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

Eukaryotic cells have evolved strategies to respond to stress conditions. For example, autophagy in yeast is primarily a response to the stress of nutrient limitation. Autophagy is a catabolic process for the degradation and recycling of cytosolic, long lived, or aggregated proteins and excess or defective organelles. In this study, we demonstrate a new pathway for the induction of autophagy. In the endoplasmic reticulum (ER), accumulation of misfolded proteins causes stress and activates the unfolded protein response to induce the expression of chaperones and proteins involved in the recovery process. ER stress stimulated the assembly of the pre-autophagosomal structure. In addition, autophagosome formation and transport to the vacuole were stimulated in an Atg protein-dependent manner. Finally, Atg1 kinase activity reflects both the nutritional status and autophagic state of the cell; starvation-induced autophagy results in increased Atg1 kinase activity. We found that Atg1 had high kinase activity during ER stress-induced autophagy. Together, these results indicate that ER stress can induce an autophagic response.

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.

SUMMARY Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

Autophagy is a degradative process conserved among eukaryotic cells. It allows the elimination of cytoplasm including aberrant protein aggregates and damaged organelles. Accordingly, it is implicated in normal developmental processes and also serves a protective role in tumor suppression and elimination of invading pathogens, whereas defects in autophagy are associated with various human diseases including cancer and neurodegeneration. Atg proteins mediate the sequestration event that occurs at the preautophagosomal structure (PAS) by catalyzing the formation of double-membrane vesicles, termed autophagosomes. In Saccharomyces cerevisiae, the integral membrane protein Atg9 that is required for autophagy cycles through the PAS. Here, we demonstrate that Atg9 shuttles between this location and mitochondria. These data support a new model where mitochondria may provide at least part of the autophagosomal lipids and suggest a novel cellular function for this well-studied organelle.