Guidelines for the use and interpretation of assays for monitoring autophagy

Daniel J. Klionsky*, Fabio C. Abdalla, Hagai Abeliovich, Robert T. Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Agnello, Patrizia Agostinis, Julio A. Aguirre-Ghiso, Hyung Jun Ahn, Ouardia Ait-Mohamed, Slimane Ait-Si-Ali, Takahiko Akematsu, Shizuo Akira, Hesham M. Al-Younes, Munir A. Al-Zeer, Matthew L. Albert, Roger L. Albin, Javier Alegre-AbarrateguiMaria Francesca Aleo, Mehrdad Alirezaei, Alexandru Almasan, Maylin Almonte-Becerril, Atsuo Amano, Ravi Amaravadi, Shoba Amarnath, Amal O. Amer, Nathalie Andrieu-Abadie, Vellareddy Anantharam, David K. Ann, Shailendra Anoopkumar-Dukie, Hiroshi Aoki, Nadezda Apostolova, Giuseppe Arancia, John P. Aris, Katsuhiko Asanuma, Nana Y.O. Asare, Hisashi Ashida, Valerie Askanas, David S. Askew, Patrick Auberger, Misuzu Baba, Steven K. Backues, Eric H. Baehrecke, Ben A. Bahr, Xue Yuan Bai, Yannick Bailly, Robert Baiocchi, Giulia Baldini, Walter Balduini, Andrea Ballabio, Bruce A. Bamber, Edward T.W. Bampton, Gábor Bánhegyi, Clinton R. Bartholomew, Diane C. Bassham, Robert C. Bast, Henri Batoko, Boon Huat Bay, Isabelle Beau, Daniel M. Béchet, Thomas J. Begley, Christian Behl, Christian Behrends, Soumeya Bekri, Bryan Bellaire, Linda J. Bendall, Luca Benetti, Laura Berliocchi, Henri Bernardi, Francesca Bernassola, Sébastien Besteiro, Ingrid Bhatia-Kissova, Xiaoning Bi, Martine Biard-Piechaczyk, Janice S. Blum, Lawrence H. Boise, Paolo Bonaldo, David L. Boone, Beat C. Bornhauser, Karina R. Bortoluci, Ioannis Bossis, Frédéric Bost, Jean Pierre Bourquin, Patricia Boya, Michaël Boyer-Guittaut, Peter V. Bozhkov, Nathan R. Brady, Claudio Brancolini, Andreas Brech, Jay E. Brenman, Ana Brennand, Emery H. Bresnick, Patrick Brest, Dave Bridges, Molly L. Bristol, Paul S. Brookes, Eric J. Brown, John H. Brumell, Nicola Brunetti-Pierri, Ulf T. Brunk, Dennis E. Bulman, Scott J. Bultman, Geert Bultynck, Lena F. Burbulla, Wilfried Bursch, Jonathan P. Butchar, Wanda Buzgariu, Sergio P. Bydlowski, Ken Cadwell, Monika Cahová, Dongsheng Cai, Jiyang Cai, Qian Cai, Bruno Calabretta, Javier Calvo-Garrido, Nadine Camougrand, Michelangelo Campanella, Jenny Campos-Salinas, Eleonora Candi, Lizhi Cao, Allan B. Caplan, Simon R. Carding, Sandra M. Cardoso, Jennifer S. Carew, Cathleen R. Carlin, Virginie Carmignac, Leticia A.M. Carneiro, Serena Carra, Rosario A. Caruso, Giorgio Casari, Caty Casas, Roberta Castino, Eduardo Cebollero, Francesco Cecconi, Jean Celli, Hassan Chaachouay, Han Jung Chae, Chee Yin Chai, David C. Chan, Edmond Y. Chan, Raymond Chuen Chung Chang, Chi Ming Che, Ching Chow Chen, Guang Chao Chen, Guo Qiang Chen, Min Chen, Quan Chen, Steve S.L. Chen, Wen Li Chen, Xi Chen, Xiangmei Chen, Xiequn Chen, Ye Guang Chen, Yingyu Chen, Yongqiang Chen, Yu Jen Chen, Zhixiang Chen, Alan Cheng, Christopher H.K. Cheng, Yan Cheng, Heesun Cheong, Jae Ho Cheong, Sara Cherry, Russ Chess-Williams, Zelda H. Cheung, Eric Chevet, Hui Ling Chiang, Roberto Chiarelli, Tomoki Chiba, Lih Shen Chin, Shih Hwa Chiou, Francis V. Chisari, Chi Hin Cho, Dong Hyung Cho, Augustine M.K. Choi, Doo Seok Choi, Kyeong Sook Choi, Mary E. Choi, Salem Chouaib, Divaker Choubey, Vinay Choubey, Charleen T. Chu, Tsung Hsien Chuang, Sheau Huei Chueh, Taehoon Chun, Yong Joon Chwae, Mee Len Chye, Roberto Ciarcia, Maria R. Ciriolo, Michael J. Clague, Robert S.B. Clark, Peter G.H. Clarke, Robert Clarke, Patrice Codogno, Hilary A. Coller, María I. Colombo, Sergio Comincini, Maria Condello, Fabrizio Condorelli, Mark R. Cookson, Graham H. Coombs, Isabelle Coppens, Ramon Corbalan, Pascale Cossart, Paola Costelli, Safia Costes, Ana Coto-Montes, Eduardo Couve, Fraser P. Coxon, James M. Cregg, José L. Crespo, Marianne J. Cronjé, Ana Maria Cuervo, Joseph J. Cullen, Mark J. Czaja, Marcello D'Amelio, Arlette Darfeuille-Michaud, Lester M. Davids, Faith E. Davies, Massimo De Felici, John F. De Groot, Cornelis A.M. De Haan, Luisa De Martino, Angelo De Milito, Vincenzo De Tata, Jayanta Debnath, Alexei Degterev, Benjamin Dehay, Lea M.D. Delbridge, Francesca Demarchi, Yi Zhen Deng, Jörn Dengjel, Paul Dent, Donna Denton, Vojo Deretic, Shyamal D. Desai, Rodney J. Devenish, Mario Di Gioacchino, Gilbert Di Paolo, Chiara Di Pietro, Guillermo Díaz-Araya, Inés Díaz-Laviada, Maria T. Diaz-Meco, Javier Diaz-Nido, Ivan Dikic, Savithramma P. Dinesh-Kumar, Wen Xing Ding, Clark W. Distelhorst, Abhinav Diwan, Mojgan Djavaheri-Mergny, Svetlana Dokudovskaya, Zheng Dong, Frank C. Dorsey, Victor Dosenko, James J. Dowling, Stephen Doxsey, Marlène Dreux, Mark E. Drew, Qiuhong Duan, Michel A. Duchosal, Karen Duff, Isabelle Dugail, Madeleine Durbeej, Michael Duszenko, Charles L. Edelstein, Aimee L. Edinger, Gustavo Egea, Ludwig Eichinger, N. Tony Eissa, Suhendan Ekmekcioglu, Wafik S. El-Deiry, Zvulun Elazar, Mohamed Elgendy, Lisa M. Ellerby, Kai Er Eng, Anna Mart Engelbrecht, Simone Engelender, Jekaterina Erenpreisa, Ricardo Escalante, Audrey Esclatine, Eeva Liisa Eskelinen, Lucile Espert, Virginia Espina, Huizhou Fan, Jia Fan, Qi Wen Fan, Zhen Fan, Shengyun Fang, Yongqi Fang, Manolis Fanto, Alessandro Fanzani, Thomas Farkas, Jean Claude Farré, Mathias Faure, Marcus Fechheimer, Carl G. Feng, Jian Feng, Qili Feng, Youji Feng, László Fésüs, Ralph Feuer, Maria E. Figueiredo-Pereira, Gian Maria Fimia, Diane C. Fingar, Steven Finkbeiner, Toren Finkel, Kim D. Finley, Filomena Fiorito, Edward A. Fisher, Paul B. Fisher, Marc Flajolet, Maria L. Florez-McClure, Salvatore Florio, Edward A. Fon, Francesco Fornai, Franco Fortunato, Rati Fotedar, Daniel H. Fowler, Howard S. Fox, Rodrigo Franco, Lisa B. Frankel, Marc Fransen, José M. Fuentes, Juan Fueyo, Jun Fujii, Kozo Fujisaki, Eriko Fujita, Mitsunori Fukuda, Ruth H. Furukawa, Matthias Gaestel, Philippe Gailly, Malgorzata Gajewska, Brigitte Galliot, Vincent Galy, Subramaniam Ganesh, Barry Ganetzky, Ian G. Ganley, Fen Biao Gao, George F. Gao, Jinming Gao, Lorena Garcia, Guillermo Garcia-Manero, Mikel Garcia-Marcos, Marjan Garmyn, Andrei L. Gartel, Evelina Gatti, Mathias Gautel, Thomas R. Gawriluk, Matthew E. Gegg, Jiefei Geng, Marc Germain, Jason E. Gestwicki, David A. Gewirtz, Saeid Ghavami, Pradipta Ghosh, Anna M. Giammarioli, Alexandra N. Giatromanolaki, Spencer B. Gibson, Robert W. Gilkerson, Michael L. Ginger, Henry N. Ginsberg, Jakub Golab, Michael S. Goligorsky, Pierre Golstein, Candelaria Gomez-Manzano, Ebru Goncu, Céline Gongora, Claudio D. Gonzalez, Ramon Gonzalez, Cristina González-Estévez, Rosa Ana González-Polo, Elena Gonzalez-Rey, Nikolai V. Gorbunov, Sharon Gorski, Sandro Goruppi, Roberta A. Gottlieb, Devrim Gozuacik, Giovanna Elvira Granato, Gary D. Grant, Kim N. Green, Aleš Gregorc, Frédéric Gros, Charles Grose, Thomas W. Grunt, Philippe Gual, Jun Lin Guan, Kun Liang Guan, Sylvie M. Guichard, Anna S. Gukovskaya, Ilya Gukovsky, Jan Gunst, Asa B. Gustafsson, Andrew J. Halayko, Amber N. Hale, Sandra K. Halonen, Maho Hamasaki, Feng Han, Ting Han, Michael K. Hancock, Malene Hansen, Hisashi Harada, Masaru Harada, Stefan E. Hardt, J. Wade Harper, Adrian L. Harris, James Harris, Steven D. Harris, Makoto Hashimoto, Jeffrey A. Haspel, Shin Ichiro Hayashi, Lori A. Hazelhurst, Congcong He, You Wen He, Marie Josée Hébert, Kim A. Heidenreich, Miep H. Helfrich, Gudmundur V. Helgason, Elizabeth P. Henske, Brian Herman, Paul K. Herman, Claudio Hetz, Sabine Hilfiker, Joseph A. Hill, Lynne J. Hocking, Paul Hofman, Thomas G. Hofmann, Jörg Höhfeld, Tessa L. Holyoake, Ming Huang Hong, David A. Hood, Gökhan S. Hotamisligil, Ewout J. Houwerzijl, Maria Høyer-Hansen, Bingren Hu, Chien An A. Hu, Hong Ming Hu, Ya Hua, Canhua Huang, Ju Huang, Shengbing Huang, Wei Pang Huang, Tobias B. Huber, Won Ki Huh, Tai Ho Hung, Ted R. Hupp, Gang Min Hur, James B. Hurley, Sabah N.A. Hussain, Patrick J. Hussey, Jung Jin Hwang, Seungmin Hwang, Atsuhiro Ichihara, Shirin Ilkhanizadeh, Ken Inoki, Takeshi Into, Valentina Iovane, Juan L. Iovanna, Nancy Y. Ip, Yoshitaka Isaka, Hiroyuki Ishida, Ciro Isidoro, Ken Ichi Isobe, Akiko Iwasaki, Marta Izquierdo, Yotaro Izumi, Panu M. Jaakkola, Marja Jäättelä, George R. Jackson, William T. Jackson, Bassam Janji, Marina Jendrach, Ju Hong Jeon, Eui Bae Jeung, Hong Jiang, Hongchi Jiang, Jean X. Jiang, Ming Jiang, Qing Jiang, Xuejun Jiang, Alberto Jiménez, Meiyan Jin, Shengkan Jin, Cheol O. Joe, Terje Johansen, Daniel E. Johnson, Gail V.W. Johnson, Nicola L. Jones, Bertrand Joseph, Suresh K. Joseph, Annie M. Joubert, Gábor Juhász, Lucienne Juillerat-Jeanneret, Chang Hwa Jung, Yong Keun Jung, Kai Kaarniranta, Allen Kaasik, Tomohiro Kabuta, Motoni Kadowaki, Katarina Kagedal, Yoshiaki Kamada, Vitaliy O. Kaminskyy, Harm H. Kampinga, Hiromitsu Kanamori, Chanhee Kang, Khong Bee Kang, Kwang Il Kang, Rui Kang, Yoon A. Kang, Tomotake Kanki, Thirumala Devi Kanneganti, Haruo Kanno, Anumantha G. Kanthasamy, Arthi Kanthasamy, Vassiliki Karantza, Gur P. Kaushal, Susmita Kaushik, Yoshinori Kawazoe, Po Yuan Ke, John H. Kehrl, Ameeta Kelekar, Claus Kerkhoff, David H. Kessel, Hany Khalil, Jan A.K.W. Kiel, Amy A. Kiger, Akio Kihara, Deok Ryong Kim, Do Hyung Kim, Dong Hou Kim, Eun Kyoung Kim, Hyung Ryong Kim, Jae Sung Kim, Jeong Hun Kim, Jin Cheon Kim, John K. Kim, Peter K. Kim, Seong Who Kim, Yong Sun Kim, Yonghyun Kim, Adi Kimchi, Alec C. Kimmelman, Jason S. King, Timothy J. Kinsella, Vladimir Kirkin, Lorrie A. Kirshenbaum, Katsuhiko Kitamoto, Kaio Kitazato, Ludger Klein, Walter T. Klimecki, Jochen Klucken, Erwin Knecht, Ben C.B. Ko, Jan C. Koch, Hiroshi Koga, Jae Young Koh, Young Ho Koh, Masato Koike, Masaaki Komatsu, Eiki Kominami, Hee Jeong Kong, Wei Jia Kong, Viktor I. Korolchuk, Yaichiro Kotake, Michael I. Koukourakis, Juan B. Kouri Flores, Attila L. Kovács, Claudine Kraft, Dimitri Krainc, Helmut Krämer, Carole Kretz-Remy, Anna M. Krichevsky, Guido Kroemer, Rejko Krüger, Oleg Krut, Nicholas T. Ktistakis, Chia Yi Kuan, Roza Kucharczyk, Ashok Kumar, Raj Kumar, Sharad Kumar, Mondira Kundu, Hsing Jien Kung, Tino Kurz, Ho Jeong Kwon, Albert R. La Spada, Frank Lafont, Trond Lamark, Jacques Landry, Jon D. Lane, Pierre Lapaquette, Jocelyn F. Laporte, Lajos László, Sergio Lavandero, Josée N. Lavoie, Robert Layfield, Pedro A. Lazo, Weidong Le, Laurent Le Cam, Daniel J. Ledbetter, Alvin J.X. Lee, Byung Wan Lee, Gyun Min Lee, Jongdae Lee, Ju Hyun Lee, Michael Lee, Myung Shik Lee, Sug Hyung Lee, Christiaan Leeuwenburgh, Patrick Legembre, Renaud Legouis, Michael Lehmann, Huan Yao Lei, Qun Ying Lei, David A. Leib, José Leiro, John J. Lemasters, Antoinette Lemoine, Maciej S. Lesniak, Dina Lev, Victor V. Levenson, Beth Levine, Efrat Levy, Faqiang Li, Jun Lin Li, Lian Li, Sheng Li, Weijie Li, Xue Jun Li, Yan Bo Li, Yi Ping Li, Chengyu Liang, Qiangrong Liang, Yung Feng Liao, Pawel P. Liberski, Andrew Lieberman, Hyunjung J. Lim, Kah Leong Lim, Kyu Lim, Chiou Feng Lin, Fu Cheng Lin, Jian Lin, Jiandie D. Lin, Kui Lin, Wan Wan Lin, Weei Chin Lin, Yi Ling Lin, Rafael Linden, Paul Lingor, Jennifer Lippincott-Schwartz, Michael P. Lisanti, Paloma B. Liton, Bo Liu, Chun Feng Liu, Kaiyu Liu, Leyuan Liu, Qiong A. Liu, Wei Liu, Young Chau Liu, Yule Liu, Richard A. Lockshin, Chun Nam Lok, Sagar Lonial, Benjamin Loos, Gabriel Lopez-Berestein, Carlos López-Otín, Laura Lossi, Michael T. Lotze, Peter Lõw, Binfeng Lu, Bingwei Lu, Bo Lu, Zhen Lu, Frédéric Luciano, Nicholas W. Lukacs, Anders H. Lund, Melinda A. Lynch-Day, Yong Ma, Fernando Macian, Jeff P. MacKeigan, Kay F. Macleod, Frank Madeo, Luigi Maiuri, Maria Chiara Maiuri, Davide Malagoli, May Christine V. 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Meyer, Chao Yu Miao, Jun Ying Miao, Paul A.M. Michels, Carine Michiels, Dalibor Mijaljica, Ana Milojkovic, Saverio Minucci, Clelia Miracco, Cindy K. Miranti, Ioannis Mitroulis, Keisuke Miyazawa, Noboru Mizushima, Baharia Mograbi, Simin Mohseni, Xavier Molero, Bertrand Mollereau, Faustino Mollinedo, Takashi Momoi, Iryna Monastyrska, Martha M. Monick, Mervyn J. Monteiro, Michael N. Moore, Rodrigo Mora, Kevin Moreau, Paula I. Moreira, Yuji Moriyasu, Jorge Moscat, Serge Mostowy, Jeremy C. Mottram, Tomasz Motyl, Charbel E.H. Moussa, Sylke Müller, Sylviane Muller, Karl Münger, Christian Münz, Leon O. Murphy, Maureen E. Murphy, Antonio Musarò, Indira Mysorekar, Eiichiro Nagata, Kazuhiro Nagata, Aimable Nahimana, Usha Nair, Toshiyuki Nakagawa, Kiichi Nakahira, Hiroyasu Nakano, Hitoshi Nakatogawa, Meera Nanjundan, Naweed I. Naqvi, Derek P. Narendra, Masashi Narita, Miguel Navarro, Steffan T. Nawrocki, Taras Y. Nazarko, Andriy Nemchenko, Mihai G. Netea, Thomas P. Neufeld, Paul A. Ney, Ioannis P. Nezis, Huu Phuc Nguyen, Daotai Nie, Ichizo Nishino, Corey Nislow, Ralph A. Nixon, Takeshi Noda, Angelika A. Noegel, Anna Nogalska, Satoru Noguchi, Lucia Notterpek, Ivana Novak, Tomoyoshi Nozaki, Nobuyuki Nukina, Thorsten Nürnberger, Beat Nyfeler, Keisuke Obara, Terry D. Oberley, Salvatore Oddo, Michinaga Ogawa, Toya Ohashi, Koji Okamoto, Nancy L. Oleinick, F. Javier Oliver, Laura J. Olsen, Stefan Olsson, Onya Opota, Timothy F. Osborne, Gary K. Ostrander, Kinya Otsu, Jing Hsiung James Ou, Mireille Ouimet, Michael Overholtzer, Bulent Ozpolat, Paolo Paganetti, Ugo Pagnini, Nicolas Pallet, Glen E. Palmer, Camilla Palumbo, Tianhong Pan, Theocharis Panaretakis, Udai Bhan Pandey, Zuzana Papackova, Issidora Papassideri, Irmgard Paris, Junsoo Park, Ohkmae K. Park, Jan B. Parys, Katherine R. Parzych, Susann Patschan, Cam Patterson, Sophie Pattingre, John M. Pawelek, Jianxin Peng, David H. Perlmutter, Ida Perrotta, George Perry, Shazib Pervaiz, Matthias Peter, Godefridus J. 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Zhao, Xiaoxiang Zheng, Boris Zhivotovsky, Qing Zhong, Cong Zhao Zhou, Changlian Zhu, Wei Guo Zhu, Xiao Feng Zhu, Xiongwei Zhu, Yuangang Zhu, Teresa Zoladek, Wei Xing Zong, Antonio Zorzano, Jürgen Zschocke, Brian Zuckerbraun

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2929 Scopus citations

Abstract

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are 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 monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Original languageEnglish
Pages (from-to)445-544
Number of pages100
JournalAutophagy
Volume8
Issue number4
StatePublished - 1 Apr 2012

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