Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

1 Guidelines for the U se and I nterpretation of Assays for Monitoring Autophagy (2 nd edition) Daniel J Klionsk y , 1,2 ,* Kotb Abdelmohsen, Akihisa Abe , Md. Jo y nal Ab edin, Hagai Abeliovich, Abraham Acevedo Ar ozena, Hiroaki Adac hi, Christopher M Adams, P eter D Adams, Khosrow Adeli, Peter J. Adhihetty , Sharon G Adler, Galila A g am, R ajesh Agarwal, Manish K Aghi, Maria Agnello, Patrizia Agostinis, Julio Aguirre-Ghiso, Slimane Ait- Si -Ali, Takahiko Akematsu, Emmanue l T Akporiaye , Moh amed Al-Rubeai, Guillermo M Albaiceta, Diego Albani, Matthew L Albert, Jesus Aldudo, Hana Algül, Mehrdad Alirezaei, Iraide Alloza, Alexandru Almasan, Emad S Alnemri, Covadonga Alonso, Dario C Altieri, Ly d ia Alvarez-Erviti, Sandro Alves, Giuseppina Amadoro, Atsuo Amano, Consuelo Amantini, Santiago Ambrosio, Amal O Amer, Mohamed Amessou, Angelika Amon, Frank A. Anania, Stig U Andersen, Usha P Andle y , Catherine K Andreadi, Nathalie Andrieu-Aba die, Alberto Anel, David K Ann, Shailendra Anoopkumar-Dukie , Hir oshi Aoki, Nadezda Apostolova, Saveria Aquila, Katia Aquilano, Koichi Araki, Eli Arama, Jun Ara y a, Al exandre Ar caro, Esperanz a Arias, Hirokazu Arimoto, Aileen R Ariosa, Thierry Arnould, I vica Arsov, Valerie Askanas, Eric Asselin, Ryuichiro Atarashi, J u lie D Atkin, L aura D Attardi, Patrick Auberger, Georg Auburger, Laure Aurelian, R icca rdo Autelli, La ur a Avag liano, Maria Laura Avanta gg iati, La ura Avagliano, Limor Avraha mi, Tiziana Bachetti, J onathan M Bac ker, Dong-Hun Bae , Ok -Nam Bae, Soo Han Bae , Eric H Baehrecke, Seung-Hoon Baek, Stephen Baghdiguian, Ag nieszka Bag niewska-Zadworna, Hua Bai, Xue -Yuan Bai, Kithi g anahalli Naray anaswam y Balaji, Walter Balduini, Andrea Ballabio, Rena Balzan, R ajkumar 2 Banerjee , Gábor Bánhegyi, Haijun B ao , Esther Barreiro, Bonnie Bartel, Alberto Bartolomé, Diane C Bassham, Maria Teresa Bassi, Robert C Bast, Jr, Henri Batoko, Maurizio Battino, Kyle Bauckman, K Ulrich Bayer, Jean-François Beaulieu, George R Bec k Jr, J David B eckham, P atrick J Bedna rski, Thomas J Beg le y, Christian Behl, Kevin E Behr ns, Elo y Bejarano, Amine Belaid, Francesca Belleudi, Gu y Berchem, Daniele Bergamasc hi, Matteo Bergami, Ben Berkhout, L aura Berliocchi, Amélie Bernard, Monique Bernard, Amanda S Bess, Sébastien Besteiro, Savita Bhalla, Sujit K Bhutia, Michele Wolfe B ianchi, Martine B iard-Piechacz y k, Claudia Bincoletto, Sara W Bird, Marc Bitoun, Guillermo A Blanc o, Heidi Kiil Blomhoff, Emili o Boa da-Romero, Stefan Böckler, Marianne B oes, Andrea Boman, Paolo Bonaldo, Jürgen Bosch, Marina Bouché, Marion Bouchecare ilh, S ebastien G Bouret, Patricia Boy a, Michaël Bo y er-Guitt aut, Peter Bozhkov, Vania MM Braga , Claudio Brancolini, Gerhard H Braus, José M Bravo-San Pedro, L isa A Brennan, Emer y H Bre snick, Patrick Brest, Marie-Agnès Bringer, Marisa Brini, Glauber C Brito, Paul S Brookes, Karen Brown, Alain Bruhat, Patricia Chakur Brum, John H Brumell, Nicola Brunetti-Pierri, Hikmet Budak, Dmitr y V Bulavin, Scott J Bultman, Geert B ult y nck, Yan Burelle, Robert E Burke, Laura Caberlotto, Ken Cadwell, Dongsheng Cai, Nadine Camougrand, Michelangelo Campane lla, Grant R Campbell, Matthew Campbell, Silvia Campello, I sabella Caniggia, Lavinia Cantoni, Lizhi Cao, Allan B Caplan, Mic hele Caraglia, Claudio Cardi nali, Sandra Morais Cardoso, Laura A Carleton, Silvia Carloni, Sven R Carlsson, Didac Carmona-Gutierrez, Le ticia AM Carneiro, Serena Ca rra, Alice Carr ier, Bernadette Carroll, Cat y Cas as, Giuliana Cassinelli, Perrine Castets, Susana Castro-Obregon, Gabriella Cavallini, I s abella Ceccherini, Fra ncesco Cecconi, Valentín Ceña, Simone Cenci, Davide Cervia, Silvia 3 Cetrullo, Hassan Chaac houay, Han-Jung Chae, Chee-Yin Chai, Edmond YW Chan, Dhyan Chandra , Ra y mon d Chuen-Chung Chang, Gopal Chakrabarti, Pallavi Chandra, Chih-Peng Chang, Saurabh Chatterjee, Yongsheng Che, Michael E Cheetham, Rajkumar Cheluvappa, Chun-Jung Chen, Gang Chen, Gang Chen, Guang-Chao Chen, Guoqiang Chen, Hongzhuan Chen, Jeff W Chen, Jian-Kang Chen, Mingzhou Chen, Peiwen Chen, Qi Chen, Quan Chen, Shang-Der Chen, Si Chen, Steve S-L Chen, Wei Ch en, Wenli Chen, Xiangmei Chen, Ye -Guang Chen, Yingyu Chen, Yongshun Chen, Yu -J en Chen, Yujie Chen, Z hen Chen, Zhong Chen, Christopher HK Cheng, Hua Cheng, Heesun Cheong, Chun He i Antonio Cheung, Hsiang Cheng Chi, Sung-Gil Chi, Fulvio Chiacchiera, Hui-Ling Chiang , Roberto Chiarelli, Mario Chiariello, L ih -Shen Chin, Gigi NC Chiu, Dong-Hyung Cho, Ssang-Goo Cho, William C Cho, Yong-Yeon Cho, Young- Seok Cho, Augustine MK Choi, Eui-J u Choi, Eu n-Kyoung Choi, Jay oung Choi, Mary E Choi, Seung- Il Choi, Tsui-Fen Chou, Salem Chouaib, Divaker Choube y , Vina y C houbey, Kuan-Chih Chow, Kamal Chowdhury, Charleen T Chu, Tsung-Hsien Chuang , Taehoon Chun, Taijoon Chung, Yong-Joon Chwae, Roberto Ciarcia, Iwona A Ciechomska, Maria Rosa Ciriolo, Mara Cirone, Joan Clària, Peter GH Clarke, Robert Clarke, Emilio Clementi, Cédric Cley rat, Miriam Cnop, Eliana M Coccia, Tiziana Cocco, Jörn Coers, Ezra EW Cohen, David Colecchia, Núria S Coll, Emma Colucci-Guyon, Sergio Comincini, Maria Condello, Graham H Coombs, Cynthia D Cooper, J Mark Cooper, Isabelle Coppe ns, Marco Corazzari, Ramon Corbalan, Mario D Cordero, Cristina Corral- Ramos, Olga Corti, Andrea Cossarizza, Paola Costelli, Safia Costes, Ana Coto-Montes, Sandra Cottet, Eduardo Couve, Lori R Covey, Carolyn B Co y ne, Mark S C ragg , Rolf J Craven, Tiziana Crepa ldi, Jose L Crespo, Ana Maria Cuervo, J ose M Cuezva, Pedro R 4 Cutillas, Mark J Czaja, Maria F Cz y z y k-Krzeska, Ruben K Dagda, Uta Dahmen, Chunsun Dai, Yun Dai, Kevin N Da lb y , L uisa Dalla Valle, Guillaume Dalmasso, Marce llo D’Amelio, Markus Damme, Arlette Darfeuille-Michaud, Victor M Darle y - Usmar, Srinivasan Da sarath y , Srikanta Dash, Crispin R Dass, Hazel Marie Davey , Lester M Davids, David Dávila, Roger J Davis, Ted M Dawson, Valina L Dawson, Paula Daza, Jackie de Be lleroche, Paul de Figueiredo, Regina Celia Bressan Queiroz de Figueiredo, José de la Fuente, Luisa De Martino, Guido RY De Meyer, Angelo De Milito, Mauro De Santi, Ja y anta Debnath, Reinhard Decha nt, Jean-P aul Dec u y pe re, Shane Deeg an, Benjamin De ha y , Barbara Del Bello, Régis Delage-Mourroux, Le a MD Delbridge, Elizabeth Delorme-Axford, Yizhen Deng, Joern Dengjel, Mela nie Denizot, Paul Dent, Vojo Deretic , Eric Deutsch, Timoth y P Devarenne, Rodne y J Devenish, Abhinav Diwan, Sabrina Di Bartolomeo, Nicola Di Daniele, Fabio Di Domenico, Alessia Di Nardo, Antonio Di Pietro, Aaron DiAntonio, Guillermo Díaz-Ara y a, Ines Díaz-Laviada, Maria T Diaz-Meco, Javier Diaz- Nido, Chad A Dicke y , Robert C Dickson, Marc Diederich, Paul Digard, Iva n Dikic, Savithrama P Dinesh-Kumar, Chan Ding , Wen-Xing Ding, Luciana Dini, Jörg HW Distler, Mojgan Djavaheri-Mergny, Kostyantyn Dmytruk, Renwick CJ Dobson, Karol Dokladny, Kelly S Doran, Gabriella D'Orazi, I sab elle Dugail, Svetlana Dokudovska y a, Massimo Donadelli, X Charlie Dong, Terrence M Donohue J r, Gera ld W Dorn II, Victor Dosenko, Liat Drucker, André du Toit, Li -Lin Du, Lihuan Du, Priyamvada Dua , Lei Du an, Michae l R Duchen, Michel A Duc hosal, I s abelle Dugail, Verónica I Dumit, Mara C Duncan, William A Dunn J r, Thomas M Durcan, Stéphane Duvezin-Caubet, Umamaheswar D uvvuri, Vina y Eapen, Darius Ebra himi-Fakhari, Arnaud Ec hard, Charles L Edelstein, Aimee L Edinge r, Ludwig Eichinge r, Tobias 5 Eisenberg, Avital Eisenberg-Lerner, N Tony Eissa, Wafik S El-Deiry, Victoria El- Khour y , Hagit Eldar -F inkelman, Enzo Emanuele, Nikolai Eng edal, Anna- Mart Enge lbrecht, Simone En ge lender, Jorrit M Enserink, Ralf Erdmann, Jekaterina Erenpre isa, Andreja Erman, R icardo E scalante, Eeva-Liisa Eskelinen, Lucile Espert, Lorena Esteban-Martínez, Thomas J Evans, Gemma Fabrias, Cinzia Fabrizi, Antonio Facchiano, Alberto Faggioni, Chunhai Fan, Daping Fan, Shengyun Fa ng, Manolis Fanto, Alessandro Fanzani, Mathias Faure, Francois B Favier, Howard Fearnhead , Massimo Federici, Erka ng Fei, Ta nia C Felizardo, Hua Fe ng, Yibin Feng, Yuchen Feng, Thomas A Ferg uson, Álvaro F Fernández , Maite G Ferna ndez-Barrena, Jose C Fernandez-Checa, Martin E Fernande z- Zapico, Elisabe tta Ferraro, Carmen Veríssima Ferreira-Halder, La szlo Fesus, Ralph Feuer, Fabienne C Fiesel, Gi useppe F ilomeni, Eduardo C Filippi- Chiela, Gian Maria Fimia, Toren Finkel, F ilomena Fiorito, Paul B Fisher, Marc Flajolet, Flavio Flamigni, Oliver Florey , Salvatore Florio, R Andres Floto, Marco Folini, Carlo Follo, Edward A Fon, France sco Fornai, Franco Fortunato, Alessandro Fraldi, Rodrigo Franco, Arnaud Franc ois, Aurélie Fra nçois, I ain DC Fra ser, Damien G Fre yssene t, Scott L Friedman, Daniel E Frigo, Dongxu Fu, José M Fuentes, J uan Fuey o, Yuuki Fujiwara, Mikihiro Fujiya , Mitsuno ri Fukuda , Simone Fulda, Carmela F usco, Matthia s Gaestel, Philippe Gailly , Malgorzata Gajewska, Gad Galili, Maria F Galindo, Lor enzo Galluzzi, Luca Ga lluzzi, Vincent Ga ly, Sam Gand y , Anand K Ganesan, Swamynathan Gane san, Fen-Biao Gao, Jian-Xin Gao, Eleonora García Véscovi, Marina García-Macia, Lore na García Nannig, Abhishek D Garg , Pramod Kumar Garg, Ricardo Gargini, N ils Christian Gassen, Da mián Gatica, Evelina Gatti, J ulie Gavard, Evr ipidis Gavathiotis, L ian g Ge, Shengfang Ge , Po -Wu Gean, Armando A Genazzani, Pascal Ge nschik, David A Gewirtz, 6 Saeid Ghavami, Eric Ghig o, Anna Maria Giammarioli, Fra ncesca Giampieri, C laudia Giampietri, Alexandra Giatromanolaki, Derrick J Gibbings, Lara Gibellini, Spencer B Gibson, Vanessa Ginet, Antonio Giordano, Flaviano Giorgini, Elisa Giovannetti, Stephen E Girardin, Alvaro Glavic, Martin Gleave, Robert M Gogal Jr, Gustavo H Goldman, Delia Goletti, Michae l S Goligorsky, Aldrin V Gomes, Hernando Gomez, Candelaria Gomez-Manzano, Rubén Gómez-Sánchez, Céline Gong ora, Pedro Gonzalez -Aleg r e, Pilar Gonzalez-Cabo, Rosa Ana González-Polo, Carlos Gorbea, Nikolai V. Gorbunov, Daphne R Goring, Adrienne M Gorman, Sharon M Gorski, Sandro Goruppi, Shino Yamada Goto, Cecilia Gotor, Roberta A Gottlieb, I ll ana Gozes, Devrim Gozuacik, Yacine Graba , Martin Graef, Giovanna E Granato, Gary Dean Grant, Steven Grant, Giovanni L uca Gravina, Douglas R Green, Alexander Greenhough, Michel T Greenwood, Benede tto Grimaldi, Fré déric Gros, Charles Grose, Jean-Francois Groulx , Paolo Grumati, Jun-Lin Guan, Kun-Liang Guan, Ba rbara Guerra, Carlos Guillen, Kailash Gulshan, Jan Gunst, Chuany ong Guo, Le i Guo, Ming Guo, Wenjie Guo, Xu -Gua ng Guo, Andrea A Gust, Åsa B Gustafsson, Ho -Shin Gwak, Albert Haas, James E Haber, Shinji Hadano, Monica Hagedorn, Andrew J Hala y ko, A nne Hamacher-Brady, Isabelle Ha mer, Qutay ba Hamid, Ester M Hammond, Feng Han, Xiao Han, J ohn A Hanove r, Malene Hansen, Masaru Harada, Ljubica Harhaji-Trajkovic, J Wade Ha rper, Abdel Halim Harrath, James Harris, Udo Hasler, Peter Hasselblatt, Kazuhisa Hasui, Robert G Hawle y , Congcong He, Cy nthia Y He, Fengtian He , Ron g-Rong He, Xian-Hui He, You-Wen He , Joan K Heath, Marie-Josée Hé bert, Gudmundur Vignir Helgason, Eliz abeth P Henske, Paul K Herman, Sonia Hernánde z-Tiedra, Claudio Hetz, P. Robin Hiesinger, Sabine Hilfiker, Bradf ord G Hill, J oseph A Hill, William D Hill, Keisuke Hino, Paul Hofman, 7 Jörg Höhfeld, Marina K Holz, Yonggeun Hong, David A Hood, Daniel Hofius, Günter U Höglinger, Jincai Hou, Chin Hsu, Li -Chung Hsu, Dong Hu, Hong-Min g Hu, Hongbo Hu, Ming Chang Hu, Yu -Chen Hu, Zhuo-Wei Hu, Fang Hua, Ya Hua, Canhua Huang, Hue y - La n Huang, Kuo-How Huang, Kuo-Yan g Hua ng, Shiqi an Hua ng, Wei-Pang Huang, Yunfei Huang , Tobias B Huber, Patricia Huebbe, Won-Ki Huh, Juha J Hulmi, Gang Min Hur, James H Hurley, Zvenyslava Husak, Sabah NA Hussain, Salik Hussain, J ung Jin Hwang, Seungmin Hwa ng, Thomas I S Hwang, Atsuhiro Ic hihara, Yuzuru Imai, Carol Imbriano, Megumi Inomata, Takeshi Into, Valentina I ovane, Juan L Iovanna, Renato V Iozzo, Nancy Y Ip, Pablo Iribarren, Yoshitaka I saka, Aleksandra J I sakovi c, Hiro y uki Ishida, Isa o Ishii, Jane E. I shma el, Ciro I sidoro, Ken-ichi Isobe, Shohreh Issazadeh- Navikas, Koji Itaha na, A nand Krishnan V Iy e r, Yotaro I zumi, Marja J äättelä, Nadia Jaber, Daniel John Jackson, William T Jackson, Ton y G eorge Jacob, Chinn aswamy Jagannath, Alkesh Jani, Bassam Janji, Paulo Roberto Jannig, Patric J J ans son, Marina Jendrach, Ju -Hong Jeon, Eui-Bae Jeung, Kailiang Jia, Lijun Jia, Hong Jiang, L iwen Jiang, Teng Jiang, Xiaoyan Jiang, Xuejun Jiang, Xuejun Jiang, Yongjun Jiang, Alberto Jiménez, Cheng Jin, Hongchuan Jin, Mei y an J in, Shengkan Jin, Umesh Kumar J inwal, Eun-Kyeong Jo, Terje Johansen, Daniel E Johnson, Gail VW Johnson, James D Johnson, Eric Jonasch, Chris Jones, Leo AB Joosten, Joaquin J ordan, Anna -Maria Joseph, Bertrand Joseph, Annie M J oubert, Dianwe n Ju, Hsueh-Fen Juan, Katrin Juenemann, Gábor Juhász, Hye Seung Jung, J ae U Jung, Yong-Keun Jung, He inz J ungbluth, Matthew J Justice, Barry Jutten, Nadeem O Kaakoush, Allen Kaasik, Tomohiro Kabuta, Katarina Kågeda l, Alon Kahana, Or Kakhlon, Dhan V Kalvakolanu, Vitaliy O Kaminsk yy , Mustapha Kandouz, Chanhee Kang, Rui Kang, Tae-Cheon Kang, Tomotake Kanki, 8 Thirumala-De vi Kanneganti, Haruo Kanno, Anu mantha G Ka nthasamy, Marc Kantorow, Maria Ka parakis- L i askos, Orsoly a Kapu y , V assiliki Karantza, Md. Razaul Karim, Parimal Karmakar, Arthur Kaser, A Murat Kaynar, Po -Yua n Ke, John H Kehrl, Kate E Keller, Oliver Kepp, Andreas Kern, Santosh Kesari , David Kessel, Robin Ketteler, Muzamil Majid Khan, Juliann G Kiang, Akio Kihara, Arianna L Kim, Cheol Hyeon Kim, Deok Ry ong Kim, Do -Hy ung Kim, Eun g Kweon Kim, Hye Youn g Kim, H y u ng-R y ong Kim, Jae-Sung Kim, Jeong Hun Kim, Jin Cheon Kim, J in Hy oung Kim, Peter K Kim, Seong Who Kim, Yong hyun Kim, Adi Kimchi, Alec C Kimmelman, J ason S King , Lorrie A Kirshenbaum, Shuji Kishi, Katsuhiko Kit amoto, Kaio Kitazato, Rudolf A Kle y , Walter T Klimecki, Jochen Klucken, Helene Knævelsrud, Erwin Knecht, Jiunn-Liang Ko, Satoru Kobaya shi, C hristelle Koec hlin-Ramonatx o, Ulrich Koenig , Young Ho Koh, Katja Köhler, Se pp D Ko hlwein, Masaaki Komatsu, Hee Jeong Kon g , Eumorphia G Konstantakou, Benjamin T Kopp, Tamas Korcsmaros, Laura Korhonen, Viktor I Korolchuk, Nadezhda V Koshkina, Michael I Koukourakis, Constantinos Koumenis, Attila L Kovács, Da isuke Ko y a , Claudine Kraft, Dimitri Krainc, Roswith Krick, J anos Kriston-Vizi, Guido Kroemer, Rejko Kruger, Nicholas T Ktistakis, Kazuyuki Kuchitsu, A Pratap Kumar , Ashok Kumar, Deepak Kumar, Dhiraj Kumar, Sharad Kumar, Hsing- Jien Kung, Atsushi Kuno, Jeff Kuret, Tino Kurz, Taeg Ky u Kwon, Yon g Tae Kw on, Albert R La Spada, Frank L afont, Tim La hm, Truong Lam, Terry H Landowski, J on D La ne, Cinzia L anzi, Louis R L apierre, J ocelyn Laporte, Gordon W Laur ie, Sergio La vandero, Le na Lavie, Kelse y B Law, Pedro A Lazo, Laurent Le Cam, Karine G Le Roch, Vijittra L eardkamolkarn, Marc L ecuit, B y ung-Hoon Lee, Che-Hsin Lee, G y un Min Lee, He -Jin L ee, Hsiny u Lee, Jae Keun Lee, Jongdae Lee, J un Hee L ee, Michael Lee, 9 Myung-Shik Lee, Sam W Lee , Seung-J ae Le e, Shiow-J u Le e, Sug Hyung Lee, Sung Sik Lee, Sung- Joon Lee, Sunhee Lee, Yong J Lee, Young H Lee, Christiaan Leeuwenburgh, Sy lvain Le fort, Renaud Legouis, Jinz hi L ei, Qun -Ying Lei, David A Leib, Gil Le ibowitz, Stéphane D Lemaire, John J L emasters, Antoinette L emoine, Guido L enz, Lilac h O L erman, Daniele Lettieri Barbato, Julia I - Ju L eu, Hing Y Leung, Beth Levine, Patrick A Lewis, Chi L i, Dan L i, Faqiang Li, Feng-J un L i, Jun L i, Ke Li, Lian L i, Min Li, Min Li, Qiang Li, Rui Li, Sheng Li, Wei L i, Xiaot ao L i, Yumin Li, J iqin Lian, Che ng y u Liang , Yulin L iao, Pawel P L iberski, Andrew P Lieberman, K y u Lim, Chang-Shen Lin, Chiou-Feng Lin, Fang Lin, Fangming L in, Fu -Ch eng Lin, Kui Lin, Kwang-Huei L in, Tianwei Lin, Wan-Wan L in, Yee-Shin Lin, Yong L in, Raf ael Linden, Dan L indholm, Lisa M Lindqvist, Lance A Liotta, Marta M Lipinski, Vitor A Lira, Michael P Lisanti, Paloma B Liton, Bo Liu, Chun-Feng Liu, Fei Liu, Hung-Jen L iu, Jianxun Liu, Jing-Jing Liu, Jing-La n Liu, Ke L iu, L eyuan Liu, Quentin Liu, Rong-Yu Liu, Shiming Liu, Wei Liu, Xian-De Liu, Xiangguo L iu, Xiao-Hong Liu, Xinfeng L iu, Xu Liu, Yule L iu, Zexian Liu, Susan E Logue , Sagar Lonial, Ben Loos, Carlos L ópez-Otín, Cristina López- Vicario, Mar Lore nte, Péter Lőrincz, Mare k Los, Michael T Lotze, Penny E Lovat, Binfeng Lu, Bo Lu, Jiahong Lu, Shemin L u, Shu y an Lu, Frédéric Luciano, Shirle y Luckhart, J ohn Milton L ucocq, Paula Ludovico, Aurelia Lugea, Nicholas W Lukacs, Julian J Lum, Anders H Lund, Hong lin Luo, Jia L uo, Shouqing Luo, Glaucia M Machado -Santelli, Gustavo Macintosh, Jeffrey P MacKeigan, Ka y F Macleod, John D MacMicking, Frank Madeo, Muniswamy Madesh, Akiko Maeda, Emilia Maellaro, Hannelore Maes, Marta Magariños, Kenneth Maiese, Tapas K Maiti, Luigi Maiuri, Maria Chiara Maiuri, Carl G Maki, Roland Malli, Walter Malorni, Na Man, Eva-Maria Mandelkow, Angelo A 16 Alexander J Whitworth, Eileen White, J L indsa y Whitton, Franziska Wild, Tom Wileman, Deepti Srinivas Wilkinson, Simon Wilkinson, Dieter Willbold, Chris Williams, Peter R Williamson, Steven S Witkin, Stepha nie E Wohlgemuth, Thomas Wollert, Ernst J Wolvetang, Esther Wong, Richard W Wong, Karen L W right, Chunlai Wu, Defeng Wu, Jian Wu, Junfang Wu, Mian Wu, William KK Wu, Zhe nlong Wu, Ramnik J Xavier, Tian Xia, Weiliang Xia , Yon g Xia, Hengyi Xiao, Shi Xiao, Wuhan Xiao, Chuan-Ming Xie, Zhiping Xie, Z hon glin Xie, Chuanshan Xu, Congfeng Xu, Feng Xu, Haoxing Xu, Hongwei Xu, Jian Xu, Jianzhen Xu, Jinxian Xu, Liang Xu, Xiaolei Xu, Yu Xue, Takahiro Yamada, Ai Yamamoto, Shunhei Yamashina, Bo Yan, Xianghua Yan, Zhen Yan, Dun-Sheng Yang, Jiin-Ming Yang, Minghua Yang, Pei-Ming Yang, Qian Yang, Sijun Yang, Wannian Yang, Wei Yua n Yang, Yi Yang, Ying Ya ng, Men g-Chao Yao, Zhe n y u Yao, Behzad Yeganeh, Long Yi, Xia o-Ming Yin, Calvin K Yip, Yeong-Min Yoo, Young H y un Yoo, S eung -Yong Yoon, Kenichi Yoshida, Tamotsu Yoshimori, Ken H Young , Huix in Yu, Jane J Yu, Jin-Tai Yu, Jun Yu, Li Yu, Zhengping Yu, J uny in g Yuan, Zhi-Min Yuan, Beatrice YJT Yue, Zheny u Yue, David N Zacks, Eld ad Zacksenhaus, Nadia Zaffaroni, Tania Zaglia, Zahra Za keri, J insheng Ze ng, Min Zeng, Qi Ze ng, Antonis S Zervos, Donna D Zhang, Fan Zhang, Guo-Chang Zhang, Guo Z hang, Hao Zhang, Hong Zhang, Hong Zhang, Hongbing Zhang, Jianhua Zhang, Jing-pu Zhang, Li Zha ng, Lin Zhang, Long Z hang, Ming-Yon g Zhang, Xiangnan Zhang, Xu Dong Zha ng, Yan Zhan g , Yingmei Zhang, Mei Z hao, Wei-Li Zhao, Xiaonan Zhao, Yan G Zha o, Yin g Z hao, Yongchao Zhao, Yu -xia Zha o, Zhendong Z hao, Dexian Zheng, Xi - Long Zheng, Xiaoxiang Z heng, Boris Zhivotovsky, Qin g Zhong , Guofei Zhou , Shufeng 17 Zhou, Xu - jie Zhou, Hongxin Zhu, Wei-Guo Zhu, Wenhua Zhu, Xia o-Feng Zhu, Shi-Mei Zhua ng, Elio Ziparo, Chr istos E Zois, Tere sa Zoladek, Wei-Xing Zong, An tonio Zorzano 1 L ife Sciences Institute, and 2 Department of Molecular, Cellular and Developmental Biology Universit y o f Michigan, Ann Arbor, MI USA ; Running title: Autophagy guidelines Keywords: autolysosome, autophagosome, flux, LC3, l y sosom e, phagophore, stress, vacuole *Corresponding author : Corresponde nce: Daniel J . Klionsky , Life Sciences I nstitut e, University of Michigan, Ann Arbor, MI 48109-2216. Tel. 734-615-6556; Fax . 734-647- 9702; Ema il: klionsky @umich.edu 18 Table of Contents Introduction A. Methods for monitoring au tophagy 1. Transmission electron microscopy ……………………………………… XX 2. Atg8/LC3 detection and quantification …………………………………. XX a. Western blotting and ubiquitin -like protein conjuga tion systems …… XX b. Turnover of LC3 -II /Atg8 – PE ………………………………………... XX c. GFP-Atg8/L C3 l y sosomal deliver y and partial proteol y sis ………….. XX d. GFP-Atg8/L C3 fluorescence microscop y …………………………….. XX e. Tandem mRFP/mCherry-GFP fluorescence microscopy …………….. XX f. Autophagic flux determination using f low and multispectral imaging cy tometr y …………………………………………………………….. XX g. Immunohistoche mistr y …………………………………………….... XX 3. SQSTM1 and related LC3 binding protein turnover assays …………….. XX 4. MTOR, AMPK and Atg1/ULK1 ……………………………………….. XX 5. Additional autophagy-related protein markers ………………………….. XX a. Atg9 ……………………………………………………………..….. XX b. A TG 12 – A TG 5 ……………………………………………………..….. XX c. ATG14 …………………………………………………………..….. XX d. ATG16L1 ………………………………………………………..….. XX e. Atg18/WIPI family ……………………………………………..….. XX f. BECN1/ Vps30/ Atg6 ………………………………………………..….. XX g. DRAM 1 …………………………………………………………..….. XX 19 h. ZFYVE1/ DFCP1 ………………………………………………..….. XX i. STX17, TECPR1 …………………………………………………… XX 6. Sphingolipids…………………………………………………………… XX 7. Transcr iptional , translational and post translationa l regulation …………………………….. XX 8. Posttranslational modification of ATG prote ins………………………... XX 9. Autophag ic protein degradation …………….………………………….. XX 10. Selective types of autophagy ……………………….………………….. XX a. The Cvt pathwa y , mitophag y , pexophag y , piecemeal microautophagy of the nucleus and nucleophagy in yeast and filamentous fungi .. XX b. Reticulophagy and ribophag y …………………………………..….. XX c. Vacuole import and degradation pathway ………………………….. XX d. Animal mitophagy and pe x ophagy ………………………..……… .. XX e. Aggrephagy ……………………………………………………..….. XX f. Allophagy …………………………………………………..…..….. XX g. Chlorophagy ……………………………………………………….. XX h. Chromatophagy ……………………………………………..…..….. XX i. Ferritinophagy ……………………………………………………… XX j. Lipophagy ……………………………………………………..….. XX k. Intraplastidial autophagy…………………………………………. XX l. RNA- silencing components………………………………………. XX m. Xenophagy. ………………………………………….…………… .. XX n. Zymophagy ……………………………………………………..….. XX 20 11. Autophagic sequestration assa y s ……………………………..……….. XX 12. Turnover of autophagic compartments ……………………… ……….. XX 13. Autophagosome-l y sosome coloca lization and dequenching assay …… XX 14. Tissue fractionation ………………………………………………..….. XX 15. Analyses in vivo …………………………………………………..….. XX 16. Clinical setting……………………………………………………….. XX 17. Cell death ……………………………………………..…………..….. XX 18. Chaperone-mediated autophagy …………………………………..….. XX B. Comments on additional m ethods …………………………………..….. XX 1. Acidotropic dyes ……………………………..……………………..….. XX 2. Autophagy inhibitors and inducers ……………. …………………..….. XX 3. Basa l autopha gy ………………………………..…………………..…. . XX 4. Experimental systems ……………………………………………..….. XX 5. Nomenclature ……………………………………………………..….. XX C. Methods and challenges of specialized topics /m odel systems ……..….. XX 1. C. elegans …………………………………………………..………… XX 2. Chicken B-lymphoid DT40 cells , retina and inner ear ………………. XX 3. Chlamydomonas …………………………………………………..….. XX 4. Drosophila …………………………………………………..………... XX 5. Ery throid cells ………… ……………………………………………… XX 6. Filamentous fungi …………………………………………………..…. XX 7. Food biotechnology …………………………………………………….. XX 8. Honeybee …………………………………………………..………….. XX 21 9. Human …………………………………………………..….. ………… XX 10. H y d ra …………………………………………………..……………... XX 11. Lepidoptera …………………………………………………..……….. XX 12. Mammals…… …………………………………………………..…….. XX 13. Marine invertebrates………………………………………………….. XX 14. Neotropical teleosts ………………………………………………….... XX 15. Odontoblasts ………………………………………………………….. XX 16. Planarians ………………………………………………………….….. XX 17. Plants …………………………………………………..…………….... XX 18. Protists ………………… …………………………………………..……. XX 19. Rainbow trout. ……………………………………………………..……. XX 20. Sea Urchin ………………………………………………………...…….. XX 21. Ticks …………………………………………………..……………. ....... XX 22. Zebrafish …………………………………………………..…………… . XX D. Noncanonical use of autophagy-related proteins ………………………..….. XX 1. LC3-a ssociated phagoc y tosis …………………………………………. XX 2. LC3- a ssociated apicoplast…… ……………………………………….. XX 3. LC3 conjuga tion s y stem for IFNG- mediated pathogen control………. XX 4. Intracellular tra fficking of bacterial pathogens………………………. XX 5. Other processes……………………………………………………….. XX E. I nterpretation of in silico assa y s for monitoring autophagy ……………… .. XX 1. Sequence comparison and comparative ge nomics approaches ……….. XX 2. Web-based re sources r elate d to autophag y …………………………… XX 22 a. The THANATOS database ………………………………………. XX b. The human autophagy database (HADb) ……… ………………… XX c. The Autophagy Database …………………… …………………… XX d. The Autophagy Reg ulator y Network (ARN) ……………………. XX e. Prediction of Atg8-famil y interacting proteins ………………….. XX f. The iLIR ser ver…………………………………………………….. XX g. The Eukar y otic Linear Motif resource (EL M)……………………. XX Conclusions and future perspectives …………………………………… . ….. XX Reference s …………………………………………………..……………..….. XX Glossary …………………………………………………..…………………. .. XX Index …………………………………………………..……………………. .. XX Abbreviations: 3-MA, 3-methyladenine; ALIS, aggresome-like induced structures; Ape1, aminopeptidase I; ARN, Autophagy Regulatory Network; Atg, autophagy-related; AV, autophagic vacuole; CLEAR, coordinated lysosoma l enhancement and regulation; CLEM, corre lative light and electron microscop y; CMA, chaperone-mediated autophagy; cryo-SXT, cryo-soft X-ray tomo g raphy; Cvt, c y to plasm- to -vacuole targeting; DQ -BSA, dequenched bovine serum albumin; e-MI , endosomal mi croa utophag y ; EBSS, Earle’s balance d salt soluti on; ER, endoplasmic r eticulum; FACS, fluorescence-a ctivated c ell sorter; GB P, guan y l ate bi nding prote in; GFP, green fluorescent protein; HKP, housekeeping prote in; HS V, herpe s simplex virus; I CD, immunogenic c ell death; IHC, immunohistochemistry ; IMP, intramembrane particle; LAMP2, lysosome-associated membrane protein 2; LAP, LC3-associated phag ocytosis; L C3, mi crotubule-associated 23 protein 1 light cha in 3 ( MAP1L C3); LN, la te nucleophagy; MDC, monodansylcadaverine; mRFP, monomeric red fluorescent protein; mtDNA, mitochondrial DNA; MTOR, mechanistic targ et of rapamycin; MVB, multivesicular body; NETs, neutrophil extracellular traps; NVJ, nucleus-vacuole junction; PAS, phag ophore assembl y sit e; PE, phosphatid y lethan olamine; P I 3K, phosphoi nositide 3- kinase; PMN, piecemeal microa utophag y of the n ucleus; PSSM, position-spec ific scoring matrix; Ptd I ns3K, phosp hatidy linositol 3 -kinase; PtdI ns3P, phosphatidylinositol 3 - phosphate; PVM, parasitophorus vacuole membrane; RBC, red blood cell; Rluc, Renilla reniformis luciferase ; ROS , rea ctive oxygen species; SD, standard deviation; SOD, superoxide dismutase; TEM, transmission electron microscopy; tfLC3, tandem fluoresce nt LC3; TORC1 , TOR complex I ; TR-FR ET, time-resolved f luorescence resonance energy tra nsfer; TVA, tubulovesicular autophag osome; UPR, unfolded protein response ; UPS, ubiquitin-proteasome system 24 In 2008 we published the first set of guidelines for standardizing research in autophag y . Since then, researc h on this top ic has continued to accelerate, and many ne w scientists have enter ed 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 acce ptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be e mphasized is that there is a difference between measurements tha t monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autoly sosomes) at an y stage of the autophagic proce ss versus those that measure f lux through the autophagy pa thway (i.e., the complete pr ocess includi ng the a mount and rate of cargo seque stered and de g raded ) . I n particular , a block in macroautophagy that results in autophag osome accumulation must be differe ntiated from stimuli that increase autophagic activity, defined as increased a utophag y induction coupled with increased deliver y to, and degrada tion within, l y sos omes (in most higher eukar y otes and some protists such as Dictyostelium ) or the vacuole (in plants and fung i). I n other words, it is especiall y importa nt that investigators new to the field understand that the appea rance of more autophag o so m es does not necessarily equate with more autophag y . I n fact, in man y c ases, autophagosomes accumulate because of a block in trafficking to l ysosomes without a concomitant cha nge in autophagosome biogenesis, whereas an increa se in autol y sosom es may reflect a reduction in degradative activity. Here, we present a se t of g uidelines for the selection and interpretation of methods for use by investigators who aim to examine macroa utophag y and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of pape rs that are focused on 25 these processes. These guidelines are not meant to be a formulaic set of rules, beca use the appropriate assays depend in part on the question being asked and the sy stem being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strong l y r ecommend the use of multiple assays to monitor autophag y . Along these lines, when attempting to bloc k autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene because individual Atg prote ins, or groups of proteins, a re involved in other cellular pathwa y s; not all Atg proteins can be used as a specific marker for an autophagic process. I n these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally , b y discuss ing the merits and limits of particula r autophag y a ssay s, we hope to encourage tec hnical innovation in t he field. Introduction Many researchers, especiall y those ne w to the field, need to determine which criteria are essential for de monstrating autophag y, e ither for the purposes of their own research, or in the capacity of a manuscript or grant review. 1 Acceptable standards are an important issue, particularly c onsiderin g that eac h of us ma y have h is/her own opinion regarding the answer. Unfortunately, the answer is in part a “moving targe t” as the field evolves. 2 T his can be extremely f rustrating for rese archers who ma y thi nk they have met those criteria, only to find out that the reviewers of their paper have different ideas. Conversely, a s a reviewer, it is tiresome to raise the same objections repe atedly, wondering why researc hers have not fulfill ed some of the basic requirements for establishing the oc currence of an autophag ic process. In addition, drugs that potentially modulate autophagy a re increasingl y being used in clinical trials, and sc reens are 32 morphologica l changes during the autophagic process ca n be followed b y TEM. The matura tion from the phagophore through the autoly sosome is a d y n amic and continuous process, 44 and, thus, the classification of compartments into discrete morpholog ical subsets can be problematic ; therefore , some ba sic guidelines are offered below . In the precee ding sections the autophagosome, the amphisome and the autolysosome were used as the terms for 3 basic stages and compartments of autophagy. It is important to make it clear that many times, when we do not want to, or cannot, differe ntiate amo ng the autophagosomal, amphisomal and a utolysosomal stage we use the term autophag ic vacuole. (Since y east studies have appeared in the autophagy field the term “autophag ic vesicle” is extensively used; b y now the two terms are used in parallel and can be considered synonyms.) Autophag osomes , also referred to as initial autophagic vacuoles ( AVi) , t y pically have a double membrane . This membrane is usuall y w ell visible as 2 parallel membrane layers ( bilayers ) separa ted b y a n arrower or wider electron-lucent cleft , when the simplest routine EM fix ation procedure is applied ( F ig. 2A ). 45 , 46 I n the case of nonselective autophagy, a utophag osomes contain cytosol and/or orga nelles appearing morp hologica ll y int act as also described above . 43 , 47 Amphisomes 48 can sometimes be identified b y the presence of small internal vesicles inside the autophagosome/autophagic vac uole (AV). 49 These internal vesicles are delivered into the lumen by f usion with multivesicular endosomes, and care shoul d there fore be taken in the identification of the organelles, especially in ce lls that produce large numbers of MVB-d erived e xosomes, such as tumor and stem ce lls. 50 Late/degradative autophag ic vacuoles / autol y sos omes (AVd or AVl ) typica ll y have onl y one limiting membr ane ; frequentl y they contain electron dense cytoplasmic material and/or organe lles at various stages of degradation ( Fig. 2A and B ). 43 , 47 33 The prese nce of l y ti c enzy mes in autolysosomes can be used to identify them. For that purpose the traditional method is to demonstrate the ac tivity of acid phosphatase by enzy me cy tochemistr y 51 or to show their presence b y im munocytochemistr y . 52 The sequential deteriora tion of c y t oplasmic structures being digested can be used for identifying a utol y sosomes b y TEM. Ev en whe n the partiall y digested and destroy ed structure cannot be recognized in itself, it can be traced back to earlier forms b y id entif y in g preceeding stage s of sequential morphological deterioration. Degradation usually leads first to i ncre ased density of still rec ognizable organelles, then to vacuoles with heterogenous density, which are becoming more and more homogenous, amorphous, mostl y d ense, but sometimes light. It must be e mphasiz ed that in addition to the autophag i c input, other (e.g., endosomal, phag osomal, chaperone-mediated) processes also carry cargo to the lysosomes. 53 , 54 Therefore, strictly spea king, we can only ha ve a mixed l y ti c compartment. Howe ver, we still ma y use the term “autolysosome” if the content a ppears to be overwhelmingly autophagic. Note that the eng ulfment of apoptotic cells also produces l y sos omes that contain cytoplasmic structure s, but in this case it orig inates from the d y in g cell. For many biologica l and pathological situations, examinati on of both early and late autophagic vacuoles y iel ds valuable data regarding the overall autophagy /lysosomal status in the cells. 13 Along these lines, it is possible to use immunocy tochemistr y to follow particula r cy tosolic proteins such as SOD1/ CuZn superoxide dismutase and CA/ carbonic anh y dras e to determine the stage of a utophag y ; the fo rmer is much more resistant to lysosomal degrada tion. 55 In some autophag y- in ducing conditions it is possible to observe multi-lamel lar membrane structures in addition to the conve ntional double-membrane autophagosomes , although t he nature of the se structures is not full y und erstood. The se multi-lamellar structures ma y indeed be 34 multiple double laye rs of phagophores 56 and positive for LC3, 57 they could be autolysosomes, 58 or they ma y for m artifact uall y durin g fixation. Special fea tures of the autophagic process ma y b e clarified by immuno-TE M with gold- labeling, 59 , 60 using antibodies, f or example, to cargo proteins of cytopla smic origin and to LC3 to verify the a utopha g ic nature of the compartment. LC3 immunogold labeling al so mak es it possible to detect novel degradative organelles within autophagy compartments. This is the case with the autophagoproteasome whe re costaining for LC3 and ubiquitin -proteasome system (UPS) antige ns occurs. The autophagoproteasome consists of sin gle- , double-, or multiple- membrane LC3-positive autophagosomes costa ining for specific components of the UPS. I t ma y be that a rich multi-enzymatic (both autophagic and UPS) ac tivit y takes pl ace within these organelles instea d of being segregated within d ifferent cell domains. Although labe ling of LC3 can be difficult, an increasing number of comme rcial antibodies are becoming available, among them good ones to visualize the GFP moiety of GFP- LC3 re porter constructs. 61 I t is important to keep in mind that LC3 ca n be associated with non - autophagic structures (see Xenophagy and Noncanonical use of autophagy-related proteins ). LC3 is involved in specialized forms of endocytosis like L C3-associated phagoc y tosis. In addition, LC3 ca n decorate vesicles dedicated to ex ocy tosis in non -conventional secretion systems (reviewed in ref. 62 ). Antibodies ag ainst an abundant cytosolic protein will result in high labeling all over the cy toplasm ; however, orga nelle markers work well. Because there are very few character ized proteins that remain associated with the completed autophagosome , the choices for confirmation of its autophagic nature are limited. Furthermore, autophagosome - associated prote ins ma y be cell type-specific. At any rate, the success of this methodology depends on the qua lit y of the antibodies and also on the TEM preparation and fix ation 35 procedures utilized. With immuno-TEM, authors should provide controls showing that labeling is specific. This may re quire a quantification of staining over differe nt cellular compartments. In addition, statistical information should be provided due to the necessity of showing only a selective number of sections. A g ain, we note that for quantitativ e data it is preferable necessary to use proper volumetric analysis ra ther than just counting numbers of sectioned objects. On the one ha nd, it must be ke pt in mind that even volumetric morphometry /st ereolog y only shows either steady state levels, or a snapshot in a changing d y namic process. Such data by themselves are not infor mative reg arding autophagic flux, unless carried out over multiple time points. Alternatively , investigation in the presence and absence of flux inhibitors can reveal the dyna mic changes in various st ag es of the autophagic pr ocess ( Fig. 20 ) . 10 , 19 , 63 , 64 , 40 On the one hand , if the turnover of autolysosomes is very rapid, a low number/volume will not necessarily be an accurate refle ction of low autopha g ic activity . On the oth er ha nd, quanti tative ana lyses indicate that autophag osome volume in man y cases does correlate with the rates of protein degrada tion. 65 - 67 One pot ential compromise is to perfor m whole cell quantification of autophagosomes using fluorescence methods, with qualitative verification by TEM, 68 to show that the change s in fluorescent puncta reflect corre sponding changes in autophagic structures. One additional ca veat with TEM, and to some extent with c onfocal fluorescence microscopy, is that the analy sis of a single pl ane within a cell ca n be misleading and ma y make the identification of a utophagic structures difficult. Confocal microscop y a nd fluorescence microscopy with dec onvolution so ftware (or with much more wor k, 3 -dimensional TEM) can be used to generate multiple/serial sec tions of the same cell to reduce this concern; however, in many cases where there is sufficient structural resolution, a naly sis of a single plane with multiple cells can suffice g iven pr actica l limitations . Newer EM tec hnologies, including focused ion beam 36 dual-beam EM, should make it much easier to apply three-dimensional analy ses. An additional methodology to assess autophagosome accumulation is correlative light and electron microscopy, CL EM, which is helpful in confirming that fluorescent structures are autophagosomes. 69 - 71 Along these lines, it is important to note that even though GFP fluoresce nce will be quenched in the acidic environment of the autolysosome, some of the GFP puncta detected by lig ht microscop y may corre spond to earl y autol y sosomes prior to GFP quenching. Th e mini Singlet Oxygen Genera tor (mi niSOG) fluore scent flavoprotein, which is less than half the size of GFP, provides an additional means to gene ticall y t ag proteins for CLEM analy sis under conditions that are particularl y suited to subsequent TEM analy sis. 72 Combinatorial assay s using tandem monom eric red fluoresce nt protein ( m RFP ) -GFP-LC3 (see Tandem mRFP/mCherry-GFP fluorescence mic roscopy ) along with static T EM imag es should help in the analysis of flux and the visualization of ca rgo structures. 73 Another technique that has prove n quite useful for anal y zin g the c omplex membrane structures that par ticipate in autophag y is t hree-dimensional electron tomograph y , 74 , 75 and cryoelectron microsc op y ( Fig. 4 ). More sophisticated, cr y o -soft X-ra y tom ograph y (cr yo-SXT) is an emerg ing imaging technique used to visualize autophagosomes. 76 Cr y o -SXT extracts ultrastructural infor mation from whole, unstained mammalian ce lls as close to the “near - native” fully-hydrated ( living) state as possible. C orre lative studies combining cr yo-fluorescence a nd cryo-SXT wor kflow (cr yo-CLXM) have been appli ed to capture earl y a utophagosomes. Finally , although onl y as an indirect measurement, the comparison of the ratio of autophagosomes to autoly s osomes b y TEM can support alterations in autophag y id entified b y other procedure s. 77 I n thi s case it is important to always compa re samples to the control of the same cell type a nd in the same growth phase, as th e autophag osome/autol y s osome ratio varies in 37 a cell context-dependent fashion, de pending on their clearance activity. It may also be necessar y to distinguish autolysosomes from teloly sosomes/late secondar y l y sosom es (the former are actively engaged in deg radation, whereas the latter have reached an end point in t he bre akdown of lumenal contents and are a lso referred to as residual bodies) because l y sosome numbers ge nerall y increase when autophagy is induced. TEM observations of platinum-carbon replicas obtained by the freeze fracture technique can also suppl y useful ultrastructural information on the autophagic process. I n quickl y frozen and fracture d cells the fracture runs preferentiall y along the hydrophobic plane of the membranes, allowing c haracterization of the limiting membranes of the different t y pes of autophagic vacuoles and visualization of their limited protein intramembrane particle s ( I MPs, or integral membra ne proteins ). Several studies have been carried out using this technique on y east, 78 as well as on mammalian cells or tissue, first on mouse exocrine pancreas, 79 then on mouse and rat liver, 80 , 81 mouse seminal ve sicle epithelium , 23 , 56 rat tumor and hea rt, 82 or canc er cell lines (e.g., brea st cancer MDA - MB -231) 83 to i nvestiga te the various phases of autophagosome matura tion , and to reveal usef ul details about the ori gin and evolution of their limiting membranes. 4, 84 - 87 The phagophore and the limiting membranes of autophagosomes contain few, or no detectable , I MPs ( Fig. 3A ,B ), when compared to other cellular membranes and to the membranes of lysosomes. In subsequent stages of the autophagic process the fusion of the autopha g osome with an endosome and a ly s osome results in i ncre ased densit y o f IMPs in the membrane of the formed autophag ic compartments (amphisomes, a utolysosomes; F ig. 3C ). 4, 23 , 78 - 81 , 88 , 89 Autolysosomes are de limit ed by a single membrane because, in addition to the engulfed material, the inner membra ne is al so degrade d b y th e l yt ic enz y mes. S imilarly , the limiting membrane of 38 autophagic bodies in yeast ( and presumably plants ) is also quickly broken down under normal conditions. Autophagic bodies can be stabilized, however, b y th e addition of pheny lmeth y lsulphon y lfl uoride (PMSF) or geneticall y b y the deletion of the yeast PEP4 gene (see The Cvt pathway, mitophagy, pexophagy and piecemeal microautophagy of the nuc leus and nucleophagy in yeast and filamentous fungi. ). Thus, another method to consider for monitoring autophagy in y east (and potentially in plants) is to count autophagic bodie s b y TEM usin g at least 2 time points. The advantage of this approach is that it can provide accurate information on flux even whe n the autophagosomes are abnormall y small. 90 , 91 Thus, although a high freque nc y of “ abnormal ” structures pr esents a c hallenge, TEM is still very helpful in anal y zin g a utopha gy. Cautionary notes: Despite the introduction of many new methods TEM mai ntains its special role in a utopha gy research. There are, however , difficulties in utilizing TEM. It is relatively time consuming , and needs technical expertise to ensure proper handling of samples in all stage s of preparation from fixation to sectioning a nd staining (contrasting). After all these criteria are met, we face the most important problem of proper identification of autophag ic structures. This is cruc ial for both qualitative and quantitative characterization , and needs considerable experienc e, even in the case of one cell type. The difficult y lies in the fac t that many subcellular c omponents may be mistaken for autophagic struc tures. For example, reviewers of manuscripts assume that almost all cy t oplasmic structures that , in the section plane , are surrounded by two (more or le ss) parallel membranes are autophagosomes. These include swollen mitochondria, plastids in plant cells, cellular interdig itations, endocy tosed apoptotic bodies, circular structures of lamellar smooth endoplasmic reticulum (ER) , and eve n areas surrounded by roug h ER . Endosomes, phagosomes and secretory vacuoles ma y h ave heteroge nous content tha t makes it possible to confuse them with autoly sosomes. Additional 39 identification problems may arise from damages caused by improper sa mple taking or fixation artifac ts . 45 , 46 , 92 , 93 Whereas fixation of in vitro samples is relativel y s traightforward, fixation of excised tissues requires care to avoid sampling a nonrepresentative , uninformative , or damaged part of the tissue. For instance, if 95% of a tumor is necrotic, TEM anal y sis of the necrotic core may not be informative, a nd if the sampling is fro m the viable rim, this needs to be specified when reported. Clearly this introduces the potential for subjectivit y because reviewers of a paper cannot request multiple image s with a careful statistical analysis with these types of samples. In addition, ex vivo samples are not t y pically randomized during processing, further comp licating the possibilit y of valid statistical anal y ses. Ex vivo t issue should be fixed immediate ly and systematically across samples to avoid changes in autophag y that ma y occur simply due to the elapsed time ex vivo. It is recommended that for tissue samples, perfusion fix ation should be used when possible. For y east, rapid freezin g tec hniques such as high pressure freezing followed by free ze substitution (i.e., dehydration at low temperature) may be particularly useful. Quantification of a utophag y b y TEM morphomet ry has been rather controversial, and unreliable procedures still continue to be used. F or the principles of reliable quantification and to avoid misleading results, excellent reviews are a vailable. 9, 94 - 96 I n line with t he basic pr inciples of morphometry we find it necessar y to emphasize here some common problems with regard to quantification. Counting autophagic vacuole pr ofiles in sections of cells gives totall y unr eliable results, partly because both c ell areas and profile areas are variable and also because the freque nc y of section profiles depends on the size of the vac uoles. There are morphometric procedures to measure or estimate the size rang e and the number of spherical objects by profiles 40 in sections; 95 however, such methods have been used in autophag y res earch only a few times. 30 , 91 , 97 , 98 Proper morphometry de scribed in the cited reviews will give us data expressed in µm 3 autophagic vacuole/µm 3 cy toplasm fo r relative volume (also called volume fraction or volume density), or µm 2 autophag ic vacuole surface/µm 3 cytopla sm for relative surface (surface densit y ). Examples of actual morphometric measurements for the charac terization of autophagic processes can be f ound in several articles. 19 , 92 , 95 , 99 ,100 I t is appropriate to note here that a change in the volume frac tion of the au tophag ic compartment ma y come from 2 sources; from the real growth of its size in a given cy toplasmic volume, or from the decrease of the cytoplasmic volume itself. To avoid this so-called “ refe rence trap ,” the reference space volume can be determined by differe nt methods. 96 ,101 If differe nt magnifications are used for measuring the autophagic vacuoles and the cytoplasm (which may be practical when autophag y is less intense) correctio n factors should always be used. In some cases, it ma y b e prudent to employ tomographic reconstructions of the TEM image s to confirm that the autophagic compartments are spherical and are not being confused with interdigitations observed between ne ighborin g cells, endomembra ne cisternae or damaged mitochondria with similar appe arance in thin-sections (e.g., see ref. 102 ), but this is obviously a time -consuming approach requiring sophisticated equipment. In addition, interpretation of tomogra phic images can be problema tic. For exa mple, starva tion-induced autophagosomes should contain cy toplasm (i.e., c y tosol and possibly organelles), but autophagosome -related structures involved in specific type s of autophag y should show the sele ctive cytoplasmic target, but may be relativel y devoid of cy toplasm. Such p rocesses include sele ctive peroxisom e or mitochondria degradation (pexophagy or mitophagy, respectively), 103 ,104 ta rgeted degrada tion of 41 pathogenic microbe s (x enopha gy), 105 -110 a c ombination of xenophagy and stress-induced mitophagy , 111 as we ll as the y e ast bios y nthetic cytoplasm- to -vacuole targe ting (Cvt) pathwa y. 112 Further more, some pathogenic microbes express membrane-disrupting factors during infection (e.g., phospholipases) that disrupt the normal double-membrane architec ture of autophagosomes. 113 I t is not even clear if the sequestering compartments used for specific organelle degra dation or x enopha gy should be termed a utophagosomes or if alt ernate terms such as pexophagosome, 114 mi tophagosome and x enophag osome should b e used, even though the membrane a nd mechanisms involved in their formation ma y be identical to those for starvation- induced autophagosome s ; for example, the double-membrane vesicle of the Cvt pathway is referre d to as a Cvt vesicle . The confusion of heterophag ic structures with autophagic ones is a major source of misinterpretation . A prominent example of this is related to apoptosis. Apoptotic bodies from neighboring cells are readil y phagocytosed by sur viving c ells of the same tissue. 1 15,1 16 Imme diatel y after phagocy tic uptake of apoptotic bodies, phagosomes ma y have double limiting membranes. The inner one is the plasma membrane of the apoptotic bod y a nd the outer one is that of the phagocy tizing cell. The earl y hete rophagic vac uole formed in this wa y ma y a ppear similar to an autophagosome or, in a later stage, an early a utol y sosome in that it contains recognizable or identifiable cy toplasmic material. A major difference, however, is that the surrounding membrane s are the thicker plasma membrane t y pe, rather than the thinner sequestration membra ne t y pe (9-10 nm, versus 7-8 nm, respectivel y ). 93 A g ood feature to distinguish betwee n autophagosomes and double plasma membrane-bound structures is the lac k of the distended empty space (charac teristic for the sequestration membranes of autophagosomes) be tween the 2 membranes of the phag oc y tic vacuoles. In addition, engulfed 48 evident in those cells that show low leve ls of LC3-II (based on western blotting) because of an intense autophagy f lux that consumes this protein , 146 or in cell lines having high leve ls of LC3- II that are tumor-derived, such as MDA- MB -231. 145 Converse l y , without careful quantifica tion the detectable formation of LC3-II is not sufficie nt evidence for autophagy. For example, homoz y gous deletion of Becn1 does not prevent the formation of LC3-II in embr y o nic stem cells even though autophagy is substantially reduced, whereas deletion of atg5 results in the complete absence of L C3-II (see Fig. 5, panel A and supplemental data in ref . 147 ). T he same is true for the ge neration of Atg8 – PE i n y east in the absence of VPS30/ ATG6 (see Fig. 7 in ref. 148 ). Thus, it is important to remember that not all of the autophagy-related proteins are required for Atg8/LC3 processing, including lipidation. 148 Vaga ries in the detection and a mounts of L C 3- I ve rsus LC3- II present technical problems. For example, LC3-I is ver y abundant in brain tissue, and the intensity of the LC3- I band ma y obs cure detection of LC3- II , unless the polyacrylamide crosslinking density is optimized , or the membra ne fraction of L C 3 is first separated from the cy tosolic fraction . 42 Converse l y , certain cell lines have much less visible LC3- I compared to LC3-II . I n addition, tissues may have as y nchronous and heterogeneous cell populations, and this variability may present challenges when analy zing LC3 b y western blotting. Second, L C3 -II also associates with the membranes of nonautophagic structure s. For example, some members of the  -pr otocadherin famil y und ergo c lustering to form intracellular tubules that emanate from ly sosomes. 149 L C3- II is recruited to these tubules, where it appears to promote or stabilize membrane e xpansion. Furth ermore , LC3 can be recruited directly to apoptotic cell-containing phag osome membranes, 150 , 151 macropinosomes, 150 the parasitophorous vacuole of Tox oplasma gondii , 152 and single- me mbrane e ntotic vacuoles, 150 as we ll as to bacteria-containing phagosome membranes under certain immune activating conditions, for 49 example, TLR-media ted stimulation in L C3-associated phagoc y tosis . 153 , 154 I mport antly, LC3 is involved in secre tor y tr afficking as it has been associated w ith secretor y granules in mast ce lls 155 and PC12 hormone secreting ce lls. 156 LC3 is also detec ted on secretor y l y sosomes in osteoblasts 157 and in amphisome-like structure s involved in mucin secretion b y goblet cells. 158 Therefore, in studies of infection of mammalian cells by bacterial pathogens , the identity of the LC3-II labelled compartment as an a utophagosome should be confirmed b y a s econd method, such as TEM. It is also worth noting that autophagy induced in response to bacterial infection is not directed solely against the bac teria but can also be a response to remnants of the phagocytic membrane. 159 S imilar cautions apply with re gard to viral infection . For example, coronaviruses induce autophagosome s during infection through the expression of nsp6; however, coronaviruses also induce the for mation of double-membrane vesicles that are coated with LC3-I, a nonlipidated form of L C3 that pla y s an autophagy-independent role in viral replication. 160 , 161 Along these lines, during herpes simplex virus (HSV) infection, a n LC3 + autophagosome-like organelle that is derived from nuclear membra nes and that contains viral proteins is observed, 162 whereas influenza virus A, via a LIR motif in its M2 protein, direc ts LC3 to the plasma membrane. 163 More over, in vivo studies have shown tha t coxsackievirus (an enterovirus) induces formation of autophagy-like vesicles in pancre atic acinar cells, together with extremely large autophagy-related c ompa rtments that have been termed megaphagosomes ; 164 the absence of Atg5 disrupts viral replication and preve nts the formation of these structures. 165 Third, caution must be exercised in ge neral when evaluating LC3 by western blotting, and appropriate standardization controls are necessar y . For example, L C3 -I ma y b e less sensitive to detection by certain anti -LC3 a ntibodies. Moreover, L C3 -I is more labile than L C3- II , being more sensitive to freezing-thawing and to degradation in SDS sample buffer . Therefore , fresh 50 samples should be boiled and a ssessed as soon as possible and should not be subjected to repea ted freeze-thaw c y cles. A general point to consider when e xamining transfected cells concerns the efficiency of transfection. A western blot will detect L C3 in the entire cell population, including those that ar e not transfected. Thus, if transfection efficienc y is too low, it may be necessary to use methods, such as fluorescence microscop y , that allow autophag y to be monitored in single cells. The critical point is that the ana l y sis of the gel shift of transfected LC3 or GFP-LC3 can be e mplo y ed to follow LC3 lipidation onl y in highly transfect able cells. 166 When dealing with animal tissues, western blotting of LC3 should be performed on frozen biopsy samples homogenized in the pre sence of g eneral protease inhibitors (C. I sidoro, personal communica tion; see also Human ). 167 Cave ats regarding detection of L C3 b y w estern blotting have been covered in a re view. 24 For example, PVDF membranes may result in a st ronger LC3-II retention than nitrocellulose membranes, possibly due to a higher aff init y for hydr ophobic proteins ( F ig. 5B ; J. Kovsan and A. Rudich, personal communication), and Triton X-100 may not efficiently solubilize LC3-II in some s y stems. 168 Heating in the pre sence of 1% SDS, or analy sis of membrane fractions, 42 may assist in the detection of the lipidated form of this protein. This observation is particularly re levant for cells with a high nucleocy toplasmic ratio, such as ly mphoc y t es. Under these constraints, direct lysis in Lae mmli loading buffer, containing SDS, just before heating, grea tl y improves LC3 detection on PVDF membranes, espec iall y when working with a small number of cells (F. Gros, unpublished observations). 1 69 Perhaps one of the most important issues concerns the quantification of c hanges in LC3- II , because this assay is one of the most widel y used in the field, but is also prone to misinterpretation . The first version of these guidelines specifically stated that the levels of LC3- II should be compared no t to LC3-I, but to ac tin (e.g., ACTB). H ere we would m odify this to 51 include other appropriate “houseke eping” protei ns (HKPs); for e xample, in some cases (both in mammals and y east) actin levels decrease when autophagy is induced, so it would be more appropriate to determine the ratio of L C3- II (or Atg8 in yeast) to another HKP control. Along these lines, i t is also worth noting the use of Coomassie Brilliant Blue , P onceau Red or Stain- Free gels 170 to detec t total ce llular proteins as an alternative to standard loading controls suc h as detecting GAPDH, actin or tubulin , which are highly e xpressed and often overloaded on gels 171 . Depending on the experiment normalizing LC3-II to a housekeeping protein may still be valid , but there are situations where this t y p e of anal y sis can be misleading . Either method (i.e., comparison of LC3-II to LC3-I versus determining the ratio of LC3- II to a HKP ) has its potential advantage s and disadvantages. For example, if the amount of L C3 - I is hig h relative to LC3- II (as in brain tissues, where the L C3 - I signal can be overwhelming), it can be difficult to quantify the change in LC3- II relative to L C3-I. U nder such a scenario, it ma y be h elpful to use gradient gels to increase the separation of LC3-I from LC3- II and/or cut away the part of the blot with LC3- I prior to the detec tion of LC3- II . I t is also always critical to ensure that ex posures of we stern blots are not saturated. Conversely , b y i gnoring the level of LC3- I in fa vor of LC3- II normalized to a HKP , the researcher ma y miss part of the overa ll picture of the cellular autophagic response. Further more, since the d y n amic range of LC3 immunoblots are generall y quite limited, we think it is imperative that other assay s be used in parallel in order to draw valid conclusions about changes in autophagy activit y . Fourth, in mammalian c ells L C3 is expressed as 4 isoforms, L C3A, LC3B, LC3B2 and LC3C, 172 , 173 which exhibit different tissue distributions and different functions , but these are ye t to be fully e lucidated. A nother point of caution along these lines is that the increase in LC3A- II versus L C3B- II levels may not display e quiv alent changes in all organisms under autophagy- 52 inducing c onditions, and it should not be assumed that L C3B is the optimal protein to monit or. 174 A key technical consideration is that the isoforms may exhibit different specificities for antisera or antibodies. Thus, it is recommende d th at investig ators report exactl y the source a nd catalog number of the a ntibodies used to detect LC3 as this mi g ht help avoid discrepancies between studies. The commerc ialized anti -L C3B antibodies also recognize L C3A, b ut do not recognize LC3C, which shares less sequence homology. It is important to note that LC3C possesses in its primary amino ac id sequence the DYKD motif that is recognized with a high affinity by anti - FLAG antibodies. Thus, the standard a nti-F L AG M2 antibody can detect and immunoprecipitat e overexpressed LC3C, and caution has to be take n in ex periments using FLAG-tagged proteins (M. Biar d-Piechacz y k and L. Espert, personal communication). Note that according to Ensembl there is no LC3C in mouse or rat. In addition, it is important to keep in mind the other subfamily of Atg8 proteins, the GABARAP subfamily (see above). 122 , 175 Certain ty p es of mitophagy induced by BN I P 3L /N IX are highly depe ndent on GABARAP and less dep ende nt on LC3 proteins. 176 , 177 Furthermore, commercial a ntibodies for GABARAP L 1 also recog nize GABARAP , 121 w hich might lea d to misinterpretation of experiments, in particula r those using immunohistochemical techniques . Sometimes t he problem with cross-reactivity of the anti-GABARAPL1 antibody c an be overcome w hen anal y z ing these proteins b y weste rn blot beca use th e isoforms can be resolved during SDS-PAGE using high concentra tion (15% ) gels, as GABARAP migrat es faster than GABARAPL1 (M. Boyer-Guittaut, personal communication). Beca use G ABARAP a nd GABARAPL1 can both be proteol y ti cally processed and lipidated, genera ting GABARAP -I or GABARAPL1-I and GABARAP-II or GABARAPL1-II, respectively, this may lead to a misassignment of the different bands. A s soon as h ighly spec ific antibodies that are able to 53 discriminate between GABARAP and GAB ARAPL1 become available, we strongl y advise their use; until then, we advise caution in interpre ting results based on the detection of thes e prote ins by western blot. In g eneral, w e advise caution in c hoosing a ntibodies for w estern blotting and immunofluorescence experiments and in interpreting results based on sta ted affinities of antibodies unless these have been clearly deter mined. As with any we stern blot , proper me thods of quantification must be used, which are, unfortunately, often not well disseminated; readers are referre d to an excellent paper on this subject (see ref. 178 ). Unlike the other members of the GAB ARAP family, almost no information is available on GABARAPL3, perhaps because it is not yet possible to differentiate betwe en GABARAPL1 and GABARAPL3 proteins, which have 94% identity. As state d by the laborator y that d escribed the cloning of the human GABARAPL1 and GABARAPL3 genes, 175 their expression patterns are a pparentl y identic al. I t is worth noting that GABARAPL3 is the only ge ne of the GABAR AP subfamily that seems to l ack a n ortholog in mice. 175 GABARAPL3 mig ht therefore be considered as a pseudogene without an intron that is derived fr om GABARAPL 1 . Hence , until new data are published, GABARAPL3 shoul d not be considered as the fourth member of the GABARA P family . F ifth , in non-mammalian species, the disc rimination of Atg8 – PE from the nonlipidated form can be complica ted b y their n early identical SDS-PAGE mobilities and the presence of multiple isoforms (e.g., there are 9 in Arabidopsis ). In yea st, it is possible to resolve Atg8 (the nonlipidated form) from Atg8 – PE by including 6 M urea in the SDS-PAGE separating gel, 179 or by using a 15% resolving gel without urea (F. Re ggiori, personal communication). Similarly, urea c ombined with prior trea tment of the samples with (or without) phospholipase D (that will remove the PE moiety) can often resolve the ATG8 spec ies in plants. 180,181 It is also possible to label cells with ra dioactive ethanolamine, followed b y auto radiogra ph y to i dentify Atg8 – PE, and 54 a C-terminal peptide can be ana l y z ed b y mass spe ctrometry to identif y the lipid modification at the terminal g l y cine residue. Special treatments are not neede d for the separation of mammalian LC3-I from LC3- II . Sixth, it is important to kee p in mind that ATG8 , and to a lesser extent LC3 , undergoes substantial transcriptional and posttranscriptional regulation. Acc ordin gly , to obtain an accurate interpretation of Atg8/LC3 protein levels it is also necessar y to monitor the mRNA levels. Without anal y zin g the c orresponding mRNA it is not possible to discriminate between changes that are strictly reflec ted in altered amounts of protein versus those that are due to changes in transcription (e.g., the rate of transcription, or the stabilit y of the message) . For example, in cells treated with the calcium ionophore A23187 or the ER calcium pump blocker thapsigargin, an obvious correlation is found betwee n the time-de pendent increase s in LC3 -I and LC3-II protein levels, as well as with the observe d increase in L C3B mRNA levels. 182 Seventh, L C3- I ca n be full y degraded by the 20S proteasome or, more problematically, processed to a form appearing equal in size to L C 3 -II on a western blot (LC3- T) ; L C 3-T was identified in HeLa cells and is devoid of the ubiquitin conjug ation domain, thus lacking its adaptor function for a utophag y .{[ Gao, 2010 #3508} Finally , we would like to point out that one general issue with regard to a n y assay is that experimental manipulation could introduce some type of stress — for example, mechanical stress due to lysis, temperature stress due to heating or cooling a sample, or ox idative stress on a microscope slide, whic h could lead to po te ntial artifacts including the induction of autophagy. 183 Special care should be taken with ce lls in suspension, as the stress resulting from centrifugation can induce autophag y . This point is no t intende d to limit the use of any specific me thodology, but rather to note that there are no perfect assay s. Therefore, it is important to verif y that the 55 positive (e.g ., treatment with rapamyc in, torin1 or other inducers) and negative (e.g., inhibitor treatment) c ontrols behave as expected in an y assay s bein g utilized. Similarly, plasmid transfec tion or nucleofection can re sult in the potent induction of autophagy (based on increases in LC3-II or SQSTM1/ p62 degra dation). I n some cell types, the amount of autophagy induce d b y transfec tion of a control empt y vector may be so high that it is virtuall y im possible to exami ne the effect of e nforced gene expression on autophag y ( B. Levine, pe rsonal communication). I t i s thus advisable to perfor m time course experiments to determine when the transfection effect returns to acceptably low levels and to use a ppropriate time-matched transfection controls (see also the discussion in GFP-Atg8/LC3 fluorescence microscopy ). This effect is ge nerall y not observed with siRNA transfec tion; however, it is an issue for plasmid ex pression construc ts including those for shRNA and for viral delivery sy s tems . The use of endotox in-free DNA reduce s, but does not eliminate, this problem. I n many cells the cationic polymer s used for DNA transfec tion, such as liposomes and pol y plex , induce large tubulovesicula r autophagosomes (TVAs) in the a bsence of DNA. 184 These structure s accumula te SQSTM1 and fuse slowly with lysosomes. Interesting l y , these TVAs appear to reduce gene delivery, which increa ses 8-10 fold in cells that are unable to make TVAs due to the absence of A TG 5. Finally , the precise composition of media components and the density of cells in culture can have profound effects on basal autophagy levels and may need to be modified empirically depe nding on the cell lines being used. Along these lines various ty pes of media, in particular those wit h differe nt serum levels (rang ing from 0-1 5%), may ha ve profound effects with regard to how cells (or organs) perce ive a fed versus starved state. Fo r example, normal ser um contains significant levels of cy tokines and hormones that likel y reg ulate the basal levels of autophagy and or its mod ulation by a dditional stress or stimuli; thus, the use of dialy z ed serum might be an alterna tive for these 56 studies. For these rea sons , the ce ll culture conditions should be full y described. It is also important to specify durati on of autophagy sti mulation, as long-term autophag y can modif y signal tra nsduction pathwa y s of importance in cell survival. 185 Conclusion : Atg8/ L C3 is o ften a n excellent marker for autophag ic structures ; however, it must be kept in mind that there are multiple LC3 isoforms, there is a second family of mammalian Atg8-like proteins (GABARAPs), and antibody affinity (for LC3 -I versus LC3-II) and specificity (for e xample, for LC3A versus L C 3B) must be considered and/or deter mined. Moreover, L C3 levels on their own do not address issues of autophagic flux. Finall y , even when flux assays are c arried out, there is a problem with the limited d y namic ran ge of LC3 immunoblots; accordingly , this method shoul d not be used by itself to a nal yze c hanges in autophagy. b. Turnover of LC3-II/Atg8 – PE . Autophagic flux is often inferred on the basis of L C3 -II turnover , mea sured b y western blot ( Fig. 5C ) 145 in the pr esence and absence of lysosomal, o r vacuolar degra dation. Ho wever, it should be cautioned that suc h LC3 assays are merely indicative of autophag ic “carrier flux”, not of actual autophagic carg o/s ubstrate flux. I t has, in fact, been observed that in ra t hepatocytes, an autophagic-l y sosomal flux of LC3-II can take place in the a bsence of an accompanying flux of cy tosolic bulk car go (N Engedal and PO Seglen, unpublished observations). The re levant parameter in L C 3 assa y s is the dif fere nce in the amount of L C3- II in the presence and absence of saturating levels of inhibitors, which can be used to examine the transit of L C3 -II throug h the autophagic pathwa y ; if flux is occurring, the amount of LC3-II will be higher in the presence of the inhibitor. 145 Ly sosomal degradation can be prevented through the use of protease inhibitors (e.g ., p epstatin A and E-64d), compounds that neutralize 57 the lysosomal pH su c h as bafilom y cin A 1 , chloroquine or NH 4 Cl , 14 ,129,186, 187 or by tr eatment with agents that block fusion of autophagosomes with lysosomes (note that bafilom y cin A 1 will ultimately ca use a fusion block as well as neutralize the pH, 188 but the inhibition of fusion may be due to a block in ATP2A/SERCA activity 189 ). 190 Alternatively , knocking down or knocking out LAMP2 re presents a genetic approach to block the fusion of autophagosomes and l y sosomes (for example, inhibiting LAMP2 in my eloid leukemic cells results in a marked increase of GFP - LC3 dot s and e ndogenous L C3-II protein compared to control cells upon autophag y induction during mye loid differentiation [ M.P. Tschan, unpublished data]). 191 This approac h, however, is only valid whe n the knockdown of L AMP2 is directed against the mRNA reg ion specific for the LAMP2B spliced varia nt, as targeting the region c ommon to the 3 variants would also inhibit chaperone-mediated autophagy, which may result in the compensatory upregulation of macroautophagy . 77 , 192, 193 Increased levels of L C 3- II in the presence of l y sos omal inhibition or interfe ring with autophagosome-lysosome fusion alone (e.g., with bafilomy cin A 1 ), may be indicative of autophagic carr ier flux, but t o assess whe ther a particular treatment alters this flux, the treatment plus bafilomyc in A 1 must be compared with results obtained with treatment alone as we ll as with bafilomyc in A 1 alone. An additive or supra-additive effect in LC3- II levels ma y indi cate that the treatment enha nces autophagic flux ( Fig. 5C ). Moreover, higher LC3-II levels with t rea tment plus bafilomyc in A 1 compared to bafilom y cin A 1 alone ma y indicate that the treatment increases the synthesis of autophagy -related m embra nes. I f the treatment by itself increase s LC3- II levels, but the treatment plus bafilomyc in A 1 does not increase LC3-II levels compared to bafilom y cin A 1 alone, this may indicate that the treatment induced a partial block in autophag ic flux. A treatment condition increasing LC3- II on its own that has no difference in LC3-II in the presence 64 solution to this problem is to follow the fluorescence of a photoactivatable version of the fluoresce nt protein, 218 which allows this assay to be performed essentially as a pulse/chase analy sis. Another alternative is to follow flux usin g GFP -LC3 fluorescence by a dding l y sosomal protease or fusion inhi bitors to cells expressing GFP- L C3 and monitoring changes in the number of puncta. In this ca se, th e pre sence of lysosomal inhibitors should increase the number of GFP - LC3-positive struc tures, an d the absence of an effect on the total number of GFP-LC3 puncta or on the percentage of cells displ ay ing numerous puncta is indicative of a defect(s) in autophagic flux. 219 The combination of protea se inhibitors (to prevent the degradation of GFP) or compounds that modify ly s osomal pH such as NH 4 Cl or chloroquine, or compounds that block fusion of autophagosomes with ly sosomes such as bafilomy cin A 1 or others (e.g., vinblastine) may be most eff ective in preventing lysosome -dependent decre ases in GFP-LC3 puncta. However, because the stabilit y of GFP is affec ted by l y sosom al pH, r esearchers may a lso consider the use of protease inhibitors whether or not l y sosomo tropic compounds or fusion inhibitors are included. ( A lthough lysosomotropic compounds should help stabilize GFP by neutralizing the pH in the ly sosome the y do not have an immediate effect on lysosomal hydr olase activit y.) Cautionary notes: The GFP -Atg8 processing assay is used routinel y to mo nitor autophagy in yeast. One cave at, however, is that this assa y i s not alwa y s carried out in a quantitative manner . F or example, western blot exposures nee d to be in the linear range. Accordingly, an enzymatic a ssa y such as the Pho8∆60 assa y ma y be preferred (see Autophagic protein degradation ), 220,221 especially when the differences in autophagic activity need to be determined precisely (note that an equivalent assa y has not been deve loped for higher eukar y otic cells) ; however, as with any e nz y m e assa y, a ppropriate caution must be used regarding for example, 65 substrate concentra tions and linearit y. The Pho8∆60 also require s a control to verif y equal Pho8∆60 expression in the diffe rent genetic backgrounds or conditi ons t o be tested; 220 d iffere nces in Pho8∆60 ex pression potentially a ffect its activity and may thus cause misinterpretation of re sults. Another issue to keep in mind is that GFP-Atg8 processing corre lates with the surface area of the inner sphere of the autophagosome, and thus provides a smaller signa l than assa ys that mea sure the volume of the autophag osome. There fore, Pgk1 -GFP processin g, 28 or the Pho8∆60 assa y are generally more se nsitive assa y s. The main limitation of the GF P-LC3 processing assay in mammalian cells is that it seems to depend on ce ll t y pe and culture conditions (N. Hosokawa and N. Mizushima, unpublished data). Apparently, GFP is more sensitive to mammalian ly sosom al h ydrolases than to the degrada tive milieu of the yeast vacuole or the l y sosomes in Drosophila . Alte rnatively, the lower pH of mammalian lysosomes relative to that of the yeast vac uole ma y contribute to differences in detecting free GFP. Under ce rtain conditions [such as Earle’s balanced salt solution (EBSS)- induced starva tion] in som e ce ll lines, when the l y sosomal pH becomes particularl y low, free GFP is undetectable because both the L C3 - II and free GFP fragments are q uickly degrade d. 216 Therefore, if this method is used it should be accompanied by immunoblotting and includ e controls to address the stability of nonlysosomal GFP such as GFP-LC3-I. It should also be noted that free GFP can be detected when cells are treated with nonsaturating dose s of inhibitors such as chloroquine, E-64d and bafilomycin A 1 . The saturating concentrations of these l y sosomal inhibitors vary in different cell lines, and it would be better to use a satura ting concentration of lysosomal inhibitors when perfor ming an autophagic flux assa y . 216 Therefore, c aution must be exercised in interpre ting the data using this assa y ; it would be helpful to combine a n analysis of 66 GFP-LC3 processing with other assa y s , such as the monitoring of endogenous LC3- II b y we stern blot. Along these lines, a caution concerning the use of the EGFP fluorescent protein for microscopy is that , this fluorophore has a relatively neutral pH optimum for fluorescence, 222 and its signal diminish es quickl y during live cell imaging due to the acidic environment of the lysosome. It is possible to circ umvent this latter problem by imaging paraformaldehyde-fix ed cultures that are maintained in a neutra l pH buffer, which retains EGFP fluorescence (M. Kleinman and J.J. Reiners, personal c ommunication). Alternativel y, it may be prefera ble to use a differe nt fluorophore suc h as monomeric re d fluorescent protein (mR FP) or mCherry , which retain fluore scence even at acidic pH. 223 On the one hand, a putative advantage of mCherry over mRFP is its enhanced photostabilit y and intensit y , wh ich are an order of magnitude higher ( and comparable to GFP), enabling acquisition of images at similar exposure setting s as are used for GFP, thus minimizing potential bias in interpre tation. 224 On the other hand, c aution is required when eva luating the localiz ation of mCherry fusio n proteins during autophagy due to the persistence of the mCherr y si gnal in acidic environments; all tagg ed proteins are prone to show enrichment in ly sosomes during nonspecific autophagy o f the cytoplasm, especially at higher expression levels. In addition, red fluore scent proteins (eve n the monomeric forms) ca n be toxic due to oligomer formation . 225 Dendra2 is an improved ve rsion of the green- to -red photoswitchable fluore sc ent protein Dendra, which is derived fr om the octocoral Dendronephthya sp . 226 D endra 2 is capable of irreversible photoconversion from a green to a r ed fluoresce nt form, but can be used also as normal GFP or RFP vector. This modified version of the fluorophore has certain properties including a monomeric state, low phototoxic activation and eff icient chromophore maturation, which make it suitable for real-time trac king of LC3 and 67 SQSTM1 ( Fig. 8 ; K. Kaarniranta, personal communication). Another alternative to mRFP or mCherry is to use the Venus variant of YFP, which is brighter than mRFP and less sensitive to pH than GFP. 227 The pH optimum of EGFP is important to consider whe n using GFP-LC3 c onstructs, as the original GFP-LC3 marker 228 uses the EGF P variant, which may result in a reduced signal upon the formation of amphisomes or autolysosomes. An additional caveat when using the photoactivatable c onstruct PA-GFP 222 is that the proce ss of activation b y photons may induce DNA damage, which c ould, in turn, induc e autophagy . Also, GFP is relatively resistant to denaturation, a nd boilin g for 5 min may be needed to prevent the folded pro tein from being trapped in the stac king gel during SDS-PAGE. As noted above ( see W estern blotti ng and ubiquiti n-like protein conjugation systems ), Atg4 c leaves the residue( s) that follow the C-terminal gl y cin e of Atg8/L C3 that will be conjugated to PE. Accordingl y , it is critical that an y chimeras be constructed with the fluorescent tag at the amino ter minus of Atg8/LC3. Finally , l y sosomal inhibition needs to be care full y c ontrolled. Prolonged inhibition of lysosomal hydrolase s (>6 h) is likel y to induce a sec ondary autophagic response triggered by the accumulated undigested autophagy cargo. This secondary autophagic response can c omplicate the analysis of the a utophag y flux, making it appear more vigorous than it would i n the absenc e of the ly sosomal inhibitors. Conclusion : The GFP-Atg8/L C3 processing assay , which monitors f ree GFP generated within the vacuole/ly sosome , is a convenient wa y to follow autophag y , but it does not work in all cell ty pes, and is not as e asy to quantify as enzyme-based assa y s. Furthermore, the assay 68 measures the flux of an autophag ic carrier, which may not necessarily be equivalent to autophagic cargo flux. d. GFP-Atg8/LC3 fluorescence microscopy . L C3B, or the protein tagged at its N terminus with a fluorescent protein such as GF P (GFP- L C3), has been used to monitor autophagy through indirect immunofluorescence or direct fluorescence microscopy ( Fig. 9 ), measured as an increase in punctate L C3 or GFP-LC3. 228 , 229 The detection of GFP-LC3/Atg8 is also useful for in vivo studies using transge nic organisms such as Caenorhabdi tis elegans , 230 Dictyostelium discoideum , 231 fi lamentous ascomy cetes, 232 -236 Ciona inte stinalis , 237 Drosophila melanogaster , 238-240 Arabidopsis thaliana , 241 Zea mays , 242 L eishmania maj or 243-246 and mice. 133 I t is also possible to use anti-LC3/Atg8 antibodies for immunocy tochemistry or immunohistochemistry (IHC) , 167,247-252 proce dures that have the advantages of detec ting the endogenous protein, obviating the need for transfection and/or the generation of a transgenic organism, as well as avo iding potential artifacts resulting from overexpression . F or example, high leve ls of overexpressed GFP- L C3 can result in its nuclea r localization, although the pr otein can still reloca te to the cytosol upon starvation. T he use of imaging cy tometr y allows rapid and quantitative measure s of the number of LC3 puncta and their relative number in individual or mixed cell t y p es, using computerized assessment, enumeration, and data display ( e.g., see ref s. 42 , 253 ). In this respect, the alterna tive use of an automated counting system may be helpful for obtaining an objective number of puncta per cell. F or this purpose, the WatershedCounting3D plug-in for Image J may be useful. 254 , 255 Monitoring the endogenous Atg8/LC3 protein obviousl y depends on the abilit y to detect it in the sy stem of interest , which is not alway s po ssible . I f th e endoge nous amount is below the 69 level of detection, the use of an e xogenous construct is warranted. In this case, it is important to consider the use of stable transformants versus transient transfe ctions. On t he one hand, stable transformants may ha ve reduced background resulting from the lower gene expression, and ar tifacts resulting from recent exposure to transfection reage nts (see below) are eliminated . Further more, with stable transformants more cells can be easily analy z ed because nearl y 100% of the population will express tagg ed LC3. On the other hand, a disadvantage of stable transfec tants is that the integration sites cannot always be pre dicted, and ex pression leve ls ma y not be optimal. Therefore, it is worth considering the use of stable episomal plasmids that avoid the problem of unsuitable integr ation. 223 A n importa nt advantage of tra nsie nt transfec tion is that this approach is bette r for ex amining the immediate effec ts of the transfected protein on autophagy ; however , the transient transfec tion approach restricts the length of time that the analy sis can be performe d, and consideration must be g iven to the induction of autophagy resulting from exposure to the transfec tion reagents (see below). One wo rd of caution is that optimizing the time of transient expression of GFP-L C3 is necessar y , as some cell t y p es (e.g., HeLa cells) may require 1 day for achieving optimal expression to visualize GFP-LC3 puncta, whereas neuronal ce ll lines such as SH- SY 5Y cells typica ll y need at least 48 h of expression prior to perfor ming GFP- LC3 punc ta anal y ses. In addition, a double transfection ca n be used (e.g., with GF P-LC3 and the protein of interest) to visuall y ta g the cells that express the prote in being examined. A disadvantage of transfecting GFP- L C3 with liposomes is that frequentl y it leads to an unstable eff icienc y of transfection, causing a reduction in the number of cells effec tivel y expressing GFP-L C3, and degrada tion of the plasmi d, thus decre asing the numbers of GFP-L C3 puncta. Stable ce lls lines ex pressing GFP- L C 3 can be generated using lentiviral s y stems and 70 efficie ntl y selected through antibiotic re sistance leading to uniform and prolonged expression levels. These stable cell lines ar e sensitive to autophagy inducers as measured b y the LC3- II /LC3- I ratio b y w estern blot, and also show increased number s of c y topl asmic GFP -LC3 puncta upon autophagic stimuli (unpublished results R. Muñoz -Moreno, R. I . Galindo, L. Barrado-Gil and C. Alonso). In conclusion, there is no simple rule for the use of stable versus transient transfections. When stable transfections are utilized throug h a nonlentiviral system it is worthwhile screening for stable clones that g ive the best signal to noise ratio ; when transient tran sfec tions are used, it is worthwhile optimizing the GFP-LC3 DNA concentration to give the best signal to noise ratio. In clones, the uniformity of ex pression of GF P- L C3 facilitates “thresholding” when scoring puncta - positive cells (see be low). However, th ere is also a need to be a wa re that a single cell clone ma y not be representative of the overall pool . Usin g a pool of multiple selected clones ma y reduce artifac ts that can arise from the selection and propagation of individual clones from a single transfec ted cell (although the use of a pool is also problematic as its composition will change over time). Another possibility is using FACS sorting to select a mixed stable population with uniform GFP-LC3 expression levels. 256 Optimization, together with including the appropriate controls (e.g., tra nsfecting GFP- LC3 G120A as a negative control), will help overcome the effects of the inhere nt variabilit y in these analyses. For accurate interpretations, it is also important to assess the level of overe xpression of the GFP- L C 3 constructs relative to endogenous LC3 by western blot. An additional use of GFP-LC3 is to monitor colocalization with a targ et during autophagy-related processes such as organelle degradation or the sequestration of pathogenic microbes. 257 - 259 Pre incubation of ce lls stably ex pressing GFP-LC3 with leupeptin can help 71 stabilize the GFP-LC3 signal during fluorescence microscop y , especiall y u nder conditions of induced autophagic flux. L eupeptin is an inhibitor of lysosomal cy steine and serine proteases and will therefore inhibit de grada tion of membrane-conjugated GFP-LC3 that is present within autolysosomes. Cautionary notes: Quantification of autophag y b y measuring GF P- LC3 puncta (or L C3 by immunofluore scence) can , depending on the method used, be more tedious than monitoring LC3-II b y western blot; however, the former ma y be more sensitive and quantitative. I d eally, it is preferable to include both assa y s and to compare the 2 sets of results. In addition, if GFP- L C3 is being quantified, it is better to determine the number of punc ta corresponding to GFP- L C3 on a per ce ll basis (or per cell area basis) rath er tha n simpl y the total number (or percentage) of cells displaying punc ta. This latter point is critical because , even in nutrient-rich conditions, cells display some ba sal level of GF P-LC3 puncta. There are, however, practical issues with counting puncta manually and reliably , especially if there are large number s per cell . Nevertheless, manual scoring ma y be more accurate than rel y ing on a so ftware program, in which case it is important to ensure that only appropriate dots a re being counted ( applicable programs include ImageJ , Imaris, and the ope n-sour ce software CellProfiler 260 ). Moreove r, when autophagosome-l y sosome fusion is blocked, large r autophagosomes are detected, possibly due to autophagosome - autophagosome fusion , or to an inability to resolve individual autophagosomes whe n the y a re present in large numbers . Although it is possible to detect changes in the size of GFP-Atg8/LC3 puncta by fluore scence mi crosc op y , it is not possible to correlate size with autophag y activit y without additional assay methods. Size determinations can be problematic b y fluor escence microscopy unless careful standardization is carr ied out , 261 and size e stimation on its own without considering punc ta number per cell is not recomme nded as a method for monitoring 72 autophagy ; however, it is possible to quantif y the fluoresc ence intensity of GFP-At g 8/LC3 at specific punc ta, which does provide a valid measure of protein recruitment. 262 In addition to autophag osome size, the number of puncta visible to the e y e will also be influenced by both the level of e xpression of GFP -L C3 in a given cell (an issue that can be avoided by ana l y zing endogenous LC3 by immunofluorescence) and b y the exposure time of the microscope, if using widefield microscopy . Anoth er way to account for differential GFP- L C3 expression levels and/or exposure is to normalize the intensity of GFP- L C3 present in the puncta to the total GFP-L C3 intensit y in the cell. This ca n be done eithe r on the population l evel 263 or individual cell level. 256 In many cell ty pes it may be possible to establish a threshold value for the number of punc ta per cell in conditions of “low” and “high” autophag y . 264 This can be tested empirically b y exposing cells to autophag y-inducing and -blocking agents. Thus, cell populations showing significa ntl y greater proportions of cells with autophagosome numbers higher than the threshold in perturbation conditions compared to the control cells could provide quantitative evidence of altered autophag y . I t is then possible t o score the population as the percentag e of cells display ing numerous autophagosomes. This approac h will onl y be feasible if the background number of puncta is relatively low. For this method, it is particularl y important to count a large number of cells and multiple representative sections of the sample . T y pi call y , it is appropriate to sc ore on th e orde r of 50 or more cells , preferabl y in at least 3 different trials, depending on the particular system a nd experiment , but the critical point is that this determination should be based on statistical power anal y sis . Accordingly , high-content imaging analy sis methods enable quantification of GFP -LC3 puncta (or overall fluorescence intensity) in thousands of cells per sa mple (e .g. see refs. 204 ,217,265 ). When using a utomated analysis methods, care must be taken to manuall y evaluate parameters used to establish background threshold 73 values for different trea tment conditions and cell types , particularl y as m an y s ystems image at lower magnifications that may be insufficient to resolve individu al puncta . Another note of caution is that treatments affecting cell morphology, leading to the “rounding - up” of cells for example, can re sult in apparent changes in the number of GFP -L C3 puncta per cell. To avoid misinterpretation of re sults due to such p otential artifacts, manua l review of cell images is highly recommende d. I f cells are rounding up due to apoptosis or mitosis, it is easy to automaticall y remove them fr om anal ysis based on nuclear morphology (using DAPI or Hoechst staining) or cell roundne ss . I f levels of autophag y in the rounded up cells are of particular intere st, images can be acquire d as z -stacks and either a nal y zed as a z -series or pr ocessed to generate maximum projection or extended depth of field images and than analy z ed. 266 To allow comparisons by other researchers attempting to repeat these experiments, it is critical that the authors also specify the baseline number of puncta that are used to define “normal” or “low” autophagy. Furthermore, the cells should be counted using unbiased procedures (e .g., using a random start point followed by in clusion of all ce lls at regular intervals), and sta tistical information should be provided for both baseline and altered conditions, as these assay s can be highl y variable. One possible method to obtain unbiased counting of GFP - LC3 puncta in a larg e number of cells is to perform mult ispectra l imaging flow cy tometr y (see Autophagic flux determination using flow and mult ispectral imaging cytometry ). 267 Multispectral imaging flow cy tometr y allows character ization of single cells within a population b y assessin g a combination of morphology and immunofluorescence patterns, thereby pro viding statistically meaningful data. 268 This method can a lso be used for endogenous LC3, and , therefore, is useful for non-tra nsfected primar y cells. 269 For adhere nt cell cultures, one caution for flow cytometry is that the techniques necessary to produce sing le cell su spensions can cause signific ant injur y to 80 autophagosomes followed by increases in late autophagosomes as an additional assurance that flux has been maintained. 299 In addition, this method can be used to monitor autophag y in high- throughput drug screening studies. 297 The quantific ation of “ yellow onl y ” (w here the y ellow signal results from merging the red a nd green channels) and “red only” dots in a stable tandem - fluoresce nt LC3-reporter cell line can be automated b y a Cellomics microsc ope that ca n be used to assess a huge population of cells (1,000 or more) over a large number of random fie lds of view. 196,300 Notably , o rganelle-specific variations of the tandem mRFP/mCherry-GFP reporter system have successfully bee n used to analyze selective type s of autophagy, such as pexophagy 301 and mitophagy 302 in ma mmalian cells. An alterna tive dual fluorescence assa y involves th e Rosella pH biosensor. This assay monitors the uptake of mater ial to the l y sosome/vac uole and complements the use of the tandem mRFP/mCherry- GF P reporter. The assay is base d upon the gene ticall y en coded dual color- emission biosensor Rosella, a fusion between a relatively pH-stable fast-maturing RFP variant, and a pH-sensitive GFP variant. When targeted to specific cellular c ompartments or fused to an individual protein, the Rosella biosensor provides information about the identity of the cellular component being delivered to the va cuole/l y soso me for degradation. I mportantl y , the pH - sensitive dual color fluoresce nce emission provides information ab out the environment of the biosensor during autophag y of various cellular components. In yeast, Rose lla has been successfully used to monitor autophagy of c y tosol , mitochondria (mitophagy ) and the nucleus (nucleophagy ). 303 -305 Furthermore , the Rosella biosensor can be used as a reporter under various conditions including nitroge n depletion-dependent induction of autophagy. 3 03,3 04 The Rosella biosensor can also be expressed in mammalian cells to follow either nonselective autophagy (cy toplasmic turnover), o r mitophagy . 304 81 Cautionary notes: The us e of tande m mRFP/mCherr y -GFP-LC3/Atg8 reporters in live imaging e xperiments can be complicated b y the m otion of LC3/Atg8 puncta. As a consequence, conventional conf ocal microscop y ma y not allow visualization of colocalize d mRFP/mCherry - GFP puncta. I n this case, GFP colocalized puncta represent newly formed autophag ic structures whereas mRFP/mCherry-onl y pun cta are ambiguous. Spinning disk confocal microscopy or rapid acquisition times may be required for imaging tandem mR FP/mCherry - GFP proteins, although these techniques require a brighter fluorescent signal associated with what may be undesirably hig her levels of transgene expression. One solution is to use the mTagRFP- mWasabi-LC3 chime ra, 306 as mTagRFP is brig hter than mRFP1 and mCherr y , and mW asabi is brighter than EGFP (J. Lin, per sonal communication). Another possibilit y is to use fix ed ce lls; however, this presents an additional conce rn: The use of tandem m RFP/mCherry-GFP relies on the quenching of the GFP signal in the acidic autolysosome; however, f ixation soluti ons are often neutra l or weak bases, which will increase the pH of the entire cell. Accordingl y , the G FP signal may be restored after fixation ( F ig. 11 ), which would cause an underestimation of the amount of signa l that corresponds onl y to RFP (i.e ., in the autoly sosome). Thus, the tissue or cell samples must be properly processed to avoid losing the acidic environment of the autol y sosom es. In addition, there may be weak fluorescence of EGFP even in an acidic environment (pH between 4 and 5). 222 , 287 There fore, it ma y be d esirable to choose a monomeric green fluorescent protein that is more ac id sensitive than EGFP for assa y ing autopha g ic flux. Another ca ution in the interpretation of the tandem fluorescent marker is that colocalization of GF P and mRFP/mCherry might also be see n in the case of impaired proteolytic degrada tion within autol y s osomes or altered l y so somal pH. Finall y , expression of tandem mRFP-GFP-LC3 is toxic to some cancer cell lines relative to GF P-LC3 or RFP- L C3 (K.S. Choi, 82 personal communica tion). The c y totoxicit y o f DsRed a nd its variants such as mRFP1 is associated with downregulation of BCL2L1 (Bcl-x L ). 307 In contrast to mRFP-GFP-LC3, overexpression of mTagRFP-mWasabi-LC3 does not appear to be toxic to HeLa cells (J. L in, personal communica tion). The Rosella assay ha s not been tested in a wide range of mammalian cell types. Accordingly, the se nsitivit y and the specificit y of the assay must be ve rified independentl y until this method has been tested more extensively and used more widel y . Finally , i t ma y be desirable to capture the dynamic behavior of autophagy in real time, to ge nerate data revealing the rate of formation and clearance of autophagosomes over time, rather than single da ta points. For ex ample, by acquiring signals from 2 fluoresce nt constructs in rea l time, the rate of change in colocalization signal as a measure of the fusion rate and rec y clin g rate between autophagosomes and ly sosomes can be a ssessed. 308 Importantly, due to the integral dyna mic relationship of autophagic flux with the onset of apoptosis and necrosis , it is advantage ous to monitor cell dea th and autophagic flux paramete rs concomitantly over time, which FRET-based reporter construc ts mak e possible. 309 In addition, as the metabolic control of a utophag y is becoming increa singly clear, highlig hting a tight network between the autophagy machinery, e nerg y se nsing pathway s and the cell’s metabolic c ircuits, 310 , 311 mi tochondrial para meters suc h as fission and fusion rate as well as the cell’s ATP dema nd should be monitored a nd correlated with autophagic flux data. This will provide a better understanding on the variability of autophagy and cell dea th susceptibilit y . Conclusion : The use of tandem fluorescent constructs, which display different emission signals depending on the environment (in particular, GF P fluorescence is sensitive to an acidic pH), provides a convenient way to monitor autophagy flux in many cell t y p es. 83 f. Autophagic flux determination using flow and multispectral imaging c ytometry . Whereas fluoresce nce microscop y, in combination with novel autophagy prob es, has permitted single cell analy sis of autophagic flux , automation for a llowing medium- to high-throughput analysis has been challenging . A number of methods has been developed that allow the determination of autophagic flux using the fluoresce nce -ac tivated cell sorter (FACS), 1 87,268, 283 ,312- 314 and commercial kits are now available f or monitoring autophag y b y flow cy tometr y . Th ese approac hes make it possi ble to capture data or, in specialized instruments, high-content , multiparametric image s of cells in flow ( at rates of up to 1,000 cells/sec for im ag ing, and higher in nonimaging flow cy tometers ), and are particularl y use ful for cells that grow in suspension. Optimization of image analy sis permits the stud y of cells with heterogeneous LC3 punc ta, thus making it possible to quantify autophag ic flux accurately in situations that might perturb nor mal processes (e.g., microbia l infection). 314 , 315 Since EGFP-LC3 is a substrate for autophagic degrada tion, total fluores ce nce intensit y of E GF P -LC3 can be used to indicate leve ls of autophagy in living mammalian cells. 312 When autophagy is i nduced, the decrease in total cellular fluoresce nce can be pre cisel y qu antified in large numbers of cells to obtain robust data. In another approach, soluble EGFP-LC3-I can be depleted from the cell b y a brief saponin extraction so that the total fluoresc ence of EGFP- L C3 then represents that of EGFP-LC3- II alone ( F ig. 12A ). 282 , 283 Since EGFP-LC3 transfection typically results in high relative levels of EGFP- LC3-I , this treatment signi fica ntly reduces the background fluorescence due to non- autophagosome-a ssociated reporter protein. B y comparing treatments in the pre sence or absence of lysosomal degrada tion inhibit ors, subtle cha nges in the flux rate of the GFP- L C 3 reporter construct ca n be detected. I f it is not desirable to treat cells with ly sosomal inhibi tors to 84 determine rates of a utophagic flux, a tandem mRFP/mCherry-EGFP-LC3 (or similar) construct can also be used for autophag ic flux measurements in FACS ex periments (see Tandem mRFP/mCherry-GFP fluorescence mic roscopy ). 313 These methods, however, require the cells of interest to be transfected with reporter constructs. Since the sa ponin ex traction method c an also be combine d with int rac ellular staining for endoge nous LC3 protein, subtle changes in autophagic flux can be mea sured without the need for reporter transfections ( F ig. 12B ). Th u s autophagic flux can be investigated in a wide variety of cell ty pes and tiss ues by using eithe r immunoflu oresce nce or flow c y tometr y methods for detection of endoge nous L C3 due to the availability of commercially available antibodies. Cautionary notes: C are must be take n when apply ing flow c y tomet r y measurements to adhere nt cells, particularly neurons and other cells with interdig itated processes, as the preparation of sing le cell suspensions entail s significant levels of plasma membrane disruption and injury that can secondaril y induc e autophagy. Users of the saponin extraction method should carefully titrate saponin concentrations and times of trea tment to ensure specific extraction of L C3 - I in their sy stems. Also, it has been observed in some cell ty pes that saponin treatment can lead to non-autopha gic aggregation of LC3, 284 which should be c ontrolled for in these assays (see GFP-Atg8/LC3 fluorescenc e microscopy ). Cell membrane pe rmeabili zation with digitonin an d extraction of the nonmembrane- bound form of LC3 allows combined staining of membrane-associated LC3- II protein and any markers f or detection of autophagy in relation to other cellular events/processes. Based on this approac h , a method for monitoring autophagy in different stages of the cell cyc le was developed. 316 Thus, the p resence of basal or starvati on-induced autophagy is detected in G 1 , S, 85 and G 2 /M phases of the cell cycle in MEFs with dox y c y c line-re gulate d ATG5 expression. I n these experiments cells were gated based on their DNA content a nd the relative intensit y of G FP - LC3-II and LC3- II expression. This approach might also be used for the detection of autophagic flux in different stages of the cell cy cle or subG 1 apoptot ic cell population by measuring accumulation of L C3- II in the presence or absence of l y sosomal inhibitors. Although GFP-LC3 can be used as a reporter f or flow c y tometr y, it is more stable (which is not necessarily idea l for flux measurements) than GFP- SQSTM1 or GF P- NB R1 (NBR1 is a selective a utophagic substrate with structural similarity to S QSTM1 317 ). GFP- SQSTM1 display s the largest magnitude change followin g the induction of autophagy by amino acid deprivation or rapamyc in treatment, and ma y thus be a better marker for following autophagic flux b y this method (confirmed in SH-SY5Y neuronal cell lines stably expressing GFP - SQSTM1 ; E.M. Valente, personal communication). 318 Conclusion : Medium- to high-throughput analy sis of autophag y is possi ble using f low and multispectral imag ing cytometry ( F ig. 13 ). The advantage of this approach is that larger numbers of ce lls can be a nalyzed with regard to GFP-L C3 puncta, cell morpholog y and/or autophagic flux, and concomitant detection of surface markers can be included, potentially providing more robust data than is achieve d with other methods. A major di sadvantag e, however, is that flow cy tometr y on ly mea sures changes in total GFP -LC3 levels, which can be subject to modification by changes in transcription/translation or by p H, and this approach cannot accurately e valuate localization ( e.g ., to autophagosomes) or lipidation (generation o f L C3- II ) without further permeabilization of the cell. 86 g. Immunohistochemistry . I mmunodetection of ATG proteins (particularl y LC3 a nd BECN1) has been reporte d as a prognostic factor in various human carcinomas, incl uding lymphoma, 167 , 319 breast carcinoma, 320 endometrial a denocarcinoma, 321 , 322 head a nd neck squamous cell carcinoma, 323- 325 hepatoce llular carcinoma, 326,327 gliomas, 328 non-small ce ll lung carc inomas, 329 pancre atic 330 and colon adenocarcinomas, 331- 333 as well as in cuta neous and uveal melanomas. 334,335 Unfortunate ly, the reported changes often reflect overall diffuse staining intensity rather than appropriatel y compartmentalized puncta. Therefore, the observation of increased levels of diffuse L C 3 staining (which may reflect a decrease in autophagy) should not be used to draw conclusions that autophagy is increased in cancer or oth er tissue samples. Importantly , this kind of assa y should be performed as rec ommended b y the Reporting Recommendations for Tumor Marke r Prognostic Studies (REMARK). 336 A s we identify new drugs for modulating aut ophagy in clinical applications, thi s ty p e of information ma y prov e useful in the identification of subgroups of patients for targe ted therap y . 337 -339 In mouse and rat ti ssues, endogenous LC3, A TG 4B, and A TG 9A hav e been detec ted b y immnohistochemical analyses using both paraff in sections and cryosections. 251,340-342 When autophagosomes are a bsent, the localization pattern of L C3 in the cells of various tissues is diffuse and cy tosolic. Moreover, intense fibrillar y staining of L C3 is detectable along dendrites of intact neurons, whereas granular staining for LC3 appears mainly in the perikarya of ne urons in CTS D- or CTS B- and CTS L -deficient mouse brains. 251 L C3 puncta are also observed in mice in the peripheral ner ves, specifically in Schwann cells af ter neurodegenerat ion. 343 I n developin g inner ear and retinal tissue in chicken, BECN1 is detected by immunofluorescenc e ; in chick retina AMBRA1 is also detec ted . 344 - 346 Finall y , in non-mammalian vertebrates, BECN1 was detected dur ing follicular atresia in the ovar y of 3 fish species usin g pa raffin sections; a punctate 87 immunostaining for BECN1 was scattered throug hout the cytoplasm of the follicular cells when they were in intense pha gocy tic activity for yolk removal (E. Rizz o, unpubli shed re sults). Cautionary notes: One problem with L C 3 I HC is that in some tissues this protein can be localized in structures other than a utophagosomes. For example, in murine hepatoc y tes and cardiomyocytes under starved c onditions, endo ge nous L C3 is d etec ted not onl y in autophagosomes but also on lipid droplets. 347 I n neurons in ATG7-deficient mice, L C3 accumulates in ubiquitin- and SQSTM1 -positive agg regates. 348 Thus, immunodetec tion of LC3 in cytopla smic granules is not sufficient to monitor autophag y in vivo. Conclusion : I t has not been clearl y d emonstrated that I HC of ATG proteins in tissues corre sponds to autopha gy activit y , and this a rea of research needs to be further explored before we ca n make specific recommendations. 3. SQSTM1 and related LC3 binding protein turnove r assays . I n addition to L C3, SQSTM1 /p62 or other receptors such as NBR1, can also be used as protein markers, at least in certain settings. 24 ,349 For example, SQSTM1 can be detec ted as puncta by IHC in cancer cells, similar to LC3. 325 The SQSTM1 protein serve s as a link between LC3 a nd ubiquiti nated substrates. 69 SQSTM1 and SQSTM1 -bound poly ubiquitinated proteins bec ome incorpora ted into the completed autophag osome and are degraded in autol y sosomes, thus serving as a n index of autophagic deg radation ( F ig. 14 ). I nhibition of autophagy correlates with increased levels of SQSTM1 in mammals and Drosophila , suggesting that steady state levels of this protein reflect the autophagic status. 342 ,350-355 Similarly , d ecreased SQSTM1 levels are associated with autophagy ac tivation. Th e phosphory l ation of SQ STM1 at Ser403 appears to regulate its role in 88 the autophagic c learance of ubiquitinated proteins, and anti-phospho- SQSTM1 antibodies can be used to detect the modified form of the protein. 281 Cautionary notes: S QSTM1 changes can be cell ty pe and context specific. In some cell types, there is no c hange in the overall amount of SQSTM1 despite strong levels of autophagy induction, verified by the tandem mRFP/mCherry-GFP-LC3 reporter as well as Atg7- and lysosome-depende nt turnover of cargo proteins (C.T. Chu, personal observation). I n other contexts, a robust loss of SQSTM1 does not correlate with increased autophagic flux as assessed by a luciferase-based measure of flux ; 206 a decrease of SQSTM1 can e ven relate to a blocka ge of autophagy due to cleavage of the protein, toge ther with ot her a utophag y pro teins, by caspases or calpains. 356 SQSTM1 may be tr anscriptionally upreg ulated under certain conditions, 274 ,357- 360 further c omplicating the interpretation of results. For example, SQSTM1 upregulation, and at least transient increase s in the amount of SQSTM1, is seen in some situations where there is an increase in autophagic f lux . 361- 363 For example, during retinoic ac id-induc ed diffe rentiation of AML cells SQSTM1 is upregulated 358 with concomitant incre ased autophagy flux. 364 Activation of a sig naling pathwa y , e.g. RAF1/Raf-MAP2K/ MEK-MAPK/ERK, can also upregulate SQSTM1 transcription. 365 SQSTM1 mRNA is also upregulated following prolonge d starvation, which can restore the SQSTM1 protein level to that before starvation. 366 ,367 Another instanc e when both mRNA and protein levels of SQSTM1 are elevated even though autophagy flux is not impaired is observed in aneuploid human and murine cells that are generated by introduction of 1 or 2 extra chromosomes. 368 , 369 Thus, appropriate positive and negative controls are needed prior to the use of SQSTM1 as a flux indicator in a particular ce llular context , and we recommend monitoring the SQSTM1 mRNA level as part of a complete analysis . 89 Of interest, SQSTM1 hypere xpression at both gene and protein levels can be observed in muscle atrophy induce d b y c ancer, though not by glucocorticoids, suggesting that the stimulus inducing a utopha gy ma y also be relevant to the differe ntial regulation of a utophagy-related proteins (F. Penna and P. Costelli, unpublished observations). O ne solution to problems relating to variations in SQSTM1 expression levels is to use a Ha loTag ® -p62 (SQSTM1) chimera. 370 The chimeric prote in can be covalentl y labeled with HaloTag ® ligands, and the loss of signal can then be monitored without interference by subsequent change s in protein s y nthe sis. Similarly, a stable cell line expressing EGFP -tagg ed SQSTM1 under the control of an inducible promoter can be used to assess the ra tes o f SQSTM1 deg radation, taking into account the limitations out lined above (see Autophagic flux de termination using flow and multispectral imaging cytometry ). 318 A similar system exists in Drosophila in which a GFP-tagge d SQSTM1 can be expressed using the UAS-GAL4 sy stem. 3 71 I t is worth noting that tetracycline can reduce autophag y l evels; therefore, the appropria te control of only tetrac y cline addition has to be included if using an inducible promoter that re sponds to thi s drug . 372 Yet another solution is to employ a radioactive pulse- chase a ssa y to measure the rates of SQSTM1 degradation. 373 SQSTM1 contains an LC3 interacting motif as well as a ubiquitin binding domain , and appears to act by linking ubiquiti nated substra tes with the autophagic ma chinery. Nonetheless, it would be prudent to keep in mind that SQSTM1 contains domains that interac t with several signaling molec ules, 374 and SQSTM1 may be part of the mechanistic target of rapamycin (M TOR) complex 1 (TORC1) . 375 Thus, it may have additional functions that need to be considered with rega rd to its role in autophagy. In the context of autophagy as a stress response, the complexit y of using SQSTM1 as an autophagy marker protein is und erscored by its capacit y to modulate the NFE2L2/ NRF2 anti-oxidant response pathway throug h a KEAP1 binding 96 The role of AMPK in a utophag y is complex and highl y dependent on both cell t y p e and metabolic conditions. Furthermore, as noted above, there are 2 isoforms of the catal y ti c subunit, PRKAA1/AMPK  1 and PRKAA2/AMPK  2, and these may ha ve distinct effects with regard to autophagy (C. Koumenis, personal communication). In liver cells, AMPK suppresses autophagy at the level of carg o sequestration , as indicated b y the rapid sequestration-inhibitory effec ts of a variety of AMPK activators, whereas it appears to stimulate autophagy in many other cell types, including fibroblasts, colon carc inoma cells and skeletal muscle. 416 -425 Autophagy - promoting effects of AMPK are most evident in cells cultured in a complete medium with serum a nd amino acids, where cargo sequestration is otherwise largely suppressed. 422 Presumably , AMPK antagonizes the autophagy -inhibitor y e ffect of amino acids (at the level of phagophore assembly) by phosphory lating proteins involved in TORC1 signaling, such as TSC2 426 and RPTOR 427 as well the TORC1 target UL K1 (see below). 428 - 430 Compound C is an effective and widely used inhibit or of a ctivated (phosphory lated) AMPK. 431,432 However, be ing a non-specific inhibitor of oxidative phosphorylation, 433 , 434 this drug has bee n observed to inhibit autophagy under conditions where AMPK is already inactive or knocked out, 435 and it has even been shown to s timulate autophagy b y an AMP-independent mechanism. 434,436 Compound C thus ca nnot be used as a stand-alone indicator of AMPK involvement. TORC1 is an autophagy-suppressive regulator that integrates growth factor, nutrient and energy signals. In most sy stems, inhibition of MTOR leads to induction of autophagy, and AMPK activity is ge nerall y antagonistic toward MTOR function. TORC1 mediates the autophagy-inhibitory effect of amino acids, which stimulate the MTOR protein kinase through a Rag GTPase dimer. I nsulin and growth factors activ ate TORC1 throug h upstream kinases 97 including AKT / protein kinase B and MAPK1/3 (ERK2/1) when the energ y supply is suffic ient, whereas energy de pletion ma y induc e AMPK-mediated TORC1 inhibition and autophagy stimulation, for example, during glucose starvation. Amino acid starvation, on the other hand, can strongly induce autophagy even in cells completely lacking AMPK catalytic activity. 437 AMPK and TORC1 regulate autophagy through coordinated phosphorylation of U LK 1. U nder glucose starvation, AMP K promotes autopha gy b y directl y a ctivating U LK 1 through phosphorylation, a lthough the ex act AMPK-mediated UL K1 phosphorylation site(s) remains unclear ( Table 2 ). 425,428-430 Under conditions of nutrient suff iciency, high TORC1 activit y prevents U LK 1 activation by phosphorylating a lternate U LK 1 residues and disrupting the interaction between U LK 1 and AMPK. There are commercially available phospho-specific antibodies that recog nize different forms of U LK 1 . For e xample, phosphor y l ation at Ser555, an AMPK site, is indicative of increased a utophagy in response to nutrient stress, whereas Ser757 is targeted by MTOR to inhibit autophagy. Even the autophag y -suppressive effects of AMPK could, conce ivabl y , b e mediated through ULK1 phosphor y lation, fo r example, at the inhibitory site Ser638. 438 AMPK inhibits MTOR b y phosphor y latin g and activating TSC2. 439 Therefore, AMPK is involved in processes that synergize to activate a utophagy, by directly activating ULK1, and indirectly impairing MTOR-dependent inhibiti on of UL K1. The identification of U LK 1 as a direct target of TORC1 and AMPK represents a signific ant step toward the definition of new tools to monitor the induction of autophagy . However, further studies directed at identifying physiolog ical substrates of U LK 1 will be essential to understand how U LK 1 activation results in initiation of the autophagy prog ram. Alon g these lines, ULK1 phosphorylates AMBRA1, 440 and the MLCK-like protein Sqa, 441 as well as ATG13 , ATG 9 and RB1CC1/ FI P200. 371 ,442-445 Fur thermore, following amino-acid starvation or MTOR inhibit ion, 98 the activated UL K1 phos phory lates BECN1 on Ser14, enhancing the activity of the complexes containing ATG14 and PIK3C3/VPS34. This BECN1 phosphory lation b y ULK1 is required for full autophag ic induction. 4 46 I n addition, U LK 1 binds to, and phosphory lates, RP TOR, leading to inhibition of TORC1. 447 Further more, ULK1 itself appears to be able to mediate inhibitor y AMPK phosphorylation to ge nerate a negative feedback loop. 448 Note that caution should be taken to use appropriate inhibitors of phosphatases (e.g, sodium fluoride, and beta- glycerophosphate) in cell lysis buffer be fore anal yzing the phosphor y lation of AMPK and UL K1 at s er ine and threonine sites. TORC1 activity c an be monitored b y followin g the phosphor y lation of its subst rates, such as EIF4EB P1 / 4E-B P1/PHAS-I a nd RPS6KB / p70S 6 kinase or the la tter’s downstream target, RP S6, for which good commerc ial antibodies are available. 449 -451 I n mammalian cells, the analy sis should focus on the phosphorylation of S6K1 at Thr389, and EIF4EBP1 at Thr37 and Thr46, which are directly phosphory lated b y TORC 1. 452 The TORC1-dependent phosphory l ation of EIF 4EBP1 can be detected as a molecular mass shift by western blot. 451 Examining the phosphorylation status of RPS6KB and EIF4EBP1 may be a better method for monitoring TORC1 activity than f ollowing the phosphor y l ation of proteins such as RP S6, because the latter is not a direct substrate of TORC1 (although RP S6 phosphory lation is a good readout for RP S6K B 1/2 activities, which are directl y dependent on M TOR), and it can also be phosphorylated by other kinases such as RPS6KA/ RSK. Furthermore , the mechanisms that determine the se lectivit y as well as the sensitivity of TORC1 for its substrates seem to be dependent on the integ rity and confi g uration of TORC1. For e xample, rapamycin strongly reduce s RP S6K B 1 phosphorylation, whereas its effect on EIF4EBP1 is m ore va riable. In the case of rapa m y cin treatment, E I F4EBP1 can be phosphorylated b y TORC1 unti l rapamy cin 99 disrupts TORC1 dimerization and its integrity, wherea s RP S6K B 1 phosphor y lation i s quickly reduce d when rapam y cin simpl y interacts with MTOR in TORC1 (see Autophagy inhibitors and inducers for infor mation on catal y tic MTOR inhibitors such as torin1). 452 Since it is likely that other inhibitors, stress, and stimuli may also affect the integrity of T ORC1 , a decrease or increase in the phosphory l ation status of one TORC 1 substrate doe s not necessarily correlate with changes in other s, includin g UL K1. Therefore, reliable antiphospho- ULK1 antibodies should be used to directly examine the phosphor ylation state of ULK1, along with additional experimental approac hes to anal y z e the role of the MTOR complex in reg ulating autophag y . The TORC1-mediated phosphory lation of AMBRA1 on Ser52 has also been described as relevant to ULK1 reg ulation and aut ophagy induction. 4 40,453 I n line with what is described for ULK1, the antiphospho-AMBRA1 antibody, c ommercially available, could be used to indirectly measure TORC1 activity. 453 Activation/assembly of the Atg1 complex in y east (composed of at lea st Atg1 -Atg13- Atg17-Atg 31 -Atg 29 ) or the U LK 1 complex in mammals (U LK 1- RB1CC1/ FIP200-A TG 13- A TG 101) is one of the first steps of autophagy induction. Therefore, activation of this complex can be assessed to monitor autophagy induction. In y e ast, dephosphorylation of Atg 13 is associated with activation/assembly of the core complex, which can be a ssessed b y immunoprecipitation or western blotting. 454 - 457 I n addition, the autophosphorylation of Atg1 at Thr226 is require d for its kinase activity and for autophagy induction; this can be detected using phospho-specific antibodies, by immunoprecipitation or western blotting ( Fig. 15 ). 458 , 459 I n Drosophila , TORC1-dependent phosphorylation of Atg1 and Atg1-dependent phosphor y lation of Atg13 can be indirectly determined by monitoring phosphory lation -induced electromobility retardation (gel shift) of protein bands in immunoblot images. 371 ,4 60, 461 Nutritional starvation 100 suppress es TORC1-mediated At g 1 phosphor y latio n, 371,460 while stimulating Atg1-mediated Atg13 phosphorylation. 371 , 460,461 I n mammalian c ells, the phosphorylation status of U LK 1 at the activating sites (Ser317, 777, 467, 555, 637, or Thr574) or dephosphor y l ation at inactivating sites (Ser638, 757 ) can be determined using phospho-specific antibodies, 429 ,430 or by western blotting. 432,462 I n general, the core complex is stab le in mammalian c ells, although, as noted above, upstream inhibitors (MTOR) or activators (AMPK) ma y int eract dynamically with it, th ereby determining the status of autophagy. One additional topic that bears on UL K1 concerns the process of L C3 -associated phag oc y tosis (see Noncanonical use of autophagy-related proteins ) . LAP is a type of phag oc y tosis in macrophages that involves the conjugation of LC3 to single-membrane pathogen-containing phagosome s, a process that promotes phagosome ac idification and fusion with lysosomes. 153 Altho ug h ULK1 is not required for LAP, in this context it is important to note that UNC-51 (the Atg1 homolog in C. elegans ) is required for apoptotic c ell corpse cleara nce (a process corre sponding to L AP) during embr y onic development in wor ms , 463 although this process is mediated by LAP in mammals , 151 and does not require UNC-51 in C . elegans Q c ell neuroblasts. 464 I n human macrophages infected with Mycobacterium tuberculosis , it has bee n shown that MORN2 is recruited at the phag osome membrane containing M. tuberculosis to induce the recruitment of L C3, and subsequent ma turation in to phag ol y soso mes. In addition, MORN2 drives trafficking of M. tuberculosis to a single-membrane compartment. Thus, in certain conditions MORN2 can be used to help to make the distinction between autophagy and LAP. 465 Cautionary notes: A decr ease in TORC1 ac tivit y is a good measure for autophagy induction; however, TORC1 activit y do es not necessaril y preclude autophagy induction because 101 there are TOR-independent mecha nisms that induce autophagy both in mammals and y e ast . 466 -470 Along these lines, whereas in most sy stems inhibiti on of MTOR leads to the induction of autophagy, there are instances in commonl y used cancer cell lines in which MTOR appears to be a positive effector. 471 Als o, MTOR suppression does not always induce autophag y , su ch as when BECN1 underg oes inhibitor y phosphor ylation by the growth factor si g naling molecules EGFR and A KT . 472 ,4 73 I n adult skeletal muscle, active M TORC1 phosphor y lates ULK1 at Ser757 to inhibit the induction of autophagosome formation. Thus, induction of autophagy requires inhibition of MTORC1 and not of MTORC2 . 474,475 There is also evidence that inhibition of MTORC1 is not sufficient to maintain autophagy flux , but require s additional activation of FOXO transcr iption fact ors for the upregulation of autophagy ge ne expression. 420 I n addition, TOR C1 is downstream of A KT ; however, oxidative stress inhibits MTOR, thus allowing autophagy, despite the c oncomitant activation of A KT . 130 Al so, persistent MTORC1 inhibition can cause downregulation of negative feedback loops on I RS -MTORC2-AKT that results in the reac tivation of MTOR C2 under conditions of ongoing starvation. 1 85,366,4 76 Along these lines, both TORC1 and autophagy can be active in spe cific cell subpopulations of yeast colonies. 470 Thus, it is necessary to be cautious in deciding how to monitor the MTOR pathway, and to verify that the pathway being a nal y z ed displays MTOR-dependent inhibition. In addition, the regulation of autophagy by MTOR can be UL K1 -independent. During mycobacter ial infection of macrophages, MTOR induces the expression of MIR155 and MIR31 to sustain the activation of the WNT5A and SHH/sonic hedgehog pathwa y s. Toge ther, these pathways contribute to the expression of lipox yg e nases and downregulation of IF NG -induced autophagy. 477 Signaling pathwa y s can be monitor ed by western blotting , and TaqMan miRNA assays are ava ilable to detect these miRNAs. 102 One problem in monitoring a ssembl y o f the U LK 1 complex is the low abundanc e of endogenous U LK 1 in m any systems, which ma kes it difficult to detect phospho-U LK 1 b y western blot ana l y sis. In addition, Atg1/ULK1 is phosphor y lat ed by multiple kinases, and the amount of phosphory lation at different sites can increase or decrease during a utophag y induction. Thus, although there is an increase in phosphory lation at the activating sites upon induction, the overall phosphorylation state s of ULK1 and ATG13 are decrease d under conditions that lead to induction of autophagy; therefore, monitoring cha nges in phosphorylation by f ollowing molecular mass shifts upon S DS-PAGE may not be infor mative. I n addition, such phosphorylation/de phosphor y lation ev ents are expected to occur relatively earl y (1 -2 h) in the signaling ca scade of autophagy. Therefore, it is necessary to optimize treatment ti me conditions. Finally , in Arabidopsis a nd possibly other eukar y otes, the ATG1 and ATG13 proteins are targets of autophagy, whic h means that their levels ma y drop substantially under conditions that induce autophagic turnover. 215 At present, the use of Atg1 /U LK1 kinase activity as a tool to monitor autophagy is limited because only a few ph y siological substrates have been identified, and the importance of the Atg1/UL K1-dependent phosphor y lation has not been determine d. Nonetheless, Atg1/U LK 1 kinase activity appears to incre ase when autophagy is induced, irrespective of the pathway leading to induction. As additional phy siological substrates of Atg1 /U LK 1 are identified , it will be possible to follow their phosphor y lation in vivo as is done with anal y ses for MTOR. Nonetheless, it must be kept in mind that monitoring changes in the activity of Atg 1/ULK1 is not a direct assay for autophag y , although such changes may correlate with autophagy activity . Further more, in some cells U L K1 h as functions in addition to autophagy, such as in axonal transport and outgrowth, and its activity state ma y thus reflec t its role in these processes. 478 -4 83 103 Accordingly, other methods as described throughout the se g uidelines should also be used to follow autophagy directly . Finally , there is not a compl ete c onsensus on the specific residues of ULK1 that are targeted by AMPK or M TOR. Similarly , apparentl y contr adictory da ta have been published regarding the association of AMPK and MTOR with the ULK1 kina se complex under diffe rent conditions. Therefore, caution should be used in monitoring ULK1 phosphory lation or the status of ULK1 association with AMPK until these issue s are resolved. Conclusion : Assay s for Atg1/ULK1 ca n provide detailed insight into the induction of autophagy, but they are not a direct measurement of the process. Similarl y , since MTOR substrates such as RP S6K B 1 a nd E I F4EBP1 are not recommended readouts for autophagy, their analy s is need s to be combined with other assa y s that directl y moni tor autophagy activity. 5. Additional autophagy-related protein markers . Although Atg8/LC3 has been the most extensively used protein for monitoring autophagy, other proteins can also be used for thi s purpose. Here, we discuss some of the more commonly used or better-characterized possibilities. a. Atg 9. Atg9 is the onl y integral membrane Atg protein that is essential for autophagosome formation in all eukary otes . Mammalian A TG 9 di splays partial coloca lization with GFP- L C3. 484 Perhaps the most unique fe ature of Atg9, however, is t hat it localizes to multiple discrete punc ta, whereas most Atg proteins are detected primarily in a single punc tum or diffusel y within the cy tosol. Yeast At g 9 ma y cy cle between the phagophore assembl y site (PAS) and peripheral reservoir s; 485 the latter corre spond to tubulovesicular clusters that are precursors to the phag ophore. 486 Anterog rade movement to the PAS is dependent on Atg11, Atg23, Atg27 a nd actin. Retrogra de movement requires Atg1 -Atg13, Atg2-Atg18 and the PtdIns3K complex I . 487 104 Mutants such as atg1∆ accumulate Atg9 exclusively at the PAS, and this phenot y p e forms the basis of the transport of Atg9 after knocking out ATG1 (TAKA) assa y . 90 In brief, this is an epistasis analy sis in which a double-mutant strain is constructed (one of the mutations being atg1∆ ) that expresses Atg 9-GFP. If the second mutated gene encodes a protein that is needed for Atg9 a nterograde transport, the double mutant will displa y multiple Atg9-GFP puncta. In contrast, if the prote in acts along with or after Atg1, all of the Atg9-G FP will be confine d to the PAS. Monitoring the localization of A TG 9 has not been use d extensively in higher eukar y otes, but this protein display s the same t ype of dependence on Atg1/ULK1 and PtdI ns3P for c y cling as seen in y east, 484 ,487 sugg esting that it is possible to follow this ATG9 as an indication of ULK1 and ATG13 function. 444 b. Atg12 – Atg5. A TG 5, A TG 12, and A TG 16 L1 , associate w ith the phagophore and have been detected by f luorescence or immunofluorescence ( Fig. 16 ). 488 ,489 E ndoge nous ATG5, ATG12 or ATG16L1 puncta formation can be f ollowed to monitor autophagy upregulation. Under physiologica l conditions, the endogenous proteins are predominantly diffusel y distributed throughout the cy toplasm. Upon induction of autophagy, for e xample dur ing starvation, there is a marked increase in the proportion of cells with punctate ATG5, ATG12 and ATG16L1. Further more, upstream inhi bitors of autophag osom e formation result in a block in this starvation- induced puncta f ormatio n, and this assa y is ver y robust in some mammalian cells. Conversel y , downstream inhibition of autophagy a t the level of autophagosome elongation, such as with inhibition of L C3/GABARAP ex pression, results in an accumulation of the phag ophore- associated ATG5, ATG12, and ATG16L1 immunofluoresce nt puncta. 490 105 A TG 12 – A TG 5 conjugation has been used in some studies to measure a utop hagy . I n Arabidopsis and some mammalian cells it appears that essentially all of the A TG 5 and A TG 12 proteins exist in the conjug ated form and the expression levels do not change , at least during short-term starva tion. 180 ,488, 489 ,491 Therefor e, monitoring A TG 12 – A TG 5 conjugation per se may not be a useful method for following the induction of autophagy . It is worth noting, however, that in some cell lines free A TG 5 can be detected, 492 sugge sting that the amount of free A TG 5 may be ce ll line-dependent ; free ATG5 levels also var y in response to stress such as DNA damage . 493 One final parameter tha t ma y b e considered is that the total amount of the A TG 12 – A TG 5 conjugate may inc rea se following prolonged starvation as has been observed in hepatocytes a nd both mouse and human fibroblasts (A.M. Cuervo, personal communication; S. Sarkar, personal communication ). c. ATG14. Yeast Atg14 is the autophagy-specific subunit of the Vps34 complex I , 494 and a human homolog, named ATG14 / ATG14L/BARKOR, has been identified. 495 - 498 ATG14 localizes primarily to phagophores . The C-terminal fragment of the protein , named the B ATS domain, is able to direct GFP and BECN1 to autophagosomes in the context of a chimeric protein. 499 ATG14-GFP or BATS-GFP detected by fluorescence microscopy or TEM can be used as a phag ophore marker prote in ; however, ATG 14 is not localized ex clusively t o phagophores, as it can also be de te c ted on mature autophagosomes as well as the ER. 499 , 500 Ac cordingly, detection of ATG14 should be carried out in co mbination with other phagophore and autophagosome markers. A good antibody that can be used to detect endogenous ATG14 is now available commercially . 112 In addition, recent data illustrate tha t direct association betwee n ceramide, a tumor suppressor sphing olipid (generated b y CERS1/ ceramide sy nthas e 1) and LC3 -II targ ets damaged mitochondria for a utophagosomal digestion in response to ceramide stress, leading to tumor suppression. 538- 540 C era mide-L C3- II binding can be detected using anti-cerami de and a nti-LC3 antibodies by immunofluoresce nce a nd confocal microscopy, co-immunoprecipitation using anti- LC3 antibody f ollowed by liquid chromato g raph y-mass spectrometry (lipidomics), or labeling cells with biotin-sphingosine to g enerate biotin-ceramide, and immunoprecipitation us ing a vidin- columns followed by western blotting to detec t LC3 -II. I t should be noted that inhibitors of cera mide generation, mutants of LC3 with altered ceramide binding (F52A or I35A), and/or G120A should be used as negative c ontrols. Conclusion: Sphingolipids are bioactive molecules that play key roles in the regulation of autophagy a t various stages, including autolysosome morphogenesis, and/or targeting phag ophores to mitochon dria for degradation mainl y via sphin golipid- L C3 association. There are also studies that implicate a role for sphingolipids in the control of upstream signal transduction pathways to reg ulate autophag y via transcriptional and/or translational mechanisms. 539 7. Transcriptional , transla tional and posttranslational regulation . The induction of autophagy in ce rtain scenarios is accompanied b y an increase in the mRNA levels of certain autophagy gene s, such as A TG 8/Lc3 , 541, 542 Atg12 , 543 and Atg14 , 544 and an autophagy - dedicated microarray wa s developed as a high-throu g hput tool to simultaneousl y monit or the transcriptional reg ulation of all genes involved in, and related to, autophag y . 545 The g ene that shows the greatest transcriptional regulation in the liver ( in response to starvation and circadian signals) is Ulk1 , but others also show more limited changes in mRNA levels including 113 Gabarapl1 , Bnip3 and, to a minor extent, Lc3B (J.D. Lin, personal communication). In several mouse and human ca ncer cell lines, ER stress and hypoxia increase the transcription of LC3 , ATG5 and ATG12 by a mecha nism involving the unfolded protein response (UPR). Similarly , a stimulus-dependent increase in LC3B expression is detec ted in neural stem cells undergoing autophagy induction. 546 Increa sed expression of A tg5 in vivo after optic ne rve axotom y in mice 547 and increa sed expression of Atg7, Becn1 and Lc3 a during neurogenesis at different embry onic stages in the mouse olfactor y bulb a re also seen. 548 L C3 and A TG 5 are not required for the initiation of autophagy , but mediate phagophore expansion and autophag osome formation. In this regard, the transcriptional induction of LC3 ma y be necessar y to replenish the LC3 protein that is turned over dur ing extensive ER stress- and h y poxia-induced autophagy. 543 , 549 Thus, assessing the mRNA levels of LC3 and other autophagy-related genes by northern blot or qRT-PCR may prov ide correlative data relating to the induction of autophag y . Downregulation of autophagy -related mRNAs has been observed in human islets under conditions of lipotoxicity 360 that impair autophagic f lux. 550 It is not clea r if these changes are sufficient to regulate autophagy, however, and therefore these are not direct measure ments. Of note, large cha nges in Atg gene transcription just prior to Drosophila salivary gland cell death (tha t is accompanied b y an increase in autophagy) are detected fo r Atg2 , Atg4 , Atg5 and Atg7 , whereas there is no significant change in Atg8a or Atg8b mRNA . 551,5 52 Autophagy is critical for Drosophila midgut cell death, which is accompanied b y transcriptio nal upre gulation of all of the Atg gene s tested, including Atg8a ( Fig. 18 ) . 240,553 Similarl y , in t he silkworm ( B ombyx mori ) larv al midgut 554 and fa t body, 555 the occurrence of autophagy is accompanie d b y an upregulation of the mRNA levels of several Atg genes . Transcriptional upregulation o f Drosophila Atg8a and Atg8b is also observed in the fat body following induction of autophag y at 114 the end of lar val development, 556 and these g enes as well as Atg2 , Atg9 and Atg18 show a more than 10-fold induction during starvation. 557 Atg5 , Atg6 , Atg8a and Atg18 are upregulated in the ovary of starved flies, 558 and an increa se in Drosophila Atg8b is observed in culture d Drosophila l(2)mbn ce lls following starvation (S. Gorski, personal communication). An upreg ulation of plant A TG 8 may be needed during the adaptation to reproductive growth; a T-D NA inserted mutation of rice A TG 8b blocked the change from vegetative growth to repr oductive growth in both homoz y gous and heteroz y gous plant lines (Ming-Yong Zhang, unpublished results ). Similarly , the upregulation of autophag y-relate d genes ( Lc3, Gabarapl1, Bnip3, Atg4b, Atg12l) ha s been docume nted at the tra nscriptional and translational level in several other species (e.g., mouse, rat, trout, Arabidopsis and maize) under conditions of ER stress, 543 and diverse types of prolonged (several day s ) catabolic situations including ca ncer cachex ia, diabetes mellitus, uremia and fa sting. 181 ,420, 559 -561 Along these line s, ATG9 and ATG16 L1 are transcriptionally upregulated upon influenza virus infection (H. Khalil, personal communication), and in C. elegans , the FOXA transcription factor PHA-4 and the TFEB ortholog (see Methods and challenges of spec ialized topics/model systems. C. elegans ) HLH-30 regulate the expression of seve ral autophag y-related gene s. 562 ,563 Such pro longe d induction of the expression of ATG genes has been thought to allow the re plenishment of critical proteins (e.g., L C3 and GABARAP) that are destro y ed during autophagosome fusion with the l y sosome. 564 The polyamine spermidine incre ases life span and induces autophagy in cultured yeast and mammalian cells, as we ll as in nematodes and flies . I n aging yeast, spermidine treatment trigge rs epigenetic deac et y lation of histone H3 through inhibition of histone acety ltran sferases, leading to significa nt upregulation of various a utophagy-related transcripts. 5 65 115 In addition to the A TG g enes, transcriptional upregulation of VMP1 (a prote in that is involved in autophagy regulation and that remains associated with the completed autophagosome) can be detec ted in mammalian cells subjected to rapam y ci n treatment or starvation, and in tissues underg oing disease-induced autophag y su ch as cancer . 566 VMP1 is an essential autophagy ge ne that is conserved from Dictyostelium to mammals, 279,567 and the VMP1 protein regulate s earl y steps of the autophagic pathwa y . 510 VMP1 is poorly expressed in mammalian cells under nutrient-normal conditions, but is highly upregulated in cells undergoing autophagy, and the expression of VMP1 induces autophag osome formation. The GLI3 transcription factor is an effector of KRAS that regulates the expression and promoter activit y of VMP1 , using the histone acetyltra nsferase EP300/p300 as a co-activator. 568 A gene regulatory ne twork, named CLEAR (coor dinated ly sosomal expression and regulation) that controls both lysosome and a utophagosome biogenesis was identified using a systems-biology approach. 569- 571 The basic helix -l oop-helix transcription fac tor EB (TFEB) acts as a master gene of the CL EAR network and positivel y r egulates the expression of both lysosomal and a utopha gy g enes, thus linking the biogenesis of 2 dist inct types of c ellular compartments that cooperate in the a utophagic pathwa y . TFEB activity is regulated b y starv ation and is controlled by both MAPK1/ ERK2- and MTOR- mediated phosphor y l ation at specific serine re sidue s; 5 69,572,5 73 thus , it ca n serve as a new tool for monitoring transcriptional regulation connected with autophagy . T FEB is phosphory lated by MTORC1 on the lysosomal surface, preventing its nuclea r translocation. A l y sosom e- to - nucleus signaling mechanism transcriptionally regulates autophag y and l ysosomal biog enesis via MTOR and TF EB. 573 A very useful rea dout of endogenous TFEB activity is the evaluation of TFEB subcellular localization, as activation of TFEB correlates with its translocation from the c y topl asm to the nucleus. This 116 shift can be monitored by immunofluorescence using antibodies against TFEB. TFEB localization may a lso be studied to monitor MTOR activity, a s in most cases TFEB nuclear localization correlate s with inhibiti on of MTOR. However, due to the low expression levels of TFEB in most cells and tissues, it may be diff icult to visualiz e the e ndoge nous protein. Thus a TFEB nuclear tra nslocation assa y was developed in a HeL a cell line stabl y transfected with TFEB-GFP. This fluorescence assay can be used to identify the conditions and factors that promote TFEB activation. 573 Similar to TFEB , the ery throid transcription factor GATA1 and its coregulator ZFPM1/FOG1 induce the tra nscription of multiple genes encoding autophagy components . T his developmentally reg ulated transcriptional response is coupled to i ncrease s in autophagosome number as we ll as the percent of cells that contain autophagosomes. 574 FOXO transcr iption factors, e speciall y FOXO1 and FOXO3, also pla y critical roles in the regulation of autophagy ge ne expression. 420 , 544 ,575 Finally , CEBPB/ C/EBP  is a transcr iption factor that regulates autophagy in response to the circ adian c y cl e. 576 Although less work has been done on post-transcriptional regulation, several studies implicate microRNAs in controlling the expression of proteins associated with autophagy . 204 ,577- 579 Cautionary notes: Most of the A TG genes do not show significant changes in mRNA levels when autophagy is induced. Even increases in LC3 mRNA can be quite modest and a re cell ty pe- and or g anism-dependent. 580 I n addition, it is generally better to follow protein levels , which, ultimately, are the significant parameter with regard to the initiation and completion of autophagy . Howe ver, A TG protein a mounts do not alwa y s change si gnificantly and the extent of in crea se is again cell t y p e- and tissue-dependent. In some cases (e.g., y e ast ATG14 ), increased 117 transcription is not acc ompanied by increased protein levels, apparently due to changes in translation efficiency under starva tion conditions (H. Abeliovich, unpubli shed data) . Finall y , changes in autophagy protein levels are not sufficient evidence of autophagy induction and must be accompanied by a dditional assa y s as described herein. Thus, monitoring changes in mRNA levels for either A TG ge nes or autophagy regulators ma y provid e some evidence supporting upregulation of the potential to underg o autopha gy , but should be used along with other methods. Another genera l caution pertains to the fact that in any cell culture s y stem mix ed populations of cells (for example, those undergoing autophag y or not) e xist sim ultaneously . Therefore, only a n average level of protein or mRNA expression can be evaluated with most methods. This means that the re sults regarding specific changes in autophagic cells could be hidden due to the bac kgr ound of the a verage data. Along these lines, ex periments using sing le- cell rea l-time PC R to examine ge ne expression in individual cardiomyocytes with and without signs of autophagy revealed that the transcription of MTOR markedl y and significa ntl y increased in autophag ic cells in intact cultures (spontaneousl y unde rgoing autophag y) a s well as in cultures treated with proteasome inhibitors to induce autophagy (V. Dosenko, personal communication). Finally , researchers need to realize that m ammalian c ell lines may have mutations that alter autophagy signaling or execution; this problem can be avoided by using primary cells. Conclusion : Although th ere are changes in ATG gene expression that coincide with, and may be nee ded for, autophag y , t his has not been carefull y studied experimentally. Therefore, a t the present time we do not recommend the monitoring of ATG gene transcription as a general readout for autophag y unless there is clear documentation that the cha nge(s) correlates with autophagy ac tivit y . 118 8. Posttranslational modification of ATG proteins . Autophagy is also controlled b y posttranslational modifications of ATG prote ins such as phosphor y lation, ubiquitination, acetylation, oxidation and cleavage, which can be monitored to analy ze the status of the process. 386 , 469, 473 ,581,582 [ad d Tang and Feng papers, and Cherr a 2010] Th e g lobal deacet y l ation of proteins, which often accompanies autophagy , can be convenientl y me asured by qua ntitative immunofluorescence with antibodies spec ific recognizing acetylate d l y sine residues. 583 Indeed, depletion of the nutrient supply causes autophagy in yeast or mammalian cells by reduc ing the nucleo-cy tosolic pool of acet y l -coenz y me A, which provides acety l groups to acet y lt ransfera ses, thus reducing the acetylation level of hundre ds of cytoplasmic and nuclear proteins. 584 A global deacety lation of cellular proteins is also observed in response to so-c alled “caloric restriction mimetics”, that is, a class of pharmacolog ical agents t hat deplete the nucleo-c y tosolic pool of acetyl-c oenz y me A, inhibit acet y ltransferases (su ch as EP300) or activate deacetylases (such as SIRT1). All these agents reduce protein acet y l ation levels in ce lls as the y i nduce autophagy . 585 One prominent ATG protein that is subjected to pro-autophagic deacety lati on is LC3. 586 , 587 . 9. Autophagic protein degradation . P rotein deg radation assay s represent a well -established methodology for measuring autophagic flux, and they allow good quantification. The general strategy is first to label cellular prote ins b y incorporation of a radioac tive ami no ac id (e.g., [ 14 C] - or [ 3 H] - leucine, [ 14 C] -val ine or [ 35 S] -methionine; a lthough valine may be preferre d ov er leucine due to the strong inhibito ry effects of the latter on autophagy), prefera bl y for a period sufficient to achieve labeling of the long-lived protei ns that best repr esent autophagic substrates, and then to follow this with a long c old-chase so that the assay starts well after labeled short-lived proteins are degraded (which occurs predominantly via the proteasome). Next, the time-dependent release of acid-soluble radioac tivit y from the labeled protein in intact cells or perfused organs is 119 measured. 2,588,589 Note th at the inclusion of the appropriate unlabeled amino acid (i.e., valine, le ucine or methionine) in the starvation medium at a concentration equivalent to that of other amino acids in the chase medium is necessary; otherwise, the released [ 14 C]-amino acid is effectively re-incorporated into cellular proteins, which results in a significant underestimation of protein degra dation. A ne w er method of quantifying a utophagic protein degradation is based on L-azidohomoalanine (AHA) labe ling. 590 When added to culture d cells, L-azidohomoalanine is incorporated into proteins during active protein s ynthesis. Af ter a click reaction between a n azide and an alkyne , the azide- containing proteins can be detected with an alk y ne -tagged fluorescent dye , coupled with flow cy tometr y . Th e turnover of spec ific proteins can also be measured in a pulse-chase regimen usin g the Tet-ON /OFF s y ste m and subsequent western blot analysis. 591 , 592 In this type of assay a considerable fraction of the measure d degradation will be non- autophagic, and thus it is important to also measure, in parallel, cell samples treated with autophagy-suppressive concentra tions of 3-M A or amino acids, or obtained from mutants missing centra l A TG co mponents (however, it is important to note that the se controls are only appropriate assum ing that nonautophagic proteol ytic activity remains unchanged, which is unlikely); these values are then subtracted from the total readouts . The complementar y app roac h of using c ompounds that block other degradative pathwa y s, such as proteasome inhibitors, may cause une xpected results and should be interpre ted with caution due to crosstalk among the degrada tive s y stems. For example, blocking proteasome function ma y activ ate a utophag y . 593 - 596 Thus, when using inhibit ors it is critical to know whether the inhibitors being used alter autophagy in the particular c ell t y pe and context being examined. I n addition, because 3 -MA could have some a utopha gy-independe nt effects in particular settings it is advisable to verify that 120 the 3- MA -se nsitive degrad ation is also sensitive to g eneral l y sosomal inhibi tors (such a s NH 4 Cl or leupeptin). The use of stable isotopes, such as 13 C and 15 N, in quantitative mass spec trometry-based proteomics allows the recording of degradation rates of thousands of proteins simultaneously . These assay s ma y be applied to autophagy-related questions enabling researchers to investigate differe ntial effects in global protein or even organelle degradation studies. 597 , 598 SILAC (stable isotope labeling with amino acids in ce ll culture) can also provide comparative information between different trea tment conditions, or between a wild type and mutant. Another assay that could be considered relies on the limited proteolysis of a betaine homocysteine meth y lt ransferase (BHMT) fusion protein. The 44-kDa full-l eng th BHMT protein is cleaved in hepatocyte amphisomes in the presence of leupeptin to ge nerate 32-kDa and 10-kD a fragments. 599 - 6 02 Accumulation of these fragments is time-dependent and is blocked b y treatment with autophagy inhibitors. A modified version of this marker, GST -BHMT, can be expressed in other cell lines where it behaves similar to the wild -type protein. 603 Other s ubstrates may be considered for similar types of a ssays. For example, the neomycin phosphotransferase II-G FP (NeoR-GFP) fusion protein is a target of autophagy . 604 Transfection of l y m phoblastoid cells with a plasmid encoding Ne oR -GF P followed b y incu bation in the prese nce of 3-MA leads to an accumulation of the NeoR-GFP protein as measured by flow cytometry . 605 A similar western blot assay is based on the degradation of a c y tosolic prot ein fused to GFP. This method has been used in yea st and Dictyostelium cells using GFP -PgkA and GFP- Tkt-1 (phosphog l y cerate kinase and transketolase, respectively). In this case the re lative amount of the free GFP and the complete fusion prote in is the relevant parameter f or quantification; although it may not be possible to detect cle ar changes in the amount of th e full-length chimera, 121 especially under conditions of limited flux. 28 , 35 As describe d above for the marker GFP- Atg8/LC3, nonsatura ting levels of l y sosomal inhi bitors are also needed in Dictyostelium ce lls to slow down the autophagic degra dation, allowing the accumulation and detection of free GFP. I t should be noted that this method monitors bulk autophag y sinc e it relies on the passive transit of a cytoplasmic mar ker to the l y sosom e. Consequently, it is important to determine that the marker is distributed homogeneously in the cy toplasm. One of the most useful methods for monitoring autophagy in S. cerevisiae is the Pho8∆60 assay. PHO8 encodes the vacuolar alkaline phosphatase, which is s y nthesi zed as a zymogen before finall y b eing transported to and activated in the vac uole. 606 A molecular genetic modification that eliminates the first 60 amino ac ids prevents the mutant (Pho8∆60) from entering the ER , leaving the zy mogen in the c y tos ol. When autophagy is induced, the mutant z y mogen is delivered to the vacuole nonselectively inside autophagosom es along with other cy toplasmic material. The resulting activation of the z y mo gen can be easily measu red b y assa y s for alkaline phospha tase. 220 To minimize ba ckground activity, it is preferable to have the gene encoding cytosolic a lkali ne phosphatase ( PHO13 ) additionall y deleted (although this is not necessary whe n assa y ing certain substrates). Cautionary notes: Measuring the degradation of long-lived proteins requires prior radiolabeling of the cells, and subsequent separation of acid-soluble from acid -insoluble radioactivity . The labeling can be done with relative ease both in cultured cells and in live animals. 2 I n cells, it is also possible to measure the relea se of an unlabeled amino acid by chromatogra phic method s, thereby obviating the need for prelabeling; 607 however, it is important to keep in mind that amino acid re lease is also regulated b y p rotein s y nthe sis, which in turn is modulated by man y dif ferent factors. In either case, one potential problem is that the released 128 machinery are required for LN, the macroautophagy-specific Atg11 and the Vps34-containing Ptd I ns3K complex I a re not. L N c an be monitored by emplo y in g a nuclear -targ eted version of the Rosella biosensor (n-Rosella) and following either its accumula tion (b y confocal microscopy), or de g radation (by immunoblotting), within the vacuole. 649 D ual labeling of cells with Nvj1-EYFP, a nuc le ar me mbrane reporter of PMN, and the nucleopla sm -targe ted NAB35- DsRed.T3 (NAB35 is a target sequence for the Nab2 RNA -binding protein, and DsRed.T3 is the pH-stable, re d fluorescent component of n-Rosella ) allows detec tion of PMN soon after the commencement of nitrogen starvation, whereas deli very to t he vacuole of the nucleoplasm reporter, indicative of LN, is observe d onl y after prolonged periods of nitrog en starvation. Few cells show simultaneous acc umulation of both reporters in the vacuole indicating PMN and LN are temporally and spatiall y separated. 649 In contrast to unice llular y easts, filamentous fun g i form an interconnected m y c elium of multinucleate hy phae containing up to 100 nuclei in a single hypha l compartment. A mycelial colony g rows b y tip extension with actively growing h y pha e at the colony mar gin surrounded b y an older, inner h y phal network that recycles nutrients to fuel the hy phal tips. B y labeling organelle markers w ith GFP it was possible to show in A. oryzae that macroautophagy mediates degrada tion of basal h y phal organelles such as peroxisomes, mitochondria and e ntire nuclei. 650 I n contrast to y east, PMN has not been observed in filamentous ascom y c etes 6 51 . In M agnaporthe oryzae germination of the condiospore and format ion of the appre ssorium is accompanied by nuclear degenera tion in the spore. 234 The deg radation of nuclei in spores requires the nonselective a utophag y machinery, wh ereas conserved components of the PMN pathwa y such as Vac8 a nd Tsc13 are disp ensable for nuclear breakdown during plant infection. 652 Nuclei are proposed to function in storage of growth-limiti ng nutrients such as phosph ate and 129 nitroge n. 653 ,654 Similar to nuclei, mitochondria a nd peroxisom es ar e also prefere ntiall y de graded in the basal hy phae of filamentous ascom y c etes. 234,650,652-655 Cautionary notes: The Cvt pathwa y ha s been demonstrated to occur only in yeast. In addition, the sequestration of prApe1 is specific, even under starvation conditions, as it involves the recog nition of the propeptide by a receptor, Atg19, which in turn interacts with the scaffold protein Atg11. 656 , 657 Thus, unless the prop eptide is removed, prApe1 is recognized as a selective substrate. Overexpression of prApe 1 saturates import b y the Cvt pathwa y , and the precursor form accumulates, but is rapidly matured upon autophagy induction. 262 I n addition, mutants such as vac8∆ and tlg2∆ accumulate prApe1 under rich conditions, but not during autophag y . 457 , 658 Accordingly, it is possible to monitor the proce ssing of pr Ape1 when overexpressed, or in certain mutant strains to follow autophagy induction. However, even the latter conditions may be misleading, a s the y do no t indicate the size of the autophagosome . The Cvt complex (prApe1 bound to Atg19) is smaller than t y pical pe roxisomes or mitochondrial fragments that are subject to autophag ic degradation. Accordingl y , particular mutants may display c omplete maturation of prApe1 under autophagy-inducing conditions, but may still have a defect in other types of selective a utophag y , as well as being unable to induce a normal level of nonspecific a utopha gy . 90 For this reason, it is good practic e to evaluate autophagosome size and number b y TEM. Actually , it is much simpler to monitor autophagic bodies (rather than autophagosomes) in y east. First, the vac uole is easil y identified, making the identification of autophagic bodies much simpler. Second, autophagic bodie s can be accumulated within the vacuole, allowing for an increased sample size. I t is best to use a strain background that is pep4∆ vps4∆ to prevent the breakdown of the autophag ic bodies, and to eliminate confounding vesicles from the 130 multivesicular body pathwa y . One caveat to the detection of autophagic bodies, however, is that they may coalesce in the vacuole lumen, making it difficult to obtain an ac curate quantification. In general, whe n workin g with y east it is preferable to use strains that have the marke r proteins integrated into the chr omosome rather than rel y in g on plasmid-based expression, because p lasmid number s can va ry from cell to cell. The GFP-At g8, or simil ar, pr ocessing assa y is easy to perform and is suitable for analysis by microscop y as well as western blotti ng ; however, particular care is needed to obta in quantitative data for GFP -Atg8, Pex14-GFP or Om45-GFP, etc. proce ssing assa y s (see cautionary notes for GFP- Atg8/LC3 lysosomal delive ry and proteolysis ). An alternative is an orga nelle targeted Pho8∆60 assay. For example, mitoPho8∆60 can be used to quantitatively mea sure mitophagy. 616 I n additi on, for the GFP-Atg8 processing assay , 2 h of s tarvation is generally sufficient to detec t a si gnificant level of free (i.e., vacuolar) GF P b y western blotting as a measure of nonselective autophag y. For selective t y pes of autophagy, the le ngth of induction needed for a clearl y detectable free GFP band will var y depending on the ra te of carg o deliver y /degradation. Usuall y 6 h of mi tophagy induction is needed to be able to detect free GF P (e.g., from Om45-GFP) b y western blot under starvation conditions, wherea s stationar y phase mitophagy ty picall y r equires 3 da y s before a free GFP band is observed. However, as with animal s y stems (se e Animal mitophagy and pexophagy ), it would be prudent to follow more than one GFP-tagged protein, as the kinetics, a nd even the occurrence of mitophagic traff icking, seems to be protein species-dependent, even within the mitochondrial matrix. 659 Care should be take n wh en choosing antibodies to assess the degree of mitochondrial protein removal by a utophag y ; the quality and clarity of the result may var y depending on the specifics of the antibody . I n testin g the efficienc y of mitophagy clearer results may be obtained 131 by using antibodies against mtDNA-encode d proteins . This experimental preca ution ma y prov e critical to uncove r subtle differences that could be missed when ev aluating the process with antibodies against nuclearly encoded, mitochondrially imported, pro teins ( M. Campanella personal communica tion). b. Reticulophagy and ribophagy . Activation of the UPR in the ER in y east induces a type of selective macroa utophagy of the ER. 660 -662 This proc ess is termed reticulophagy to b e consistent with the terms pexophagy and mitophag y . 662 Reticulophagy- associated autophagosomes are lamellar-membraned structures that conta in ER proteins. I n theor y, since reticulophag y is selective, it should be able to sequester parts of the ER that are damaged, and eliminate protein aggrega tes that cannot be removed in other ways. Reticulophagy ma y also serve to limit the UPR, by reducing the ER to a normal level after a particular stress condition has ended. Some of the mutated dy sferlin protein, L GMB2B/Miyoshi ty pe muscle d ystrophy, which accumulates in the ER, is deg raded b y E R-stress mediated reticulophagy. 663 I n addition to activation of the UPR (particularly upregulation of HSPA5/GRP 78 and phosphory lation of EIF2S1/ eIF2  359 , 664 -666 ) , Ptd I ns3P and its binding proteins could be good markers for reticulophag y . 532 Autophagy is also used for the se lective removal of ribosom es. 667 This proce ss can be monitored by western blot, following the ge neration of free GFP from Rpl25 -GF P or the disappearance of ribosomal subunits such as Rps3. Vacuolar localization of Rpl25 -GFP can also be seen by fluorescence microscopy. c. Vac uole import and de gradation pathway . I n yeast, g luconeogenic enz y mes such as fructose- 1,6-bisphosphatase (Fbp1, also called FBPase), malate dehydrogenase (M dh 2), isocitrate lya se 132 (Icl1) a nd phosphoenolp y r uvate carbox y kinase (Pck1) c onstitute the cargo of the vacuole import and degra dation (Vid) pathway. 668 These e nz y mes are induced whe n y e ast cells are glucose starved (grown in a medium containing 0.5% glucose and potassium ace tate). Upon replenishing these cells with fresh g lucose ( a m edium containing 2% g lucose), these enz y m es are degraded in either the proteasome 669 - 671 or the vac uole 668,672 depe nding on the duration of starvation. Following g lucose replenishm ent after 3 days glucose starvation, the gluconeogenic enz y mes are delivered to the vacuole for deg radation. 673 These e nzymes are sequestere d in specialized 30- to 50-nm vesicles ca lled Vid vesicles. 674 Vid vesicles ca n be purified b y fract ionation and gradient centrifuga tion; western b lotting analysis using antibodies against organelle markers a nd Fbp1 , and the subsequent verification of fractions by EM facilitate their identification. 674 Fur thermore, the amount of marker proteins in the cytosol compared to the Vid vesicles can be examined by differe ntial centrifugation. I n this case, y e ast cells are lysed and subjected to differe ntial centrifuga tion. The Vid vesicle-enriched pellet fraction and the c y tosolic supernatant fraction are examined with antibodies against Vid24, Vid30, Sec28 and Fbp1. 675 -6 77 Vid/endosomes containing their cargo aggregate around endoc y ti c ve sicles forming on the plasma membrane and are relea sed into the c ytoplasm. The V id/endosomes can be purified by frac tionation and dens ity gra dient centrifugation. 6 78 The fra ctions containing purified Vid/endosomes can be identified by western blot anal y sis using antibodies against Vid24, Fbp1 and the endosomal marker Pep 12. The distribution of Vid vesicles containing cargo destined for endosomes, and finally for the vacuole , can be ex amined using FM 4-64, a lipophilic d y e that stains endocy tic compartments and the va cuole limiting membrane. 679 I n these experiments, starved yeast cells are replenished with fr esh glucose and FM 4-64, and cel ls are c ollected at appropriate time points for examination by fluorescen ce microscop y. 676 Th e site of deg radation 133 of the cargo in the vac uole can be determined b y st udying the distribution of Fbp1-GFP, or other Vid cargo markers in wild-type and pep4∆ cells. 680 Cells can also be examined for the distribution of Fbp1 at the ultrastructural level by immuno-T EM . 678 As actin patch polymer iz ation is require d for the deliver y of cargo to the vacuole in the Vid pathway, distribution of Vid vesicles con taining cargo and actin patches can be e xamined b y actin staining (with phalloidin conjuga ted to rhodamine) using fluorescen ce microscopy. 678 The distribution of GFP tagged protein and ac tin is exa mined by fluorescent microscop y . GFP - Vid24, Vid30-GFP and Sec28-GFP colocalize with actin during prolonged glucose starvation and for up to 30 min following g lucose replenishment in wild-t y pe cells; however, colocalization is less obvious by the 60 min time point. 675, 678 d. Animal mitophagy and pe xophagy . There is no consensus at the present time with regard to the best method for monitoring mitophag y in animals . As with any or g anelle-specific form of autophagy, it is necessary to demonstrate: i ) increased leve ls of autophago somes containing mitochondria, ii) maturation of these autophagosomes to culminate with mitochondrial degrada tion, which can be blocked b y spe cific inhibitors of autophagy or of l y sosom al degrada tion, and iii) whe ther the c hanges are due to selective mitopha gy or incre ased mitochondrial degradation during generalized autophagy. Techniques to address each of these points have been reviewed. 40 , 681 Ultrastructura l anal y sis at earl y ti me points can be used to establish selective mitophagy, although a maturation inhibitor ma y be n ee ded to trap early autophagosomes with recognizable cargo ( F ig. 20 ). Dep ending on the use of specific imaging techniques, dyes for living cells or antibodies for fixed cells have to be c hosen. In any case, transfection of the autophagosomal 134 marker GFP-LC3 to monitor the initiation of mitophagy , or RFP- L C3 to assess mitophag y progression, and visualization of mitochondria ( independent of their mitochondrial membrane potential) makes it possible to determine the association of these 2 cellular components. Qualitatively , t his ma y appear as fluorescence colocalization or as ring s of GFP -LC3 surrounding mitochondr ia in higher resolution images. 682 , 683 For live cell imag ing microscop y , mitochondria should be labeled by a matrix -targeted f luorescent protein transfection or by mitochondria-specific dies. When using matr ix -targe ted fluorophores for certain cell lines (e.g., SH -SY5Y), it is important to allow at le ast 48 h of transient expression for sufficie nt targeting /import of mitochondrial GFP/RFP prior to anal y zin g mitophagy. Among the MitoTracker probes are lipophi lic ca tions that include a chloromethyl group and a fluorescent moiety. They concentrate in mitochondria due to their negative charge a nd react with the r educed thiols present in mitochondrial matrix proteins. 684 - 686 After this rea ction the probe can be fixed and remains in the mitochondria independe nt of alt ere d mitochondrial function or mitochondrial membrane potential. 686 - 688 This method can thus be used whe n cells remain healthy as the dye will remain in the mitochondria and is re tained after fixation, although, as s tated above , accumulation is depe ndent on t he membra ne potential. Antibodi es that specifica ll y reco gnize mitochondrial proteins such as VDAC, TOM M 20 or COX4I1 (cy tochorome c oxidase subunit IV isoform I ) may be used to visualiz e mitochondr ia in immunohistochemical experimental procedures. 689 , 690 I n neuronal cells, stabilized PINK1 on the mitochondrial outer membr ane that accumulates in response to certa in forms of acute mitochondrial damage is also a useful ma rker because it differentiates betwee n h ealthy mitochondria a nd those that have lost their membrane potential. Redistribution of ca rdiolipin to the outer mitochondrial membrane acts as an elimination signal for mitophagy in mammalia n ce lls, including primary neurons, and an 135 ANXA5 binding assa y fo r externalized cardiolipin can also be considered a good marker for damaged mitochondria and earl y mitopha gy. 125 Coloca lization analyses of mitochondria and autophagosomes provide an indication of the degree of autophagic sequestration. T EM can be used to demonstrate the presence of mitoc hondria within autophagosomes (referred to as mitophagosomes during mitophagy), a nd this can be coupled with bafilomycin A 1 tre atment to prevent fusion with the ly sosome. 40 To quantify early mitophag y , the p ercentage of LC3 puncta (endogenous, RF P- or G FP-L C3 puncta) that colocalize with mitochondria and the number of colocalizing LC3 punc ta per cell — as assessed b y confocal microscop y — in response to mitophagic stimuli can be employ ed as well. I n addition, the percentage of lysosome s that colocalize with mitochondria ca n be used to quantif y l ysosomal-mediated d elivery of mitochondria. Overall, it is important to quantif y mitophag y at various st ag es (initiation, progression, and late mitophag y ) to identif y sti muli that elicit this process. 691, 692 The fusion process of mitophagosomes with hydrolase-containing l y sosom es represents the next step in the degradation process. To monitor the a mount of fused organelles via live cell imaging microsc op y , Mit oTracker® Green FM and Ly soTracker® Red DND-99 ma y be used to visualize the fusion process ( Fig. 21 ). Inde pendent of the cell-t y pe specific concentration used for both dy es, we recommend ex chang ing MitoTracker® Green FM with normal medium (prefera bl y phenol-free and CO 2 independent to reduce unwanted autofluorescence) after incubation with the dy e, whereas it is best to maintain the LysoTracker® Red staining in the incubation medium during the acquisition of images. Given that these f luor esce nt d y es are extremely sensitive to photobleaching, it is critical to pe rform live cell mitophagy ex periments via confocal microscopy , preferabl y b y using a spinnin g disc c onfocal microscope for long-term imaging e xperiments. For imm uncy tochemical experiments, antibodies specific for 136 mitochondrial proteins and an antibody against LAMP1 (lysosomal-associated membrane protein 1 ) can be used. Overlapp ing signa ls appear as a m erged c olor and can be used as indicators for successful fusion of autophagosomes that contain mitochondria with l y soso mal structures. 693 To measure the correlation between 2 variables by imaging techniques, such as the coloca lization of 2 differe nt stainings, we recommend some form of correlation anal y sis to assess the value corre lating with the stren gth of the association. This may use, for example, Image J softwa re, or other coloca lization scores that can be derived from consideration not onl y of pixel colocalization, but also from a determina tion that the structures have the appropriate shape. During live-ce ll imaging, the 2 structures (autophagosome s and mitochondria) should move together in more than one fra me. Mitophag y can also be quantitatively monitored using a mitochondria-targe ted version of the pH- dependent Keima protein. 694 The pe ak of the excitation spectrum of the protein shifts from 440 nm to 586 nm when mitochondria are delivered to ac idic lysosomes, which allows ea s y quantification of mitophagy ( Fig. 22 ). However, it should be noted that long e xposure ti me of the spe cimen to intense laser light lead to a similar spectra l change. Finally, a mitochondrially-targeted version of the tandem mCherry-GFP fluorescent reporter (see Tandem mRFP/mCherry-GFP fluorescence microscopy ) using a targeting sequence from the mitochondrial membrane protein FIS1 302 ,695 can be used to monitor mitophagy f lux . 695 The third and last step of the degra dation process is the monitoring of the amount of remaining mitochondria b y anal y zin g the mitochondrial mass. This final step provides the opportunity to deter mine the efficienc y of degradation of d y sfun ctional, aged or impaired mitochondria. Mitochondrial mass ca n either be measured by a FACS technique using MitoTracker® Green F M or MitoTracker Deep Red FM , 686 on a single cell basis, by either live cell imag ing or immunc ytoche mistry (using antibodies specifically raised aga inst different 137 mitochondrial proteins ) . Alternativel y , mi tochondrial content in re sponse to mitophagic stimuli (in the presence a nd absence of autophag y inhibitors to assess the contribution of mitophag y ) in live or fixed cells can be quantified at the single cell level as the perce ntage of c y tosol occupi ed by mitochondrial-specific fluorescent pixels using NIH I mageJ. 692 I mmunoblot analysis of the levels of mitochondrial proteins from different mitochondrial subcompar tments is valuable for validating the data from FACS or microscopy studies , and it should be noted that outer mitochondrial membrane proteins in particular can be degra ded b y the proteasome, especiall y in the context of mitochondrial depolarization. 696,697 EM can a lso be used to verif y loss of entire mitochondria, and PCR (or fluorescence microsc op y ) to qu antif y mitochon drial DNA (mtDNA) . A reliable e stimation of mtDNA can be performed b y r eal -time PCR of the MT -ND2 (mitochondrially encoded NADH dehydrogenase 2) gene expressed as a ra tio of mtDNA:nuclear DNA by normalizing to that of TERT ge nomic DNA. 698 The spectrophotometric measurement of the activity of CS (citrate s y nthase), a mitochondrial matrix enz y me of the Krebs cy cle , which remains hig hl y constant in these organelles and is considered a reliable marker of their intracellular content, can also be used to estimate the mitochondrial mass . 698 In addition to monitoring the steady state levels of different steps of mitophagy — whe ther by single-cell ana l y ses of L C3 mi tochondrial c olocalization or b y immunobl otting for mitochondrial markers — investig ation of the mitophag ic flux is needed to determine whether mitophagy is impaired or ac tivated in response to stimuli, and at which steps . Therefore, appropriate trea tment (pharmacological or siRNA- mediated knoc kdown of ATG genes) ma y b e applied to preve nt mitoc hondrial degra dation at dist inct steps of the proce ss. A re cent method using flow cy tometr y in combination with autophagy and mitopha gy inhibitors has been developed to determine mitophagy flux using MitoTrac ker probes. 686 144 which appear ne xt to the sperm DNA, membranous organelles and mitochondria (labeled with CMXRos or mitochondria-targeted GFP) within 15 to 30 min post -fertilizat ion, can be used to verify the a utopha g ic nature of the degradation. TEM can a lso be utilized to demonstrate the presence of mitochondr ia within autophagosomes in the early embry o. Conclusion : There are many assays that can be used to monitor specific t ype s of autophagy, but ca ution m ust be used in choosing an appropriate marker(s). The potential role of other degradative pa thways for an y individual organelle or cargo marker should be conside red, and it is advisable to use more than one marker or technique. g. Chlorophagy . Besides functioning a s the primar y energy suppliers for plants, chloroplasts repre sent a major source of fix ed c arbon and nitroge n to be remobilized from senescing leaves to storage orga ns and newl y de veloping tissues. As such, the turnover of these organelles has long been considered to occur via an autophagy-type mecha nism. But, while the detection of chloroplasts within autophag ic bod y-like vesicles or within vacuole-like compartments has been observed for decades, only recentl y has a direct connection between chloroplast turnover and autophagy be en made through the anal y sis of atg mutants combined with the use of fluorescent ATG8 reporters. 733 , 734 I n fact, it is now clear that chlorophagy, the selective degradation of chloroplasts by macroautophagy, can occur via several routes, including the encapsulation of whole chloroplasts, or the budding of chloroplast material into small distinct autophag ic vesicles called Rubisco-containing bodies ( RCBs) and ATI1 plastid-associated bodes (ATI-PS), which then transport chloroplast carg o to the vacuole. 733 ,735 Chloroplasts produce long tubes called stromules that project out from the org anelles outer membrane. Recent studies su ggest that stromules are part of the chlorophagy process, b y which the stromule tips presumably containing 145 unwanted or damaged chloroplast material are e ngulfed by autophagic membranes using ESCRT I I endoc y totic m achinery that de pends on ATG8. 736 The appeara nce of RCBs is tig htl y linked with leaf carbon status, indicating that chlorophagy through RBCs represents an important route for recy cling plant nutrients provided in plastid stores. h. Chromatophagy . Autophagy has been known for its pro-survival role in cells under metabolic stress and other conditions. However, excessively induced autophagy ma y be cy totoxic and ma y lead to cell death. Chromatophagy (chromatin-specific autophagy) comes into view as one of the autophag ic responses that can contribute to cell death. 737 Chromatophagy can be seen in cells during nutrient depletion, such as arginine starvation, and its phenotype consists of giant-autophagosome formation, nucleus membrane rupture and histone-associa ted-chromatin/DNA leakage t hat is capture d b y autophagosome s. 737 Arginine starva tion can be achieved b y adding PA DI ( peptid y l arginine deiminase) to remove arginine from c ulture medium, or b y using arginine-dropout medium. The degrada tion of leaked nuclear DNA/chromatin can be observed b y fluorescence microscopy ; with GFP- LC3 or anti-L C3 antibod y , and Ly soTracker Red o r anti-LAMP1, multiple giant autophagosomes or autoly sosomes containing leaked nu clear DNA can be detected. I n addition, the chromatophagy-related autophagosomes also contain parts of the nuclear outer-membr ane, including NUP98 (nucleoporin 98kDa), indicating that the process involves a fusion event. 737 i. Ferritinophagy. This is a selective f orm of autophag y that functions in int ra cellular iron processing. 738 I ron is recruited to ferritin for storage and to prevent generation of free radical 146 iron. 739,740 To release iron from ferritin, the iron bound form is sequestered withi n an autophagosome. 741 Fusio n with a ly sosome leads to breakdown of ferritin and release of iron. Further more, iron can be acidified in the l y sosome, convertin g it from an inactive state of Fe +3 to Fe +2 . 742,743 I ron can be detected in the autol y sosom e via TEM. 742 Coloca lization of iron with autolysosomes may also be determined utiliz ing calce in AM to tag iron. 742 ,744 NCOA4 is a ca rgo rece ptor that recruits ferrit in to the autophag osome. 7 38 j. Lipophagy . The spec ific macroautophagic degradation of lipid droplets represents another type of selective autophagy. 745 L ipoph ag y requir es the core a utophagic machiner y and can be monitored by f ollowing trigl y ceride content, or total lipid levels using B ODIPY 493/503 or HCS LipidTOX neutral lipid stains with fluoresc ence microscopy, c ell staining with Oil Red O, the cholesterol dye f ilipin III,{[ Koenig, 2015 #3509} or ideally label-free techniques such as CARS or SRS microscopy. BODIPY 493/503 should be used with caution, however, when performing costains (especially in the green and red spectra) because this commonl y u sed fluorescent marker of neutral lipids is highly susceptible to bleed- th rough into the other fluorescence channels (hence often y ielding false positives), unlike the LipidTOX stain that has a narrow emission spectrum. 746 TEM ca n also be used to monitor lipid dro plet size and number , as well as lipid droplet-associa ted double-membrane structures, which cor respond to autophagosomes . 7 45,747,7 48 The master transcriptional reg ulator of autophag y TFEB positively regulate s lipophagy and promotes fatty acid  -oxidation, thus providing a regulatory link betwee n different lipid degrada tion pathwa y s. Accordingl y , TFEB overexpression rescues fat accumulation and metabolic sy ndrome in a diet -induced model of obesit y . 749 ,7 50 The reg ulation of expression of lipid droplet reg ulators (s uch as the perilipin family ) and of autophag y ada ptors (such as the 147 T BC 1D1 famil y ) during starvation and disease is one of several areas in this topic that deserves further e xploration. 751 -753 Cautionary notes: Wit h rega rd to changes in the cellular neutra l lipid content, th e presence and potential activation of cy toplasmic lipases that are unrelated to l y sosomal degrada tion must be considered. k. Intraplastidial a utophagy . Intraplastidial autophagy is a process whereby plastids of some cell types adopt autophagic functions, eng ulfin g and digesting portions of the cy toplasm. These plastids are character ized b y formation of invaginations in their double -membrane envelopes that eventually generate a cy toplasmic compartment within the plastidial stroma, isolated from the outer cytoplasm. W. Nagl (1977) coined the term plastolysome to define this special plastid type. 754 I nitiall y , the engulfed cytoplasm is identical to the outer cy topl asm, containing ribosomes, vesicles and even larg er organelles. Lytic activity was demonstrated in these plastids, in both the cy toplasmic compartment and the stroma. Therefore, it was sugg ested that plastolysomes digest themselves toge ther with their c y topl asmic c argo, and transform into lytic vacuoles. Intap lastid ial autophagy h as been reported in plastids of suspensor cells of Phaseolus coccineus 754 a nd Phaseolus vulgaris , 755 where plastids transfor med into autophag ic vacuoles during the senescence of the suspensor. This proce ss was also demonstrated in petal cells of Dendrobium 756 and in Br assica napus microspores experimentally induce d towards embry ogenesis. 757 All these reports established a clear link between these plastid transformations and their engagement in autophagy. At pre sent, descriptions of this process are limited to few, specialized, plant cell ty pes. However, pictures of cytoplasm-containin g plastids in other plant cell ty pes have been occasionall y publi shed, although the authors did not make a n y mention of 148 this special plastid type . For example, pictures ha ve bee n published of ferti le and Og u-INRA male sterile tetra ds of Br assica napus where a few plastids contain round compartments filled with cytopla sm, 758 and of Phaseolus vulgaris root cells where some plastids contained cy toplasm portions. 759 Possibly , this process is not as rare as initially thou ght, but auth ors have only paid attention to it in those cell type s where it is particularly frequent. l. RNA-silencing components. Several components of the RNA-silencing machinery are selectively deg raded b y autophagy in different organisms. This was first shown for the plant AGO1 /ARGONAUTE1 protein, a ke y compon ent of the Arabidopsis RNA-induced silencing complex (RISC) that af ter ubiquit in ation by a virus encoded F-box protein is targeted to the vacuole. 760 AG O1 colocalizes with Arabidopsis A TG8a-positive bodies a nd its degradation is impaired by various drugs such as 3-MA and E64d, or in Arabidopsis mutants in which autophagy is compromised such a s the TOR-overex pressing mutant line G548 or the atg7-2 mutant allele (P. Genschik, unpublished data). Moreover, this pathwa y also degrade s AGO1 in a nonviral context, especially whe n the production of miRNAs is im paire d. In m ammalian c ells, not only the main miRNA effec tor AGO2, but also the miRNA -proce ssing enz y me DICER1, is degrade d as a miRNA- free e ntit y b y selective aut ophagy . 761 Chemical inhibitors of autophagy (bafilomy cin A 1 and chloroquine) and, in He L a cells, deplet io n of key autophagy components ATG5, ATG6 or ATG7 using short interfering RNAs, blocks the degradation of both proteins. Electron microsc op y sho ws that D I CER1 is associ ated with membrane-bound structures having the hallmarks of a utophagosomes. Moreover, the selectivit y of DICER1 and AGO2 degrada tion might depend on the autophagy receptor CA LCOCO2/NDP52, at least in these cell types. Finally , in C. elegans , A IN-1, a homolog of ma mmalian TNRC 6A/GW182 that intera cts with 149 AGO and media tes silencing, is also degraded by autophagy. 762 AI N -1 colocalizes with SQST -1 that acts as a receptor for autophag ic degradation of ubiquit in ated protein agg regates and also directly interacts with Atg8/LC3 contributing to cargo specificity. m. Xe nophagy . The macroautophag y pathway has emerge d as an important cellular factor in both innate and ada ptive immunit y . Man y in vitro and in vivo studies have demonstrated tha t ge nes encoding macroautophagy components are required for host defense against infe ction b y bacteria, parasites and viruses. Xenophagy is often used as a term to de scribe autophag y of microbial pathogens, mediating their capture and deliver y to lysosomes for degrada tion. Since xenophagy presents an immune defense, it is not surpr ising that microbial pathogens have evolved strategies to overcome it. The inte ractions of such pathogens with the autophag y s ystem of host cells are complex and have been the subject of several excellent reviews. 1 05-110, 763 - 768 Here we will make note of a f ew ke y consid erations when study ing interactions of microbial pathogens with the autophagy system. Importantly, autophagy should no long er be considered as strictly antibacter ial, and several studies have described the fact that autophagy ma y serve to either re strict or promote bacterial replication both in vivo 769 and in vitro (reviewe d in refs. 770,771 ) . LC3 is commonly used as a marker of macroautophagy. However, studies have established that L C3 can promote phagosome maturation independentl y of macroa utophag y through LC3-associated phag oc y tosis (see cautionar y notes in Atg8/LC3 detection and quantification and Noncanonical use of autophagy-related proteins ). Other studies show that macroautophagy of Salm onella Typhimurium is depende nt on Atg9, an essential macroautophagy gene, whereas LC3 recruitment to bacteria does not require Atg9. 772 I n contrast, 150 macroautophagy of these bacteria requires either gl y can-dependent binding of galectin-8 to damaged membranes and subsequent recruitme nt of the cargo receptor CALCOCO2/NDP52 773 or ubiquitination of target proteins (not y et identifi ed) and recruitme nt of 3 ubiqui tin-binding rece ptor proteins, SQSTM1, 774 CAL C OCO2/ NDP52 775 and OPTN. 776 Therefore, the c urrentl y available criteria to differentiate L AP from macroautophagy include: i) LAP involves L C 3 recruitment to ba cteria in a manner that requires reactive ox y gen species (ROS) production b y an NADPH oxidase. I t should be noted that most cells express at least one member of the NADPH oxidase (also known as NOX) famil y . Targeting expression of the common p22 p hox subunit is an effective way to disrupt t he NOX NADPH oxidases. Scavenging of ROS by antioxidants such as resveratrol and a lpha-toc opherol is also an e ffective wa y to inhibi t L AP. In contrast, N- acetylcysteine , which raises cellular glutathione levels, does not inhibit LAP. 777 ii) Macroa utopha gy of bacteria requires A TG 9, while L AP apparently does not. 772 iii) L AP involves single-membrane structures. For L AP, CLEM (with L C3 as a marker) is expected to show single-membrane structures that are LC3 + with LAP. 1 53 I n contrast, macroautophagy is expected to genera te double-memb rane structures surrounding cargo (which may include single me mbrane phag osomes, giving rise to triple-membrane struct ures 772 ). I t is anticipated t hat more spec ific markers of LAP will be identified as these phagosomes are further characterized. Nonmotile Listeria monocytogene s can be tar g eted to double-membrane autophagosomes upon antibiotic treatment, 778 which indicates that macroa utophagy serves as a cellular defense to microbes in the cytosol. Howe ver, subsequent studies have revealed that macroa utopha gy can also target pathoge ns within phagosomes, damaged phagosomes or the c y tosol. Therefore, when studying microbial interactions by EM , man y structures can be visualized, with any number of membranes encompassing microbes, all of which may be LC3 + . 779 As discussed above , single- 151 membrane struc tures that are LC3 + ma y arise through LAP, and we cannot rule out the possibilit y that both LAP and ma croautophagy may operate at the same time to target the same phag osome. Viruses can also be targeted by autophagy, a nd in turn can act to inhibit autophagy. For example, infection of a cell by influenza and dengue viruses 780 or enforced expression of the hepatitis B virus C protein 781 have profound consequences for autophagy , as viral proteins such as NS4A stimulate autophagy and protect the infected cell against apoptosis, thus exten ding the time in which the virus can replica te. C onverse l y , the HSV I CP34.5 protein inhibits autophagy by targeting BECN1 . 782 While the impact of I C P34.5’s targeting of BECN1 on virus re plication in cultured permissive cells is minimal, it has a significant impact upon pathoge nesis in vivo, most likel y throu gh interfering with activation of CD4 + T ce lls , 783,784 and throug h cell-intrinsic antiviral effects in neur ons. 785 Also, viral B CL 2, encode d b y la rge DNA viruses, are able to inhibit autophagy interacting with B ECN1 515 through their BH3 homolog y domain. An e xample of these include murine herpes virus 68, 786 Kaposi sa rcoma herpes virus and African swine fever virus (ASFV) vB CL 2 homologs. 787 ASFV e ncodes a protein homologous to HSV I CP34.5 , which, similar to its herpes virus counterpart, inhibits the ER stress response ac tivating PPP1/protein phosphatase1; however, in contrast t o HSV I CP34.5 it does n ot interact with BECN1. ASFV vB CL 2 strongl y inhibits both autophagy (reviewed in ref. 788 ) and apoptosis. 789 HIV utilizes the initial, nondegr adative stages of autophagy to promote its replication in macrophages. In a ddition, the H I V-1 protein Nef acts as an anti-autopha gic maturation fa ctor protecting the virus from degradation b y ph ysically blocking BECN1. 790 -792 Autophagy contributes to limiting viral pathoge nesis in H I V- 1 nonprogre ssor-infected pat ients by targeting viral components for degradation. 793 152 Care must be take n in determining the role of autophagy in virus replica tion, as some viruses such as vaccinia use double-membrane structures that form independent ly of the autophagy mac hiner y . 794 Similarly , d engue virus replication, which a ppears to inv olve a double- membrane c ompartment, require s the ER rather than autophagosomes, 795 where as coronaviruses use a nonlipidated version of L C3 (see Atg8/LC3 detection and quantification ). 161 Yet another type of varia tion is seen with hepatitis C virus, which requires BECN1, ATG4B, ATG5 and ATG12 for initiating replication, but does not require these proteins once an infection is established. 79 6 Finally , it is important to realize that there ma y be other macroautophag y -like pathways that have yet to be characterized. For example, in response to cytotoxic stress (treatment with etoposide), autophag osomes are formed in an A TG 5- a nd A TG 7-independent manner. 25 While this does not rule out involvement of other macroautophagy reg ulators/components in the formation of these autophag osomes, it does establish t hat the c anonical macroa utophag y p athway involving L C3 conjugation is not involved. I n contrast, R AB 9 is required for th is alternative pathway, potentially providing a useful marker f or anal y sis of these structures. Returning to xenophagy, M. tuberculosis can be targeted to autophag osomes in an A TG 5-independe nt manner. 797 F urthermore , up to 25% of intracellular Salmonella typhimurium are observed in multi-lamellar membrane structures re sembling autophagosomes in Atg5 -/- MEFs. 774 These findings indicate that an alternate macroautophagy pathwa y is relevant to host -pathogen interactions. Moreover, differe nces are observed that depend on the cell type being studied. Yersinia pseudotuberculosis is targeted to autophag osomes where the y c an replicate in bone marrow-derived macrophages, 798 where as in RAW264.7 and J774 cells, bac teria are targeted 153 both to autophagosome s, and L C 3-negative, single-membrane vacuoles (F. Lafont, personal communication). One key c onsideration has recentl y em erge d in stud y in g xenophagy. Whereas the basal autophagy flux in most cells is essential for their survival, infectin g pathogens can selectivel y modulate antibacter ial autophag y (i.e., x enophagy) without influencing basal autophagy. This may help pathogens en sure prolonged cellular survival. Thus, in the case of xenophag y it would be prudent to monitor substrate ( pathogen)-specific autopha gy flux to understand the true nature of the perturbation of infecting patho g ens on autophag y (D Kumar, personal communication). Further more, this consideration particularl y limits the sensitivity o f LC3 w estern blots for use in monitoring autophagy regulation. n. Zymophagy . Zymophagy Zy mophagy refers to a specific mechanism of crinophag y (i.e., selective degrada tion of secretor y gra nules b y autophag y ) , which eliminates pancreatitis - activated zy mogen granules in the pancreatic acinar c ells and, thus, prevents deleterious effect s of prematurely activated and intracellularly released proteoly tic enz y m es, when impairment of secre tor y function occurs. 7 99 Therefor e, z y mopha gy is considered to be a protective mechanism implemented to sustain secre tor y homeosta sis and to mitigate pancreatitis. Crinophagy processing of secre tor y granules similar to the defined z ymophagy was reported in activated secre tor y P aneth cells of the crypts of Lieberkuhn in the small int estine. 800 Note that one of the major functions of Paneth cells is to prevent translocation of intestinal bacteria by secreting hydr ol y tic enz y mes and antibac terial peptides to the crypt lumens. The presence of similar it y in mechanisms of degrada tion of secretor y granules in these 2 different t y pes of secretory cells 256 b. The human autophagy database (HADb). The human autophagy database, developed in the La borator y o f Experimental Hemato-Oncol ogy (Luxembourg), lists over 200 human genes/proteins related to autophagy . 545 These entries have been manually collected from the biomedica l literature and other online resources 545 and there is curre ntl y no information that the initiall y publishe d list has been fur ther updated. For each gene there e xists information on its s equence, transcripts and isofor ms (including exon boundaries) as we ll as links to ex ternal re sources. HADb provides ba sic search and browsing functionalities and is publicly available online at the URL http://autophag y .lu/ . c. The Autophagy Database. The Autophagy Database is a multiface ted online resource providing infor mation for proteins related to autophag y and their homologs across several eukaryotic species, w ith a focus on functional and structural data. 1298 It is deve loped b y the National Institute of Genetics (Japan) under the Targeted Proteins Research Program of the Ministry of E ducation, C ulture, Sports, Scienc e and Technology (MEXT) (http://www.tanpaku.org/). This resource is regularly upda ted and as of August 2014 contained information regarding 312 reviewed protein entries; when additional data regarding orthologous/homol og ous proteins from more than 50 eukaryotes is considered, the total number of entries rea ches approximatel y 9,000. In addition to the browse functionalities offe red under the “Protein List” and the “Homologs” menus, an instance of the NCBI -B LAST softwa re facilitates se quence -base d queries against the database entries. Moreover, interested users ma y download the gene list or the a utophag y dump files licensed under a Creative Commons Attribution-ShareAlike 2.1 Japan L icense. The 257 Autophagy Database is p ublicly available online at the URL http://www.tanpaku.org /autophag y /ind ex.html . d. The Autophagy Regulatory Network (ARN). A recent addition to the web -based resources re levant to autophag y research is the A utophagy Reg ulator y Network (ARN), developed a t the Eötvös Loránd Universit y and Semmelweis Universit y (Hungary) in collaboration with the I nstitut e of F ood Research and The Ge nome Anal y sis Centre (TGAC, United Kingdom). Maintanence and hosting the ARN resource is sec ured at TGAC until at least 2019. ARN is an integrated systems-level resource aiming to collect and provide an interac tive user interface enabling access to validated or pre dicted protein- protein, transcr iption factor -gene and miRNA-mRNA interactions related to autophag y in human. 1299 ARN contains data f rom 26 resources, including an in-house extensive manual cura tion, the dataset of the Ch I P -MS stud y of B ehrends et al., 416 ADB and ELM. As of June 2015, a total of more than 14,000 proteins and 386 miRNAs are included in ARN, including 38 core autophagy prote ins and 1 13 predicte d regulators. Importantl y , all autophagy-related pro teins are linked to major signaling pathwa y s. A flex ible — in terms of both content and for mat — download functionalit y enables users to locally use the ARN data under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. The autophag y reg ulator y network resource is publicly available online at the URL http://autophagy-regulation.org. e. Prediction of Atg8-fam ily interacting proteins . Being centra l components of the autophagic core mac hiner y , Atg8-famil y members (e.g., LC3 and GABARAP in 258 mammals) and their interactome ha ve attracted a lot of interest. 416 ,130 0,1301 Dur ing the last decade, a number of proteins have been shown to intera ct with Atg8 homologs via a short linear peptide; depending on conte xt, different research groups have described this peptide as the LC3-interacting region (LIR) 276 , the L C3 recognition sequence (LRS) 591 , or the Atg8-fa mil y interacting motif (AIM) 13 02 . Rec ently, two independe nt efforts resulted in the first online a vailable tools for identification of these motifs (LIR-motifs for brevity) in c ombination with ot her se quence features, which may sig nif y inte resting targets for f urther validation. f. The iLIR server. The iLIR server is a spec ialized web server that scans an input se quence for the presence of a degenerate version of LI R, th e extended LIR -motif (xLI R). 13 03 Currently , th e server also reports additional matches to the “canonical” LIR motif (WxxL), de scribed b y the simple regular e xpression x(2) -[WFY]-x(2)-[ LI V]. Kalvari and colleag ues have also compiled a position -spec ific scoring matrix (PSSM) based on validated insta nces of the LIR motif, demonstrating that many of the fa lse positive hits (i.e., spurious matches to the xLIR motif) a re eliminated when a PSSM score >15 is sought. In addition, iLI R also overla y s the afore mentioned results to segments t hat reside in or are a djacent to disordered regions and are likel y to form stabiliz ing interactions upon binding to another g lobular protein as predicted by the ANCHOR package. 1304 A combination of a n x L IR matc h with a high PSSM score (>13) and/or an overlap with an ANCHOR segment is shown to give re liable predictions. 1 303 It is worth mentioning that, intentionally , iLIR does not provide explicit predictions o f functional LI R-motifs but rather displa y s all the above information accompanie d b y a graphical 259 depiction of query matches to known protein domains and motifs; it is up to the user to interpret the iLI R output. As mentioned in the original iLIR publication, a limitation of this tool is that it does not handle any non-canonical LIR-motifs at present. The iLIR server is jointly developed b y the Univ ersity of Warwick and Universit y of C y prus and is free l y available online at the URL http://repeat.biol.ucy.ac.cy /iLIR. g. The Eukaryotic Linear Motif resource (ELM) . The Euka ryotic Linear Motif resource (EL M) 13 05 is a g eneric resource f or examining functional sites in proteins in the form of short linear motifs, which have been manually c urated from the literature. Sophisticated filters based on known (or predicted) query fea tures ( such as taxonomy, subcellular localization, structural context) are used to nar row down the results lists, which can be very long lists of potential matches due to the short lengths of ELMs. This resource has incorporated 4 entries related to the LIR-motif (si nce May 2014; http://elm.eu.org/infos/news.html), while another 3 are being evaluated as candidate ELM additions ( Table 3 ). Again, the EL M resource displays matches to any motifs and users are left with the decision as to which of them are worth studying further. ELM is developed/maintained by a consortium of Eur opean groups coordinated b y t he European Molecular Biology Laboratory (EMBL) and is freely available online at the URL http://elm.eu.org. Conclusions and future perspective s There is no que stion that research on the topic o f autophagy has expanded dramaticall y since the publication of the first set of guidelines. 1 To help keep track of the field we have published a 260 glossary of autophag y -related molecules and processes , 1306,1307 and now incl ude the glossary as part of these g uidelines. With this continued influx of new researchers we think it i s critica l to try to def ine standards for the field. Accordingly, we have highlighted the uses and ca veats of an expanding set of rec ommended methods for monitoring macroautophag y in a wide range of s y stems ( Table 4 ). I mportantl y , inv estigators need to determine whether the y a re evaluating lev els of early or late autophagic c ompartments, or autophagic f lux. I f the question being asked is whether a particular condition change s autophagic flux (i.e., the rate of delivery of autophagy substrates to lysosomes or the vacuole, followed b y degradation), then assessment of steady state levels of autophagosomes (e .g., b y c ounting GFP-LC3 puncta, monitoring the amount of LC3- II without examining turnover, or by sin g le time point electron micrographs) is not sufficient as an isolated approac h. I n this case it is also necessar y to directly measure the flux of autophag osomes and/or autophagy cargo (e .g., in wild -ty pe cells compared to autophagy-deficient cells, the latter ge nerated b y treatment with an autophag y inhi bitor or re sultin g from A TG ge ne knockdowns). Collectively, we strongl y recommend the use of multiple assay s whenever possible, rather than rely ing on the results from a single method. As a final re minder, we stated at the beginning of this article that this set of g uidelines is not meant to be a for mulaic compilation of rules, beca use the appropriate assays depend in par t on the question being ask ed and the sy stem being used. R ather , these guidelines are presented primarily to emphasize ke y issues th at need to be addre ssed such as the differenc e between measuring autophagy components, and flux or substrate c learance; the y a re not meant to constrain imaginative a pproaches to monitorin g autophagy . I ndeed, it is hoped that new methods for monitoring a utopha gy will continue to be developed, and new findings may alter our view of 261 the current assays. Similar to the proc ess of autophag y , this is a dyna mic field, and we need to remain flexible in the standards w e apply. Acknowledgments In a ra pidl y expanding and highl y d yna mic field such as autophagy, it is possible that some authors who should have been include d on this manuscript have been missed. D.J.K. ex tends his apologies to researc hers i n the field of autophagy who, due to oversight or any other reason, could not be included on this manuscript. This work was supported in part by National Institutes of Health, including Public Health Service gra nt GM053396 to D.J .K. Due to space and other limitations, it is not possible to include all other sources of financ ial support. 262 Table 1. Genetic and pharmac ological regulation of autophagy. 1 Method Comments 1. 3-methy ladenine A PtdIns3K inhibitor that effectivel y blocks an early stage of autophagy by inhibiting the class III PtdIns3K, but not a specific a utopha gy inhibitor. 3-MA also inhibit s the class I Ptd I ns3K and can thus, at suboptimal concentra tions in long-term experiments, promote autophagy in some systems, as well as affec t cell survival through AK T and other kinases. 3-MA does not inhibit BECN1-independent autophagy. 2. 10-NCP 10- (4′ -N-diethy lamino)but y l) -2-chlorophenoxazine; an AKT inhibitor that induces autophagy in neurons. 1 049 3. 17- AAG An inhibitor of the HSP90-CDC37 chaperone complex ; induces autophagy in certain sy stems (e.g., neurons), but impairs starvation-induced autophagy a nd mitophag y in others by promoting the turnover of ULK1. 41 0 4. Akti-1/2 An allosteric inhibitor of AKT1 and AKT2 that promotes autophag y in B- cell ly mphoma. 1308 5. AR7 AR7 was developed as a highly potent and se lective enhancer of CMA through antagonizing RARA/RAR α ; AR7 is the first small molecule de veloped to selectively 1 This table is not meant to be complete, a s there are man y compounds and genetic methods that regulate a utophag y , and new ones are being discovered routinel y . 263 stimulate CMA without affe cting macroautophagy . 1309 6. ARN5187 Ly s osomotropic compound with a dual inhibitor y activity against the circadian regulator NR1D2/REV-ERB  and autophag y . 1 310 7. ATG4 C74A An active site mutant of ATG4 that is defective for autophagy. 1311 8. Bafilomycin A 1 A V-ATPase inhibitor that causes an increase in lysosomal/vac uolar pH, and , ult imately , blocks f usion of autophagosomes with the vacuole ; the latter ma y r esult from inhibition of ATP2A/SERCA . Mauvezin, 20 15 #3456} 9. Calcium An autophagy activator that ca n be released from ER or lysosomal stores under stress conditions; however, calcium can also inhibit autophagy . 1090 10. Chloroquine, NH 4 Cl Ly s osomotropic compounds that elevate/neutralize the ly sosomal/vacuolar pH. 11. DFMO α -difluoromethylornithine, an irreve rsible inhibitor of ODC1 (ornithine decarbox y lase 1) that blocks spermidine synthesis and ATG gene expression. 12. E-64d A membrane-permeable cysteine protease inhibitor that ca n 264 block the activity of a subset of l y sosom al hydrolases; should be used in combination with pepstatin A to inhibit lysosomal protein degradation. 13. ESC8 A cationic estradiol derivative that induces autophagy and apoptosis simultaneousl y b y down regulating the MTOR kinase pathway in breast cancer cells. 14. Everolimus An inhibitor of MTORC1 that induces both autophagy and apoptosis in B-cell l y mp homa primary culture s. 1308 15. Fumonisin B1 An inhibitor of ceramide s y nthesis t hat interfere s with macroautophagy . 16. Gene d e letion This method provides the most direct evidence for the role of an autophag ic compon ent; howeve r, more than one gene involved in autophagy should be targeted to avoid indi rec t effects. 17. HMOX1 induction Mitophagy a nd the formation of iron -containing cytoplasmic inclusions and corpora am y l acea are accelerated in HMOX1 -transfected rat astroglia and astrocytes of GFAP. HMOX1 transgenic mice. Heme- derived ferrous iron and carbon monoxide, products of the HMOX1 reaction, promote macroautophagy in these cells. 1312-1314 18. Knockdown This method (including miRNA, RNAi, shRNA and siRNA) can be used to inhibit gene expression and provides 265 relativel y direct evidence for the r ole of an autophagic component. However, the efficiency of knockdown varies, as does the stability of the targe ted protein. In addition, more than one gene involved in autophagy should be targeted to avoid misinterpreting indirec t effects. 19. KU -0063794 An MTOR inhibitor that binds the cataly tic site an d activates a utophag y . 297 ,1315 20. Le upeptin An inhibitor of cy steine, serine and threonine proteases that can be used in combination with pepstatin A and/or E- 64d to block lysosomal protein degra dation. Leupeptin is not membrane permea ble, so its effect on cathepsins ma y depend on endocy tic activit y . 21. microRNA Can be used to re duce the levels of target mRNA(s) or block translation. 22. MLN4924 A small molecule inhibitor of NAE (NEDD8 activating enzyme); 1316 induces autophagy by blockage of MTOR signals via DEPTOR and the HIF 1A-DD IT4/REDD1- TSC1/2 axis as a result of inactivation of cullin-RI NG ligases. 1317 - 1319 23. NAADP - AM Activates the lysosomal two por e channel and induces autophagy. 1074 24. NED-19 Inhibits the ly sosomal two-pore channel and NAADP - in duced a utophag y . 1074 272 the LI R moti f that binds to Atg8 protein famil y members to mediate processes invol ved in autophagy. LI G_A I M [WY]..[ILV] Atg8-family interacting motif (AI M) found in Atg19, SQSTM1/ p62, A TG 4B and CALR/calreticulin, involved in autophagy - related processes. Candidate LI G_ LI R WxxL or [W YF]xx[LI V] LC3-interac ting region (LI R) mig ht link ubiquitinated substrates that should be degra ded to the autophag y -related proteins in the phag ophore membrane. Candidate LI G_GABARAP W.FL GABA A receptor binding to clathrin and CAL R; possibl y linked to trafficking. Candidate 273 Table 4 . Recommended methods for monitoring autophagy. Method Description 1. Electron microsc op y Quantitative elec tron microscop y , immuno- TEM ; monitor autophagosome number, volume , and content/cargo 2. Atg8/LC3 we stern blotting Western blot. The analysis is carried out in the absence and presence of ly sosomal protease or fusion inhibitors to monitor flux ; an increase in the L C3- II amount in the presence of the inhibitor is us ually indicative of flux 3. GFP-Atg8/L C3 l y sosomal deliver y and Western blot +/- l y sosom al fusion or proteoly sis degrada tion inhibitors; the generation of free GFP indicates lysosomal/vac uolar delivery 4. GFP-Atg8/L C3 fluorescence microscop y Fluore scence microscop y, F ACS to monitor vacuolar/lysosomal loca liz ation. Also, increase in punctate GFP-Atg8/LC3 or Atg18/WI PI , and live time-lapse fluoresce nce microscop y to track the dyna mics of GFP-Atg8/ LC3-positive structures 274 5. Tandem mRFP/mCherry-GFP fluorescence Flux can be monitored as a dec rease in microscopy, Rosella green/red (ye llow) fluorescence (phagophore s, autophagosomes) and an increase in red fluorescence (a utol y sosomes) 6. Autophag osome quantifi cation Flow c y tometr y 7. SQSTM1 and related LC3 binding protein The amount of SQSTM1 increases when turnover autophag y is i nhibited and dec reases when autophag y is i nduced , but the potential impact of transcriptional/translational regulation or the formation of insoluble aggregates should be addressed in individual experimental systems 8. MTOR, AMPK and Atg1/ULK1 kinase activity Western blot, immunoprecipitation or kinase assa y s 9. W I P I fluor escence microscopy Quantitative fluore scence analysis using endogenous WI PI protei ns, or GF P- or M y c- tagged versions. Suitable for hig h - throughput imaging procedure s. 10. Bimolecular fluorescence complementation Can be used to monitor protein-protein interaction in vivo 11. FRET Interaction of LC3 with gang liosides to monitor autophagosome formation 12. Transcriptional and translational regulation Northern blot, or qRT-PCR, autophagy- 275 dedicated micr oarra y 13. Autophagic protein degradation Turnover of long-lived proteins to monitor flux 14. Pex 14-GF P, GFP-Atg8, Om45-GFP, A range of assays can be used to monitor mitoPho8∆60 selective types of autophagy . Th ese t y pic ally involve proteoly tic maturation of a resident enzyme or de g radation of a chimera, whic h can be f ollowed enz y m atically or b y western blot 15. Autophagic sequestration assa y s A ccumulation of cargo in autophagic compartments in the presence of ly sosomal protease or fusion inhibitors b y bioch emical or multilabel fluorescence techniques 16. Turnover of autophagic compartments Electron microscopy with morphometry /stereolog y at different time points 17. Autophagosome-l y sosome coloca lization Fluore scence microscopy and dequenching assa y 18. Sequestration and processing assays Chimeric RFP fluorescence a nd processing, in plants and light and electron microscop y 19. Tissue fractionation Centrifugation, western blot and elec tron microscopy 20. Degradation of endogenous lipofuscin Fluorescence microscopy 276 277 Refere nces 1. 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