TY  - JOUR
TI  - Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
AU  - Daniel J. Klionsky
AU  - Kotb Abdelmohsen
AU  - Akihisa Abe
AU  - Md. Joynal Abedin
AU  - Hagai Abeliovich
AU  - Abraham Acevedo‐Arozena
AU  - Hiroaki Adachi
AU  - Christopher M. Adams
AU  - Peter D. Adams
AU  - Khosrow Adeli
AU  - Peter J. Adhihetty
AU  - Sharon G. Adler
AU  - Galila Agam
AU  - Rajesh Agarwal
AU  - Manish K. Aghi
AU  - Maria Agnello
AU  - Patrizia Agostinis
AU  - Patricia V. Aguilar
AU  - Julio A. Aguirre‐Ghiso
AU  - Edoardo M. Airoldi
AU  - Slimane Ait‐Si‐Ali
AU  - Takahiko Akematsu
AU  - Emmanuel T. Akporiaye
AU  - Mohamed Al‐Rubeai
AU  - Guillermo M. Albaiceta
AU  - Chris Albanese
AU  - Diego Albani
AU  - Matthew L. Albert
AU  - Jesús Aldudo
AU  - Hana Algül
AU  - Mehrdad Alirezaei
AU  - Iraide Alloza
AU  - Alexandru Almasan
AU  - Maylin Almonte-Beceril
AU  - Emad S. Alnemri
AU  - Covadonga Alonso
AU  - Nihal Altan‐Bonnet
AU  - Dario C. Altieri
AU  - Silvia Álvarez
AU  - Lydia Alvarez‐Erviti
AU  - Sandro Alves
AU  - Giuseppina Amadoro
AU  - Atsuo Amano
AU  - Consuelo Amantini
AU  - Santiago Ambrosio
AU  - Ivano Amelio
AU  - Amal O. Amer
AU  - Mohamed Amessou
AU  - Angelika Amon
AU  - Zhenyi An
AU  - Frank A. Anania
AU  - Stig Uggerhøj Andersen
AU  - Usha P. Andley
AU  - Catherine Andreadi
AU  - Nathalie Andrieu‐Abadie
AU  - Alberto Anel
AU  - David K. Ann
AU  - Shailendra Anoopkumar‐Dukie
AU  - Manuela Antonioli
AU  - Hiroshi Aoki
AU  - Nadezda Apostolova
AU  - Saveria Aquila
AU  - Katia Aquilano
AU  - Koichi Araki
AU  - Eli Arama
AU  - Agustı́n Aranda
AU  - Jun Araya
AU  - Alexandre Arcaro
AU  - Esperanza Arias
AU  - Hirokazu Arimoto
AU  - Aileen Ariosa
AU  - Jane L. Armstrong
AU  - Thierry Arnould
AU  - Ivica Arsov
AU  - Katsuhiko Asanuma
AU  - Valerie Askanas
AU  - Éric Asselin
AU  - Ryuichiro Atarashi
AU  - Sally S. Atherton
AU  - Julie D. Atkin
AU  - Laura D. Attardi
AU  - Patrick Auberger
AU  - Georg Auburger
AU  - Laure Aurelian
AU  - Riccardo Autelli
AU  - Laura Avagliano
AU  - Maria Laura Avantaggiati
AU  - Limor Avrahami
AU  - Suresh Awale
AU  - Neelam Azad
AU  - Tiziana Bachetti
AU  - Jonathan Backer
AU  - Dong-Hun Bae
AU  - Jae‐sung Bae
AU  - Ok‐Nam Bae
AU  - Soo Han Bae
AU  - Eric H. Baehrecke
AU  - Seung‐Hoon Baek
AU  - Stephen Baghdiguian
AU  - Agnieszka Bagniewska‐Zadworna
PY  - 2016
JO  - Autophagy
DO  - 10.1080/15548627.2015.1100356
UR  - https://doi.org/10.1080/15548627.2015.1100356
AB  - In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, 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 forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays 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 autophagyrelated 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 specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
ER  -