Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
- Authors: Klionsky, D.; Abdelmohsen, K.; Abe, A.; Abedin, M.; Abeliovich, H.; Acevedo Arozena, A.; Adachi, H.; Adams, C.; Adams, P.; Adeli, K.; Adhihetty, P.; Adler, S.; Agam, G.; Agarwal, R.; Aghi, M.; Agnello, M.; Agostinis, P.; Aguilar, P.; Aguirre-Ghiso, J.; Airoldi, E.; Ait-Si-Ali, S.; Akematsu, T.; Akporiaye, E.; Al-Rubeai, M.; Albaiceta, G.; Albanese, C.; Albani, D.; Albert, M.; Aldudo, J.; Algül, H.; Alirezaei, M.; Alloza, I.; Almasan, A.; Almonte-Beceril, M.; Alnemri, E.; Alonso, C.; Altan-Bonnet, N.; Altieri, D.; Alvarez, S.; Alvarez-Erviti, L.; Alves, S.; Amadoro, G.; Amano, A.; Amantini, C.; Ambrosio, S.; Amelio, I.; Amer, A.; Amessou, M.; Amon, A.; An, Z.; Anania, F.; Andersen, S.; Andley, U.; Andreadi, C.; Andrieu-Abadie, N.; Anel, A.; Ann, D.; Anoopkumar-Dukie, S.; Antonioli, M.; Aoki, H.; Apostolova, N.; Aquila, S.; Aquilano, K.; Araki, K.; Arama, E.; Aranda, A.; Araya, J.; Arcaro, A.; Arias, E.; Arimoto, H.; Ariosa, A.; Armstrong, J.; Arnould, T.; Arsov, I.; Asanuma, K.; Askanas, V.; Asselin, E.; Atarashi, R.; Atherton, S.; Atkin, J.; Attardi, L.; Auberger, P.; Auburger, G.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avantaggiati, M.; Avrahami, L.; Awale, S.; Azad, N.; Bachetti, T.; Backer, J.; Bae, D.; Bae, J.; Bae, O.; Bae, S.; Baehrecke, E.; Baek, S.; Baghdiguian, S.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X.; Bailly, Y.; Balaji, K.; Balduini, W.; Ballabio, A.; Balzan, R.; Banerjee, R.; Bánhegyi, G.; Bao, H.; Barbeau, B.; Barrachina, M.; Barreiro, E.; Bartel, B.; Bartolomé, A.; Bassham, D.; Bassi, M.; Bast, R.; Basu, A.; Batista, M.; Batoko, H.; Battino, M.; Bauckman, K.; Baumgarner, B.; Bayer, K.; Beale, R.; Beaulieu, J.; Beck, G.; Becker, C.; Beckham, J.; Bédard, P.; Bednarski, P.; Begley, T.; Behl, C.; Behrends, C.; Behrens, G.; Behrns, K.; Bejarano, E.; Belaid, A.; Belleudi, F.; Bénard, G.; Berchem, G.; Bergamaschi, D.; Bergami, M.; Berkhout, B.; Berliocchi, L.; Bernard, A.; Bernard, M.; Bernassola, F.; Bertolotti, A.; Bess, A.; Besteiro, S.; Bettuzzi, S.; Bhalla, S.; Bhattacharyya, S.; Bhutia, S.; Biagosch, C.; Bianchi, M.; Biard-Piechaczyk, M.; Billes, V.; Bincoletto, C.; Bingol, B.; Bird, S.; Bitoun, M.; Bjedov, I.; Blackstone, C.; Blanc, L.; Blanco, G.; Blomhoff, H.; Boada-Romero, E.; Böckler, S.; Boes, M.; Boesze-Battaglia, K.; Boise, L.; Bolino, A.; Boman, A.; Bonaldo, P.; Bordi, M.; Bosch, J.; Botana, L.; Botti, J.; Bou, G.; Bouché, M.; Bouchecareilh, M.; Boucher, M.; Boulton, M.; Bouret, S.; Boya, P.; Boyer-Guittaut, M.; Bozhkov, P.; Brady, N.; Braga, V.; Brancolini, C.; Braus, G.; Bravo-San Pedro, J.; Brennan, L.; Bresnick, E.; Brest, P.; Bridges, D.; Bringer, M.; Brini, M.; Brito, G.; Brodin, B.; Brookes, P.; Brown, E.; Brown, K.; Broxmeyer, H.; Bruhat, A.; Brum, P.; Brumell, J.; Brunetti-Pierri, N.; Bryson-Richardson, R.; Buch, S.; Buchan, A.; Budak, H.; Bulavin, D.; Bultman, S.; Bultynck, G.; Bumbasirevic, V.; Burelle, Y.; Burke, R.; Burmeister, M.; Bütikofer, P.; Caberlotto, L.; Cadwell, K.; Cahova, M.; Cai, D.; Cai, J.; Cai, Q.; Calatayud, S.; Camougrand, N.; Campanella, M.; Campbell, G.; Campbell, M.; Campello, S.; Candau, R.; Caniggia, I.; Cantoni, L.; Cao, L.; Caplan, A.; Caraglia, M.; Cardinali, C.; Cardoso, S.; Carew, J.; Carleton, L.; Carlin, C.; Carloni, S.; Carlsson, S.; Carmona-Gutierrez, D.; Carneiro, L.; Carnevali, O.; Carra, S.; Carrier, A.; Carroll, B.; Casas, C.; Casas, J.; Cassinelli, G.; Castets, P.; Castro-Obregon, S.; Cavallini, G.; Ceccherini, I.; Cecconi, F.; Cederbaum, A.; Ceña, V.; Cenci, S.; Cerella, C.; Cervia, D.; Cetrullo, S.; Chaachouay, H.; Chae, H.; Chagin, A.; Chai, C.; Chakrabarti, G.; Chamilos, G.; Chan, E.; Chan, M.; Chandra, D.; Chandra, P.; Chang, C.; Chang, R.; Chang, T.; Chatham, J.; Chatterjee, S.; Chauhan, S.; Che, Y.; Cheetham, M.; Cheluvappa, R.; Chen, C.; Chen, G.; Chen, G.; Chen, G.; Chen, H.; Chen, J.; Chen, J.; Chen, M.; Chen, M.; Chen, P.; Chen, Q.; Chen, Q.; Chen, S.; Chen, S.; Chen, S.; Chen, W.
- Publication year: 2016
- Type: Review essay (rassegna critica) (Articolo in rivista)
- OA Link: http://hdl.handle.net/10447/166323
Abstract
In 2008 we published the fi rst set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the fi eld. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between meas urements that monitor the num- bers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the com- plete process including the amoun t and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accu mulation must be differentiated from stimuli that increas e autophagic activity, de fi ned as increased autophagy induction coupled with in creased delivery to, and degra- dation within, lysosomes (in most higher eukaryotes and some pro- tists such as Dictyostelium ) or the vacuole (in plants and fungi). In otherwords,itisespeciallyimportantthatinvestigatorsnewtothe fi eld understand that the appearance of more autophagosomes does not necessarily equate with mo re autophagy. In fact, in many cases, autophagosomes accumu late because of a block in traf fi cking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may re fl ect a reductionindegradativeactivity .Itisworthemphasizingherethat lysosomal digestion is a stage of au tophagy and evaluating its com- petence is a crucial part of the evaluation of autophagic fl ux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appro- priate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no indi- vidual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy- related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a speci fi c marker for an autophagic process. In these guidelines, we con- sider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the fi eld