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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field

[Abstract unavailable

Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.

No abstract

Autophagy is a potent intracellular degradation process with pivotal roles in health and disease. Atg8, a lipid-conjugated ubiquitin-like protein, is required for the formation of autophagosomes, double-membrane vesicles responsible for the delivery of cytoplasmic material to lysosomes. How and when Atg8 functions in this process, however, is not clear. Here we show that Atg8 controls the expansion of the autophagosome precursor, the phagophore, and give the first real-time, observation-based temporal dissection of the autophagosome formation process. We demonstrate that the amount of Atg8 determines the size of autophagosomes. During autophagosome biogenesis, Atg8 forms an expanding structure and later dissociates from the site of vesicle formation. On the basis of the dynamics of Atg8, we present a multistage model of autophagosome formation. This model provides a foundation for future analyses of the functions and dynamics of known autophagy-related proteins and for screening new genes.