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

KLIONKY D; ROMANO PS

AUTOPHAGY

LANDES BIOSCIENCE

Año: 2016 vol. 12 p. 1 - 122

1554-8627

In 2008 we published the first set of guidelines for standardizingresearch in autophagy. Since then, research on this topichas continued to accelerate, and many new scientists haveentered the field. Our knowledge base and relevant new tech-2070 nologies have also been expanding. Accordingly, it is importantto update these guidelines for monitoring autophagy in differentorganisms. Various reviews have described the range ofassays that have been used for this purpose. Nevertheless, therecontinues to be confusion regarding acceptable methods to2075 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 numbersor volume of autophagic elements (e.g., autophagosomes orautolysosomes) at any stage of the autophagic process versus those2080 that measure flux through the autophagy pathway (i.e., the completeprocess 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 increased2085 autophagy induction coupled with increased delivery to, and degradationwithin, lysosomes (inmost higher eukaryotes and some protistssuch as Dictyostelium) or the vacuole (in plants and fungi). Inother words, it is especially important that investigators new to thefield understand that the appearance of more autophagosomes2090 does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in traffickinto lysosomes without a concomitant change in autophagosomebiogenesis, whereas an increase in autolysosomes may reflect areduction in degradative activity. It is worth emphasizing here thatlysosomal digestion is a stage of autophagy and evaluating its com- 2095petence is a crucial part of the evaluation of autophagic flux, 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 for 2100reviewers 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 appropriateassays depend in part on the question being asked andthe system being used. In addition, we emphasize that no indi- 2105vidual 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 delete or 2110knock down more than one autophagy-related gene. In addition,some individual Atg proteins, or groups of proteins, areinvolved in other cellular pathways so not all Atg proteins canbe used as a specific marker for an autophagic process. In theseguidelines, we consider these various methods of assessing 2115autophagy and what information can, or cannot, be obtainedfrom them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technicalinnovation in the field.