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

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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 macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy 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 macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). 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 these 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 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 verify an autophagic response.

Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin- and LC3-binding protein "p62" regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific.

The biochemical properties of beclin 1 suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis. We show here that beclin 1 ؊/؊ mutant mice die early in embryogenesis and beclin 1 ؉/؊ mutant mice suffer from a high incidence of spontaneous tumors. These tumors continue to express wild-type beclin 1 mRNA and protein, establishing that beclin 1 is a haploinsufficient tumor suppressor gene. Beclin 1 ؊/؊ embryonic stem cells have a severely altered autophagic response, whereas their apoptotic response to serum withdrawal or UV light is normal. These results demonstrate that beclin 1 is a critical component of mammalian autophagy and establish a role for autophagy in tumor suppression. They both provide a biological explanation for recent evidence implicating beclin 1 in human cancer and suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.A utophagy is a complex catabolic program for lysosomal degradation of proteins and other subcellular constituents. It is often activated in response to nutrient deprivation, when it leads to recycling of organelles and other cytoplasmic substances to provide metabolic precursors. Genetic analysis in yeast has identified a large number of genes required for autophagy and has begun to place them into an ordered pathway that is required for the response to starvation (1-3). Failure to activate autophagy in response to nutrient deprivation, or its constitutive activation in response to stress, can lead to cell death. For this reason, autophagy is sometimes referred to as a second form of programmed cell death. Autophagy and apoptosis are often activated together in response to stress (4, 5). Although autophagy has been shown to be activated in response to starvation, and in many neurodegenerative conditions, its role in normal development and tissue homeostasis has not been determined.Beclin 1 is the mammalian orthologue of the yeast Apg6͞ Vps30 gene. It can complement the defect in autophagy present in apg6 yeast strains and stimulate autophagy when overexpressed in mammalian cells (6). Beclin 1 can also bind to Bcl-2, an important regulator of apoptosis (7). Beclin 1 is monoallelically deleted in human breast and ovarian cancers and is expressed at reduced levels in those tumors (6,8). In the nervous system, beclin 1 is present in a complex bound to glutamate receptor ␦2 (GluR␦2), whose constitutive activation in the lurcher mouse leads to the activation of autophagy and death of cerebellar Purkinje cells (9). To understand the role of beclin 1 and autophagy in development and tissue homeostasis, we have generated and analyzed beclin 1 Ϫ/Ϫ mutant mice. Our results demonstrate that beclin 1 is essential for early embryonic development and is a haploinsufficient tumor suppressor gene, and they show that beclin 1 is not required for apoptosis but that it is necessary for autophagy. Taken together, these results demonstrate that beclin 1 and autophagy are critical for ma...