SNARE Dance

There is little doubt that humans rely on vision as their primary sensory input. However, various studies indicate that audiovisual combinations of data presentation actually enhance the ability of the learner to comprehend the information. We present an example of a musical-biological interface that provides an audible demonstration of SNARE protein function in the process of macroautophagy.     

SNARE Dance: A musical interpretation of Atg9 transport to the tubulovesicular cluster

There is little doubt that humans rely on vision as their primary sensory input. However, various studies indicate that audiovisual combinations of data presentation actually enhance the ability of the learner to comprehend the information. We present an example of a musical-biological interface that provides an audible demonstration of SNARE protein function in the process of macroautophagy.     

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.     

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.     

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

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.     

The vacuole vs. the lysosome

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

Editorial: The vacuole vs. the lysosome: When size matters

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

Autophagy and Human Disease

As a conserved cellular degradative pathway in eukaryotes, autophagy relieves cells from various types of stress. There are different forms of autophagy, and the ongoing studies of the molecular mechanisms and cellular functions of these processes are unraveling their significant roles in human health. Currently, the best-studied of these pathways is macroautophagy, which is linked to a range of human disease. For example, as part of the host immune defense mechanism, macroautophagy is activated to eliminate invasive pathogenic bacteria; however, in some cases bacteria subvert this process for their own replication. Autophagy also contributes to endogenous major histocompatibility complex class II antigen presentation, reflecting its role in adaptive immunity. In certain neurodegenerative diseases, which are associated with aggregation-prone proteins, macroautophagy plays a protective role in preventing or reducing cytotoxicity by clearance of the toxic proteins; however, the autophagy-dependent processing of some components correlates with the pathogenesis of certain myopathies. Finally, autophagy acts as a mechanism for tumor suppression, although some cancer cells use it as a cytoprotective mechanism. Thus, a fundamental paradox of autophagy is that it can act to promote both cell survival and cell death, depending on the specific conditions.     

Genetic aberrations in macroautophagy genes leading to diseases

The catabolic process of macroautophagy, through the rapid degradation of unwanted cellular components, is involved in a multitude of cellular and organismal functions that are essential to maintain homeostasis. Those functions include adaptation to starvation, cell development and differentiation, innate and adaptive immunity, tumor suppression, autophagic cell death, and maintenance of stem cell stemness. Not surprisingly, an impairment or block of macroautophagy can lead to severe pathologies. A still increasing number of reports, in particular, have revealed that mutations in the autophagy-related (ATG) genes, encoding the key players of macroautophagy, are either the cause or represent a risk factor for the development of several illnesses. The aim of this review is to provide a comprehensive overview of the diseases and disorders currently known that are or could be caused by mutations in core ATG proteins but also in the so-called autophagy receptors, which provide specificity to the process of macroautophagy. Our compendium underlines the medical relevance of this pathway and underscores the importance of the eventual development of therapeutic approaches aimed at modulating macroautophagy.     

Involvement of macroautophagy in the dissolution of neuronal inclusions

Ubiquitinated inclusions are a common feature of many neurodegenerative conditions. We have proposed that, at least in part, such inclusions may be formed due to dysfunction of the proteasome. We have modeled such proteasomal dysfunction by applying pharmacological inhibitors to cultured embryonic rat cortical neurons. This treatment leads to neuronal death and formation of ubiquitin/-synuclein-positive cytoplasmic inclusions. At late time points following proteasomal inhibition such inclusions are no longer discerned. Instead, many neurons accumulate small ubiquitinated aggregates, which may represent remnants of the inclusions. In this work we have examined a potential mechanism for inclusion dissolution. Electron microscopy images showed activation of macroautophagy at late time points after proteasomal inhibition. Labeling with LysoTracker Red, a dye that accumulates in acidic compartments, or immunostaining for the lysosomal enzyme Cathepsin D, showed an increase in globular staining. Cathepsin D co-localized partially with small ubiquitinated aggregates, but not inclusions. Application of an inhibitor of macroautophagy or of the vacuolar ATPase led to an increase in the number of inclusions and a decrease in small aggregates, whereas an activator of autophagy had the opposite effects. There was no significant change in apoptotic death following these manipulations. We conclude that, following proteasomal inhibition of cultured cortical neurons, there is activation of macroautophagy and of the lysosomal pathway. This activation results in dissolution of ubiquitinated inclusions into small aggregates, without directly impacting neuronal cell death. These data further support the idea that in this model inclusions and neuronal cell death are independent processes.     

Autophagy takes an alternative pathway

ATG5 and ATG7 are considered as essential molecules for induction of macroautophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stresses. Although lipidation of LC3 is accepted to be a good indicator of macroautophagy, it did not occur during the ATG5/ATG7-independent alternative macroautophagy. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of the phagophore with vesicles derived from the trans-Golgi and late endosomes. Mammalian macroautophagy can occur via at least two different pathways, which are an ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.     

Molecular machinery of macroautophagy and its deregulation in diseases

Macroautophagy maintains cellular homeostasis through targeting cytoplasmic contents and organelles into autophagosomes for degradation. This process begins with the assembly of protein complexes on isolation membrane to initiate the formation of autophagosome, followed by its nucleation, elongation and maturation. Fusion of autophagosomes with lysosomes then leads to degradation of the cargo. In the past decade, significant advances have been made on the identification of molecular players that are implicated in various stages of macroautophagy. Post-translational modifications of macroautophagy regulators have also been demonstrated to be critical for the selective targeting of cytoplasmic contents into autophagosomes. In addition, recent demonstration of distinct macroautophagy regulators has led to the identification of different subtypes of macroautophagy. Since deregulation of macroautophagy is implicated in diseases including neurodegenerative disorders, cancers and inflammatory disorders, understanding the molecular machinery of macroautophagy is crucial for elucidating the mechanisms by which macroautophagy is deregulated in these diseases, thereby revealing new potential therapeutic targets and strategies. Here we summarize current knowledge on the regulation of mammalian macroautophagy machineries and their disease-associated deregulation.     

Aminopeptidase-resistant peptides are targeted to lysosomes and subsequently degraded

Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of the proteasome and the effect of its impairment on aggregation have been intensively studied, little is known about how cells deal with peptides that show resistance to degradation by aminopeptidases. Here, we introduced peptidase-resistant peptides into living cells and show that these peptides rapidly and irreversibly accumulate into puncta in the perinuclear region of the cell. Accumulation appears to be independent of peptide sequence but is less efficient for longer peptides. The puncta colocalize with autophagosomal and lysosomal markers, suggesting that these peptides end up within lysosomes via macroautophagy. Surprisingly, the peptides still accumulate within lysosomes when macroautophagy is impaired, suggesting a trafficking route independent of macroautophagy. Upon lysosomal uptake, peptides are degraded, suggesting that cells can clear peptidase-resistant proteasomal products by an alternative pathway, which targets them to lysosomes.     

Does autophagy have a license to kill mammalian cells?

Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.     

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.     

Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus

Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the interplay between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-alpha (TNF-alpha). Atg5-/- mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-alpha in concert with decreased ATP levels. Fas/TNF-alpha treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-alpha, Atg5-/- cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.     

Why do we need to regulate autophagy (and how can we do it)? A cartoon depiction

In a recent issue of this journal I attempted to explain the purpose of macroautophagy/autophagy to a non-specialist audience through the use of cartoons. In the present article, I am continuing this approach by considering the topic of autophagy regulation—why does the cell need to modulate the autophagic response, and what are the basic morphological mechanisms that can be used to attain different levels of autophagy activity?     

To save or degrade: balancing proteasome homeostasis to maximize cell survival

Autophagic degradation of proteasomes (termed proteaphagy) is a conserved mechanism by which cells eliminate excess or damaged particles. This clearance is induced rapidly when organisms are starved for nitrogen and, because proteasomes are highly abundant, their breakdown likely makes an important contribution to the amino acid pools necessary for survival. By contrast, our recent studies found that proteasomes are not degraded in response to carbon starvation, even though bulk macroautophagy is similarly activated. Instead, carbon starvation induces sequestration of proteasomes into membrane-less cytoplasmic condensates previously termed proteasome storage granules (PSGs), which protect proteasomes from autophagic degradation. Preserving proteasomes in PSGs enhances the ability of yeast cells to recover from a variety of stresses, implying that rapid remobilization of stored proteasomes when conditions improve is advantageous to cell fitness. Consequently, the choice of whether to save or degrade proteasomes can profoundly impact cell survival.     

Macroautophagy in Immunity and Tolerance

Autophagy and proteasomal degradation constitute the two main catabolic pathways in cells. While the proteasome degrades primarily short-lived soluble proteins, macroautophagy, the main constitutive autophagic pathway, delivers cell organelles and protein aggregates for lysosomal degradation. Both the proteasome and macroautophagy are attractive effector mechanisms for the immune system because they can be used to degrade foreign substances, including pathogenic proteins, within cells. Therefore, both innate and adaptive immune responses use these pathways for intracellular clearance of pathogens as well as for presentation of pathogen fragments to the adaptive immune system. Because, however, the same mechanisms are used for the steady-state turnover of cellular self-components, the immune system has to be desensitized not to recognize these. Therefore, proteasomal degradation and macroautophagy are also involved in tolerizing the immune system prior to pathogen encounter. We will discuss recent advances in our understanding how macroautophagy selects self-structures in the steady state, how presentation of these on major histocompatibility complex class II molecules leads to tolerance and how macroautophagy assists both innate and adaptive immunity. This new knowledge on the specialized functions of the metabolic process macroautophagy in higher eukaryotes should allow us to target it for therapy development against immunopathologies and to improve vaccinations.