamber, Bruce A.
AU - Bassham, Diane C.
AU - Bergamini, Ettore
AU - Bi, Xiaoning
AU - Biard-Piechaczyk, Martine
AU - Blum, Janice S.
AU - Bredesen, Dale E.
AU - Brodsky, Jeffrey L.
AU - Brumell, John H.
AU - Brunk, Ulf T.
AU - Bursch, Wilfried
AU - Camougrand, Nadine
AU - Cebollero, Eduardo
AU - Cecconi, Francesco
AU - Chen, Yingyu
AU - Chin, Lih Shen
AU - Choi, Augustine
AU - Chu, Charleen T.
AU - Chung, Jongkyeong
AU - Clarke, Peter G H
AU - Clark, Robert S B
AU - Clarke, Steven G.
AU - Clavé, Corinne
AU - Cleveland, John L.
AU - Codogno, Patrice
AU - Colombo, María I.
AU - Cotomontes, Ana
AU - Cregg, James M.
AU - Cuervo, Ana Maria
AU - Debnath, Jayanta
AU - Demarchi, Francesca
AU - Dennis, Patrick B.
AU - Dennis, Phillip A.
AU - Deretic, Vojo
AU - Devenish, Rodney J.
AU - Di Sano, Federica
AU - Dice, J. Fred
AU - DiFiglia, Marian
AU - Dinesh-Kumar, Savithramma
AU - Distelhorst, Clark W.
AU - Djavaheri-Mergny, Mojgan
AU - Dorsey, Frank C.
AU - Dröge, Wulf
AU - Dron, Michel
AU - Dunn, William A.
AU - Duszenko, Michael
AU - Eissa, N. Tony
AU - Elazar, Zvulun
AU - Esclatine, Audrey
AU - Eskelinen, Eeva Liisa
AU - Fésüs, László
AU - Finley, Kim D.
AU - Fuentes, José M.
AU - Fueyo, Juan
AU - Fujisaki, Kozo
AU - Galliot, Brigitte
AU - Gao, Fen Biao
AU - Gewirtz, David A.
AU - Gibson, Spencer B.
AU - Gohla, Antje
AU - Goldberg, Alfred L.
AU - Gonzalez, Ramon
AU - González-Estévez, Cristina
AU - Gorski, Sharon
AU - Gottlieb, Roberta A.
AU - Häussinger, Dieter
AU - He, You Wen
AU - Heidenreich, Kim
AU - Hill, Joseph A
AU - Høyer-Hansen, Maria
AU - Hu, Xun
AU - Huang, Wei Pang
AU - Iwasaki, Akiko
AU - Jäättelä, Marja
AU - Jackson, William T.
AU - Jiang, Xuejun
AU - Jin, Shengkan
AU - Johansen, Terje
AU - Jung, Jae U.
AU - Kadowaki, Motoni
AU - Kang, Chanhee
AU - Kelekar, Ameeta
AU - Kessel, David H.
AU - Kiel, Jan A K W
AU - Hong, Pyo Kim
AU - Kimchi, Adi
AU - Kinsella, Timothy J.
AU - Kiselyov, Kirill
AU - Kitamoto, Katsuhiko
AU - Knecht, Erwin
AU - Komatsu, Masaaki
AU - Kominami, Eiki
AU - Kondo, Seiji
AU - Kovács, Attila L.
AU - Kroemer, Guido
AU - Kuan, Chia Yi
AU - Kumar, Rakesh
AU - Kundu, Mondira
AU - Landry, Jacques
AU - Laporte, Marianne
AU - Le, Weidong
AU - Lei, Huan Yao
AU - Lenardo, Michael J.
AU - Levine, Beth
AU - Lieberman, Andrew
AU - Lim, Kah Leong
AU - Lin, Fu Cheng
AU - Liou, Willisa
AU - Liu, Leroy F.
AU - Lopez-Berestein, Gabriel
AU - López-Otín, Carlos
AU - Lu, Bo
AU - Macleod, Kay F.
AU - Malorni, Walter
AU - Martinet, Wim
AU - Matsuoka, Ken
AU - Mautner, Josef
AU - Meijer, Alfred J.
AU - Meléndez, Alicia
AU - Michels, Paul
AU - Miotto, Giovanni
AU - Mistiaen, Wilhelm P.
AU - Mizushima, Noboru
AU - Mograbi, Baharia
AU - Monastyrska, Iryna
AU - Moore, Michael N.
AU - Moreira, Paula I.
AU - Moriyasu, Yuji
AU - Motyl, Tomasz
AU - Münz, Christian
AU - Murphy, Leon O.
AU - Naqvi, Naweed I.
AU - Neufeld, Thomas P.
AU - Nishino, Ichizo
AU - Nixon, Ralph A.
AU - Noda, Takeshi
AU - Nürnberg, Bernd
AU - Ogawa, Michinaga
AU - Oleinick, Nancy L.
AU - Olsen, Laura J.
AU - Ozpolat, Bulent
AU - Paglin, Shoshana
AU - Palmer, Glen E.
AU - Papassideri, Issidora
AU - Parkes, Miles
AU - Perlmutter, David H.
AU - Perry, George
AU - Piacentini, Mauro
AU - Pinkas-Kramarski, Ronit
AU - Prescott, Mark
AU - Proikascezanne, Tassula
AU - Raben, Nina
AU - Rami, Abdelhaq
AU - Reggiori, Fulvio
AU - Rohrer, Bärbel
AU - Rubinsztein, David C.
AU - Ryan, Kevin M.
AU - Sadoshima, Junichi
AU - Sakagami, Hiroshi
AU - Sakai, Yasuyoshi
AU - Sandri, Marco
AU - Sasakawa, Chihiro
AU - Sass, Miklos
AU - Schneider, Claudio
AU - Seglen, Per O.
AU - Seleverstov, Oleksandr
AU - Settleman, Jeffrey
AU - Shacka, John J.
AU - Shapiro, Irving M.
AU - Sibirny, Andrei
AU - Silva-Zacarin, Elaine C M
AU - Simon, Hans Uwe
AU - Simone, Crisfiano
AU - Simonsen, Anne
AU - Smith, Mark A.
AU - Spanel-Borowski, Katharina
AU - Srinivas, Vickram
AU - Steeves, Meredith
AU - Stenmark, Harald
AU - Stromhaug, Per E.
AU - Subauste, Carlos S.
AU - Sugimoto, Seiichiro
AU - Sulzer, David
AU - Suzuki, Toshihiko
AU - Swanson, Michele S.
AU - Tabas, Ira
AU - Takeshita, Fumihiko
AU - Talbot, Nicholas J.
AU - Tallóczy, Zsolt
AU - Tanaka, Keiji
AU - Tanaka, Kozo
AU - Tanida, Isei
AU - Taylor, Graham S.
AU - Taylor, J. Paul
AU - Terman, Alexei
AU - Tettamanti, Gianluca
AU - Thompson, Craig B.
AU - Thumm, Michael
AU - Tolkovsky, Aviva M.
AU - Tooze, Sharon A.
AU - Truant, Ray
AU - Tumanovska, Lesya V.
AU - Uchiyama, Yasuo
AU - Ueno, Takashi
AU - Uzcátegui, Néstor L.
AU - Van Der Klei, Ida
AU - Vaquero, Eva C.
AU - Vellai, Tibor
AU - Vogel, Michael W.
AU - Wang, Hong Gang
AU - Webster, Paul
AU - Wiley, John W.
AU - Xi, Zhijun
AU - Xiao, Gutian
AU - Yahalom, Joachim
AU - Yang, Jin Ming
AU - Yap, George
AU - Yin, Xiao Ming
AU - Yoshimori, Tamotsu
AU - Yu, Li
AU - Yue, Zhenyu
AU - Yuzaki, Michisuke
AU - Zabirnyk, Olga
AU - Zheng, Xiaoxiang
AU - Zhu, Xiongwei
AU - Deter, Russell L.
N1 - Funding Information: an autophagy inhibitor or resulting from Atg gene knockdowns). oversight or any other reason, could not be included on this manu-Collectively, we strongly recommend the use of multiple assays script. This work was supported by National Institutes of Health whenever possible, rather than relying on the results from a single Public Health Service grant GM53396 to D.J.K. Due to space and method. other limitations, it is not possible to include all other sources of As a final reminder, we stated at the beginning of this review financial support. that this set of guidelines is not meant to be a formulaic set of References rules, because the appropriate assays depend in part on the question 1. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a being asked and the system being used. Rather, these guidelines are decade. Nat Rev Mol Cell Biol 2007; 8:931-7. presented primarily to emphasize key issues that need to be addressed human. Autophagy 2007; 3:181-206.2.Klionsky DJ, Cuervo AM, Seglen PO. Methods for monitoring autophagy from yeast to Bioscience. such as the difference between measuring autophagy components, 3. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004; 36:2491- and flux or substrate clearance; they are not meant to constrain imag-502. inative approaches to monitor autophagy. Hopefully, new methods tocytic protein degradation by vinblastine, leupeptin or a lysosomotropic amine. Exp Cell Kovács AL, Reith A, Seglen PO. Accumulation of autophagosomes after inhibition of hepa- for monitoring autophagy will continue to be developed, and new Res 1982; 137:191-201. findings may alter our view of the current assays. For example, one Seglen PO. Regulation of autophagic protein degradation in isolated liver cells. In: area that shows promise is the use of nanoparticles as tools for moni-Academic Press, 1987:369-414.Glaumann H, Ballard FJ, eds. Lysosomes: Their Role in Protein Breakdown. London: toring autophagy,208 as they could be used in EM (e.g., providing de Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol 1966; 28:435-92. contrast by using different sizes and shapes of nanoparticles) or tion. Trends Cell Biol 1994; 4:139-43.Dunn WA, Jr. Autophagy and related mechanisms of lysosome-mediated protein degrada- to follow autophagLic faluxn ind liveinsg c ells (e.g., relying on the stable Gordon PB, Seglen PO. Prelysosomal convergence of autophagic and endocytic pathways. fluorescence of quantum dots) allowing the tracking of autophago-Biochem Biophys Res Commun 1988; 151:40-7. somes and amphisomes. Similar to the process of autophagy, this Dunn WA, Jr. Studies on the mechanisms of autophagy: formation of the autophagic vacu- is a dynamic field, and we need to remain flexible in the standards Ishihara N, Hamasaki M, Yokota S, Suzuki K, Kamada Y, Kihara A, Yoshimori T, Noda ole. J Cell Biol 1990; 110:1923-33. we apply. T, Ohsumi Y. Autophagosome requires specific early Sec proteins for its formation and
PY - 2008/2/16
Y1 - 2008/2/16
N2 - Research in autophagy continues to accelerate,1and 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.2,3There 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.
AB - Research in autophagy continues to accelerate,1and 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.2,3There 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.
KW - Autolysosome
KW - Autophagosome
KW - Flux
KW - Lysosome
KW - Phagophore
KW - Stress
KW - Vacuole
UR - http://www.scopus.com/inward/record.url?scp=38949108670&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=38949108670&partnerID=8YFLogxK
U2 - 10.4161/auto.5338
DO - 10.4161/auto.5338
M3 - Review article
C2 - 18188003
AN - SCOPUS:38949108670
SN - 1554-8627
VL - 4
SP - 151
EP - 175
JO - Autophagy
JF - Autophagy
IS - 2
ER -