Macrophages rapidly transfer pathogens from lipid raft vacuoles to autophagosomes

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH(+) Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediatezly resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pretreated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.     

Macrophages Rapidly Transfer Pathogens from Lipid Raft Vacuoles to Autophagosomes

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH⁺ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediately resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pre-treated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.     

Nondegradative role of Atg5-Atg12/ Atg16L1 autophagy protein complex in antiviral activity of interferon gamma

Host resistance to viral infection requires type I (α/β) and II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8-processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes

Autophagy normally involves the formation of double-membrane autophagosomes that mediate bulk cytoplasmic and organelle degradation. Here we report the modification of single-membrane vacuoles in cells by autophagy proteins. LC3 (Light chain 3) a component of autophagosomes, is recruited to single-membrane entotic vacuoles, macropinosomes and phagosomes harbouring apoptotic cells, in a manner dependent on the lipidation machinery including ATG5 and ATG7, and the class III phosphatidylinositol-3-kinase VPS34. These downstream components of the autophagy machinery, but not the upstream mammalian Tor (mTor)-regulated ULK-ATG13-FIP200 complex, facilitate lysosome fusion to single membranes and the degradation of internalized cargo. For entosis, a live-cell-engulfment program, the autophagy-protein-dependent fusion of lysosomes to vacuolar membranes leads to the death of internalized cells. As pathogen-containing phagosomes can be targeted in a similar manner, the death of epithelial cells by this mechanism mimics pathogen destruction. These data demonstrate that proteins of the autophagy pathway can target single-membrane vacuoles in cells in the absence of pathogenic organisms.     

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.     

Atg9 Trafficking in Yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and non-selective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element.5 Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.

Addendum to:

Atg9 Sorting from Mitochondria is Impaired in Early Secretion and VFT Complex Mutants in Saccharomyces cerevisiae

F. Reggiori and D.J. Klionsky

J Cell Sci 2006: 119:2903-11     

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.     

Atg9 trafficking in the yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and nonselective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element. Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.     

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

Atg1 kinase regulates early and late steps during autophagy

The notion that phosphorylation constitutes a major mechanism to induce autophagy was established 15 years ago when a conserved Atg1/ULK kinase family was identified as an essential component of the autophagy machinery. The key observation was that starved atg1Δ cells lack autophagosomes in the cytosol and fail to accumulate autophagic bodies in the vacuole. Although many studies have revealed important details of Atg1 activation and function, a cohesive model for how Atg1 regulates the autophagic machinery is lacking. Our recent findings identified conserved steps of temporal and spatial regulation of Atg1/ULK1 kinase at both the PAS and autophagosomal membranes, suggesting that Atg1 not only promotes autophagy induction, but may also facilitate late stages of autophagosome biogenesis.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

Hierarchal Autophagic Divergence of Hematopoietic System

Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.     

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double-membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy-related (Atg) genes identified so far, there are two ubiquitin-like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7--an E1-like protein--and Atg10--an E2-like protein. Similarly, Atg7 and Atg3 are the respective E1-like and E2-like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12-Atg5 and Atg8 localize to the developing autophagosome. The Atg12-Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8-phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.     

Dual roles of Atg8-PE deconjugation by Atg4 in autophagy

Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8-PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8-PE and its deconjugation.