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

Abdelmohsen, Kotb; Abe, Akihisa; Abedin, Md Joynal; Abeliovich, Hagai; Acevedo Arozena, Abraham; Adachi, Hiroaki; Adams, Christopher M; Adams, Peter D; Adeli, Khosrow; Adhihetty, Peter J; Adler, Sharon G; Agam, Galila; Agarwal, Rajesh; Aghi, Manish K; Agnello, Maria; Agostinis, Patrizia; Aguilar, Patricia V; Aguirre-Ghiso, Julio; Airoldi, Edoardo M; Ait-Si-Ali, Slimane; Akematsu, Takahiko; Akporiaye, Emmanuel T; Al-Rubeai, Mohamed; Albaiceta, Guillermo M; Albanese, Chris; Albani, Diego; Albert, Matthew L; Aldudo, Jesus; Alg?l, Hana; Alirezaei, Mehrdad; Alloza, Iraide; Almasan, Alexandru; Almonte-Beceril, Maylin; Alnemri, Emad S; Alonso, Covadonga; Altan-Bonnet, Nihal; Altieri, Dario C; Alvarez, Silvia; Alvarez-Erviti, Lydia; Alves, Sandro; Amadoro, Giuseppina; Amano, Atsuo; Amantini, Consuelo; Ambrosio, Santiago; Amelio, Ivano; Amer, Amal O; Amessou, Mohamed; Amon, Angelika; An, Zhenyi; Anania, Frank A; Andersen, Stig U; Andley, Usha P; Andreadi, Catherine K; Andrieu-Abadie, Nathalie;...

Authors

Kotb Abdelmohsen

Akihisa Abe

Md Joynal Abedin

Hagai Abeliovich

Abraham Acevedo Arozena

Hiroaki Adachi

Christopher M Adams

Peter D Adams

Khosrow Adeli

Peter J Adhihetty

Sharon G Adler

Galila Agam

Rajesh Agarwal

Manish K Aghi

Maria Agnello

Patrizia Agostinis

Patricia V Aguilar

Julio Aguirre-Ghiso

Edoardo M Airoldi

Slimane Ait-Si-Ali

Takahiko Akematsu

Emmanuel T Akporiaye

Mohamed Al-Rubeai

Guillermo M Albaiceta

Chris Albanese

Diego Albani

Matthew L Albert

Jesus Aldudo

Hana Alg�l

Mehrdad Alirezaei

Iraide Alloza

Alexandru Almasan

Maylin Almonte-Beceril

Emad S Alnemri

Covadonga Alonso

Nihal Altan-Bonnet

Dario C Altieri

Silvia Alvarez

Lydia Alvarez-Erviti

Sandro Alves

Giuseppina Amadoro

Atsuo Amano

Consuelo Amantini

Santiago Ambrosio

Ivano Amelio

Amal O Amer

Mohamed Amessou

Angelika Amon

Zhenyi An

Frank A Anania

Stig U Andersen

Usha P Andley

Catherine K Andreadi

Nathalie Andrieu-Abadie

Alberto Anel

David K Ann

Shailendra Anoopkumar-Dukie

Manuela Antonioli

Hiroshi Aoki

Nadezda Apostolova

Saveria Aquila

Katia Aquilano

Koichi Araki

Eli Arama

Agustin Aranda

Jun Araya

Alexandre Arcaro

Esperanza Arias

Hirokazu Arimoto

Aileen R Ariosa

Jane L Armstrong

Thierry Arnould

Ivica Arsov

Katsuhiko Asanuma

Valerie Askanas

Eric Asselin

Ryuichiro Atarashi

Sally S Atherton

Julie D Atkin

Laura D Attardi

Patrick Auberger

Georg Auburger

Laure Aurelian

Riccardo Autelli

Laura Avagliano

Maria Laura Avantaggiati

Limor Avrahami

Suresh Awale

Neelam Azad

Tiziana Bachetti

Jonathan M Backer

Dong-Hun Bae

Jae-sung Bae

Ok-Nam Bae

Soo Han Bae

Eric H Baehrecke

Seung-Hoon Baek

Stephen Baghdiguian

Agnieszka Bagniewska-Zadworna

Daniel J Klionsky



Abstract

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that
there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete
process 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 increased
autophagy 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 many
cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. 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 for
reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple
assays to monitor autophagy. 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.

Journal Article Type Article
Acceptance Date Sep 22, 2015
Online Publication Date Jan 21, 2016
Publication Date Jan 21, 2016
Deposit Date Mar 11, 2016
Journal Autophagy
Print ISSN 1554-8627
Electronic ISSN 1554-8635
Publisher Taylor & Francis
Peer Reviewed Peer Reviewed
Volume 12
Issue 1
Pages 1-222
DOI https://doi.org/10.1080/15548627.2015.1100356
Keywords LC3; autolysosome; autophagosome; chaperone-mediated autophagy; flux; lysosome; macroautophagy; phagophore; stress; vacuole
Public URL http://researchrepository.napier.ac.uk/id/eprint/9656
Publisher URL http://dx.doi.org/10.1080/15548627.2015.1100356