Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

KLIONSKY, D.; OTROS; COLOMBO, M. I.; OTROS

Autophagy

LANDES BIOSCIENCE

Año: 2008 vol. 4 p. 1 - 25

1554-8635

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor autophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure autophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in autophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, the lysosome (in most higher eukaryotes) or vacuole (in plants and yeast). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate macroautophagy-related processes. This set of guidelines is 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 strongly recommend the use of multiple assays to verify an autophagic response, and emphasize that no individual assay is guaranteed to be the most appropriate one in every situation.