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

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Abstract

In 2008 we published thefirst set of guidelines for standardiz-ing research in autophagy. Since then, research on this topichas continued to accelerate, and many new scientists haveentered thefield. Our knowledge base and relevant new tech-nologies have also been expanding. Accordingly, it is importantto update these guidelines for monitoring autophagy in differ-ent organisms. Various reviews have described the range ofassays that have been used for this purpose. Nevertheless, therecontinues to be confusion regarding acceptable methods tomeasure 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 num-bers or volume of autophagic elements (e.g., autophagosomes orautolysosomes) at any stage of the autophagic process versus thosethat measureflux through the autophagy pathway (i.e., the com-plete process including the amount and rate of cargo sequesteredand degraded). In particular, a block in macroautophagy thatresults in autophagosome accumulation must be differentiatedfrom stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degra-dation within, lysosomes (in most higher eukaryotes and some pro-tists such asDictyostelium) or the vacuole (in plants and fungi). Inotherwords,itisespeciallyimportantthatinvestigatorsnewtothefield understand that the appearance of more autophagosomesdoes not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in traffickingto lysosomes without a concomitant change in autophagosomebiogenesis, whereas an increase in autolysosomes may reflect areductionindegradativeactivity.Itisworthemphasizingherethatlysosomal digestion is a stage of autophagy and evaluating its com-petence is a crucial part of the evaluation of autophagicflux, orcomplete autophagy.Here, we present a set of guidelines for the selection andinterpretation of methods for use by investigators who aim toexamine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiquesof papers that are focused on these processes. These guidelinesare not meant to be a formulaic set of rules, because the appro-priate assays depend in part on the question being asked andthe system being used. In addition, we emphasize that no indi-vidual assay is guaranteed to be the most appropriate one inevery situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of thepotential for pleiotropic effects due to blocking autophagythrough genetic manipulation, it is imperative to target by geneknockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, orgroups of proteins, are involved in other cellular pathwaysimplying that not all Atg proteins can be used as a specificmarker for an autophagic process. In these guidelines, we con-sider these various methods of assessing autophagy and whatinformation can, or cannot, be obtained from them. Finally, bydiscussing the merits and limits of particular assays, we hope toencourage technical innovation in thefield
Original languageEnglish
Pages (from-to)1-222-222
Number of pages240
JournalAutophagy
Volume12
Publication statusPublished - 2016

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All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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