Guidelines for the use and interpretation of assays for monitoring autophagy

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.     

Guidelines for the use and interpretation of assays for monitoring autophagy

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.     

The machinery of macroautophagy

Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.     

There is more to autophagy than induction: regulating the roller coaster

Considerable attention has been paid to the topic of autophagy induction. In part, this is because of the potential for modulating this process for therapeutic purposes. Of course we know that induced autophagy can also be problematic—for example, when trying to eliminate an established tumor that might be relying on autophagy for its own cytoprotective uses. Accordingly, inhibitory mechanisms have been considered; however, the corresponding studies have tended to focus on the pathways that block autophagy under noninducing conditions, such as when nutrients are available. In contrast, relatively little is known about the mechanisms for inhibiting autophagy under inducing conditions. Yet, this type of regulation must be occurring on a routine basis. We know that dysregulation of autophagy, e.g., due to improper activation of Beclin 1 leading to excessive autophagy activity, can cause cell death.¹ Accordingly, we assume that during starvation or other inducing conditions there must be a mechanism to modulate autophagy. That is, once you turn it on, you do not want to let it continue unchecked. But how is autophagy downregulated when the inducing conditions still exist?Key words: Atg1, autophagosome, flux, lysosome, macroautophagy, phagophore, regulation, stress, TOR, Ulk1, vacuoleOne possibility for downregulating macroautophagy is suggested by Tom Neufeld’s lab, which showed that p70S6 kinase is a positive regulatory factor for autophagy in the Drosophila fat body.² Accordingly, inhibition of TOR activity ultimately results in decreased p70S6K function, which in turn downregulates autophagy. An alternate suggestion from Fred Meijer and Patrice Codogno is that p70S6K acts in part by negatively regulating the class I PtdIns 3-kinase.³ In this scenario, when TOR is inhibited the decrease in p70S6K activity results in the eventual reactivation of the class I PtdIns3K, which then stimulates TOR and downregulates autophagy.Further insight into this question is provided by a relatively recent study from Adi Kimchi’s lab. The conserved protein DAP1 is an mTOR substrate that inhibits macroautophagy. In nutrient-rich conditions, active mTORC1 inhibits DAP1 so that the latter has no effect on autophagy. The inhibition of mTORC1 during nutrient starvation results in the dephosphorylation and activation of DAP1, and the subsequent inhibition of macroautophagy, which limits the magnitude of autophagy-dependent degradation.^4,5Another mechanism of regulation is indicated in studies by Li Yu and colleagues who showed that autophagy is downregulated through mTOR reactivation in an autophagy-dependent manner that requires protein degradation in autolysosomes.⁶ This negative feedback mechanism provides another simple means of self-regulation whereby the nutrient levels within the cell dictate whether autophagy needs to be maintained or shut down. A study described in this issue of the journal provides further support for this mechanism, demonstrating that autophagy can be downregulated during starvation in yeast.⁷ Shin and Huh found that TOR activity is recovered during prolonged starvation, and that this again depends on autophagy (see Fig. 1 in the Autophagic Flux section, p. 803). These studies suggest that autophagy may cycle on and off repeatedly during starvation as nutrient supplies are consumed and then resupplied, ensuring that autophagy is maintained at optimal, and not excessive, levels. The latter mechanism, however, cannot explain how autophagy is regulated during other types of stress, suggesting that multiple control systems are involved.In closing, we introduce a new category of papers and a new section to the journal that we are calling Resource and Autophagic Flux, respectively. Resource papers will provide information that may be useful to the autophagy community, but that may not have specific mechanistic information, such as may occur with large-scale screens. For example, in this issue, see the paper from Marja Jäättelä’s lab that describes the use of a human kinome siRNA library to identify new kinases that regulate macroautophagy. Finally, we have chosen the name Autophagic Flux for the new section because it encompasses the full spectrum of the autophagic process. The schematic summary in Figure 1 of that section highlights the paper by Shin and Huh that we mention here. Our intention is to provide schematic highlights of most of the research papers in the Autophagic Flux section, providing readers with a quick overview and summary of the key point(s) of the study. We hope you find this useful; we welcome feedback.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a1?/?) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a12/2) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.