When an autophagosome or an amphisome fuse with a lysosome, the resulting compartment is referred to as an autolysosome. Some people writing papers on the topic of autophagy use the terms “autolysosome” and “autophagolysosome” interchangeably. We contend that these words should be used to denote 2 different compartments, and that it is worthwhile maintaining this distinction—the autophagolysosome has a particular origin in the process of xenophagy that makes it distinct from an autolysosome.
amphisome; lysosome; phagophore; stress; xenophagy
Autophagy is a fundamental cell biological process whereby eukaryotic cells form membranes in the cytoplasm to sequester diverse intracellular targets. Although significant progress has been made in understanding the origins of autophagosomal organelles, the source of lipids that support autophagic membrane formation remain an important open question.
Here we show that lipid droplets as cellular stores of neutral lipids including triglycerides contribute to autophagic initiation. Lipid droplets, as previously shown, were consumed upon induction of autophagy by starvation. However, inhibition of autophagic maturation by blocking acidification or using dominant negative Atg4C74A that prohibits autophagosomal closure, did not prevent disappearance of lipid droplets. Thus, lipid droplets continued to be utilized upon induction of autophagy but not as autophagic substrates in a process referred to as lipophagy. We considered an alternative model whereby lipid droplets were consumed not as a part of lipophagy but as a potential contributing source to the biogenesis of lipid precursors for nascent autophagosomes. We carried out a screen for a potential link between triglyceride mobilization and autophagy, and identified a neutral lipase, PNPLA5, as being required for efficient autophagy. PNPLA5, which localized to lipid droplets, was needed for optimal initiation of autophagy. PNPLA5 was required for autophagy of diverse substrates including degradation of autophagic adaptors, bulk proteolysis, mitochondrial quantity control, and microbial clearance.
Lipid droplets contribute to autophagic capacity by enhancing it in a process dependent on PNPLA5. Thus, neutral lipid stores are mobilized during autophagy to support autophagic membrane formation.
The now well-accepted innate immunity paradigm that autophagy acts as a cell-autonomous defense against intracellular bacteria has its key origins in studies with Mycobacterium tuberculosis, an important human pathogen and a model microorganism infecting macrophages. A number of different factors have been identified that play into the anti-mycobacterial functions of autophagy, and recent in vivo studies in the mouse model of tuberculosis have uncovered additional anti-inflammatory and tissue-sparing functions of autophagy. Complementing these observations, genome wide association studies indicate a considerable overlap between autophagy, human susceptibility to mycobacterial infections, and predisposition loci for inflammatory bowel disease. Finally, recent studies show that autophagy is an important regulator and effector of IL-1 responses, and that autophagy intersects with type I interferon pathology-modulating responses.
ER-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and diseases. Here, we identify UVRAG, a Beclin1-binding autophagic factor, as a PI(3)P-binding protein that depends on PI(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-Beclin-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide, and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.
UVRAG; RINT-1; phospholipid; Golgi-to-ER traffic; COPI; Atg9; Autophagy
Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key pro-inflammatory cytokine, IL-1β, induced autophagy leading to autophagic killing of mycobacteria in macrophages and this IL-1β activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.
Autophagy (macroautophagy) is often defined as a degradative process and a tributary of the lysosomal pathway. In this context, autophagy carries out cytoplasmic quality control and nutritional functions by removing defunct or disused organelles, particulate targets and invading microbes, and by bulk digestion of the cytoplasm. However, recent studies indicate that autophagy surprisingly affects multiple secretary pathways. Autophagy participates in extracellular delivery of a number of cytosolic proteins that do not enter the conventional secretory pathway via the Golgi apparatus but are instead unconventionally secreted directly from the cytosol. In mammalian cells, a prototypical example of this manifestation of autophagy is the unconventional secretion of a major proinflammatory cytokine, IL-1β. This review examines the concept of secretory autophagy and compares and contrasts the role of autophagy in secretion of IL-1α and IL-1β. Although IL-1α and IL-1β have closely related extracellular inflammatory functions, they differ in intracellular activation, secretory mechanisms, and how they are affected by autophagy. This example indicates that the role of autophagy in secretion is more complex, at least in mammalian cells, than the simplistic view that autophagosomes provide carriers for unconventional secretion of cytosolic proteins.
Autophagy; alarmins; inflammasome; calpain
Autophagy is a cell biological process ubiquitous to all eukaryotic cells, often referred to as a catabolic, lysosomal degradative pathway. However, current studies in mammalian systems suggest that autophagy plays an unexpectedly broad biogenesis role in protein trafficking and secretion. Autophagy supports alternative trafficking pathways for delivery of integral membrane proteins to the plasma membrane and affects secretion, including the constitutive, regulated and unconventional secretion pathways. Autophagy-based unconventional secretion, termed here ‘autosecretion’, is one of the pathways enabling leaderless cytosolic proteins to exit the cell without entering the ER-to-Golgi secretory pathway. In this review, we discuss the emerging underlying mechanisms of how autophagy affects different facets of secretion. We also describe the physiological roles of autosecretory cargos that are often associated with inflammatory processes and also play a role in the formation of specialized tissues and in tissue remodeling, expanding the immediate sphere of influence of autophagy from the intracellular to the extracellular space.
Autophagy; autosecretion; inflammasome; cystic fibrosis; IL-1; GRASP
Recent genome-wide association studies have linked polymorphisms in two atophagy genes, Atg16L1 and IRGM, with Crohn’s Disease. Now, experiments with Atg16L1 transgenic mice indicate multiple roles for autophagy in inflammatory bowel disease via effects on Paneth cells, a runaway inflammasome, and the proinflammatory cytokine IL-1b.
Tuberculosis is one of the world's most prevalent infectious diseases. The causative agent, M. tuberculosis, asymptomatically infects more than 30% of the world population and causes 8 million cases of active disease and 2 million deaths annually. Its pathogenic success stems from its ability to block phagolysosome biogenesis and subsequent destruction in the host macrophages. Recently, our laboratory has uncovered autophagy as a new means of overcoming this block and promoting the killing of mycobacteria. Here we describe the methods to study autophagy during M. tuberculosis infection of macrophages. The described assays can be used to investigate and identify factors important for autophagic elimination of mycobacteria that could potentially provide new therapeutic targets to defeat this disease.
Autophagy is a fundamental eukaryotic process with multiple cytoplasmic homeostatic roles, recently expanded to include unique standalone immunological functions and interactions with nearly all parts of the immune system. Here, we review this growing repertoire of autophagy roles in innate and adaptive immunity and inflammation. Its unique functions include cell-autonomous elimination of intracellular microbes facilitated by specific receptors. Other intersections of autophagy with immune processes encompass effects on inflammasome activation and secretion of its substrates including IL-1β, effector and regulatory interactions with Toll-like and Nod-like receptors, antigen presentation, naïve T cell repertoire selection, and mature T cell development and homeostasis. Genome wide association studies in human populations strongly implicate autophagy in chronic inflammatory disease and autoimmune disorders. Collectively, the unique features of autophagy as an immunological process and its contributions to other arms of the immune system represent a new immunological paradigm.
Mycobacterium tuberculosis parasitizes host macrophages and subverts host innate and adaptive immunity. A number of cytokines elicited by the tubercle bacilli have been recognized as mediators of mycobacterial clearance or pathology in tuberculosis. Surprisingly, interleukin-1β (IL-1β), a major pro-inflammatory cytokine activated by processing upon assembly of a specialized protein complex termed the inflammasome, has not been implicated in host-pathogen interactions in tuberculosis. Here, we show that M. tuberculosis prevents inflammasome activation and IL-1β processing, and that a functional M. tuberculosis zmp1 gene is required for this process. Infection of macrophages with the zmp1 null M. tuberculosis triggered activation of caspase-1/IL-1β inflammasome, resulting in increased secretion of IL-1β, enhanced mycobacterial phagosome maturation into phagolysosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered the previously masked role for IL-1β in control of M. tuberculosis, and the existence of a mycobacterial system that prevents IL-1β/inflammasome activation.
Autophagy is a rapidly expanding field in the sense that our knowledge about the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. Similarly, the vocabulary associated with autophagy has grown concomitantly. This fact makes it difficult for readers, even those who work in the field, to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors or chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, or the role of accessory machinery or structures that are associated with autophagy.
autophagy; definitions; glossary; lexicon; terms
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.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
Autophagy is rapidly developing into a new immunological paradigm. The latest links now include overlaps between autophagy and innate immune signaling via TBK-1 and IKKα/β, and the role of autophagy in inflammation directed by the inflammasome. Autophagy's innate immunity connections include responses to pathogen and damage associated molecular patterns including alarming such as HMGB1 and IL-1β, Toll-like receptors, Nod-like receptors including NLRC4, NLRP3 and NLRP4, and RIG-I-like receptors. Autophagic adaptors referred to as SLRs (sequestosome 1/p62-like receptors) are themselves a category of pattern recognition receptors. SLRs empower autophagy to eliminate intracellular microbes by direct capture and by facilitating generation and delivery of antimicrobial peptides, and also serve as inflammatory signaling platforms. SLRs contribute to autophagic control of intracellular microbes, including Mycobacterium tuberculosis, Salmonella, Listeria, Shigella, HIV-1 and Sindbis viruses, but act as double edged sword and contribute to inflammation and cell death. Autophagy roles in innate immunity continue to expand vertically and laterally, and now include antimicrobial function downstream of vitamin D3 action in tuberculosis and AIDS. Recent data expand the connections between immunity related GTPases and autophagy to include not only IRGM but also several members of the Gbp (guanlyate-binding proteins) family. The efficacy with which autophagy handles microbes, microbial products and sterile endogenous irritants governs whether the outcome will be with suppression of or with excess inflammation, the latter reflected in human diseases that have strong inflammatory components including tuberculosis and Crohn's disease.
The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunological process with a wide array of roles in immunity. The immunological functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunological effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a step-wise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-I-like receptors (RLRs), damage-associated molecular patterns (DAMPs) such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors (SLR) that act as autophagy adapters, immunity related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.
autophagy; dendritic cells; T cells; AIDS; bacterial; inflammatory bowel disease
adaptors; autophagy; cargo; mitophagy; stress; xenophagy
In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.
autophagy; tuberculosis; ribosome; ubiquitin; antimicrobial peptides
Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-γ)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.
Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes host macrophages where it persists in immature phagosomes by avoiding their maturation into phagolysosomes. The mechanisms of how M. tuberculosis inhibits phagolysosome biogenesis have been researched in detail and the maturation block at least partially depends on the manipulation of host phosphoinositide interconversions, with phosphatidylinositol 3-phosphate (PI3P) being a central target since it has been shown to be required for phagolysosome biogenesis. PI3P earmarks intracellular organelles for binding and assembly of effector molecules that interact with PI3P or its derivatives, including Class E Vps proteins such as Hrs and ESCRT components, early endosome antigen 1, which are required for sequential protein and membrane sorting within the endosomal and, by extension, phagosomal systems. In a search of a cellular mechanism that can bypass the tubercule bacillus-imposed PI3P block, researchers have uncovered a new general bactericidal process, autophagy, which can eliminate intracellular pathogens. This is a new, rapidly growing field replete with possibilities for novel, previously untried immunologic and pharmacologic interventions applicable not only to TB but to other stubborn bacterial, parasitic and viral diseases.
autophagy; macrophage; phagosome; phosphoinositide; Rab; tuberculosis
Mammalian target of rapamycin (mTOR) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles 1, 2 intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates 3-8. Here we report that lysosomal positioning coordinates anabolic and catabolic responses to changes in nutrient availability by orchestrating early plasma membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, while starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH (pHi). Lysosomal positioning regulates mTORC1 signalling, which, in turn, influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting both at the initiation and termination stages of the process. Our findings provide a fundamental physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
Autophagy is a key cytoplasmic biomass and organellar quality and quantity control pathway of the eukaryotic cell. It is particularly suited to capture and degrade large, multi-macromolecular cytosplasmic targets earmarked for degradation or turnover. Typical autophagic cargos represent large swaths of cytosol as a source of energy and anabolic precursors at times of growth restrictions imposed by the absence of growth factors, nutrient limitation or hypoxia. Autophagy is the only effective mechanism for removal of whole organelles such as leaky or surplus mitochondria, disposal of potentially toxic protein aggregates too large for proteasomal removal, and elimination of intracellular microbes including bacteria, protozoa and viruses. Recent studies have shown that human immunodeficiency virus (HIV) is targeted for eliminated by autophagy but that this is countered by the viral protein Nef. Here we review these relationships and underscore the untapped potential of autophagy as a druggable antiviral process.
IRGM, a human immunity related GTPase, confers autophagic defense against intracellular pathogens by an unknown mechanism. Here we report the unexpected mode of IRGM action. IRGM showed differential affinity for mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd but not IRGMb splice isoforms caused mitochondrial depolarization and autophagy-independent but Bax/Bak-dependent cell death. By acting on mitochondria IRGM confers autophagic protection or cell death, explaining IRGM action both in defense against tuberculosis and in damaging inflammation in Crohn's disease.
Dendritic cells (DCs) in mucosal surfaces are early targets for human immunodeficiency virus-1 (HIV-1). DCs mount rapid and robust immune responses upon pathogen encounter. However, immune response in the early events of HIV-1 transmission appears limited, suggesting that HIV-1 evade early immune control by DCs. We report that HIV-1 induces a rapid shutdown of autophagy and immunoamphisomes in DCs. HIV-1 envelope activated the mammalian target of rapamycin pathway in DCs, leading to autophagy exhaustion. HIV-1-induced inhibition of autophagy in DC increased cell-associated HIV-1 and transfer of HIV-1 infection to CD4+ T cells. HIV-1-mediated downregulation of autophagy in DCs impaired innate and adaptive immune responses. Immunoamphisomes in DCs engulf incoming pathogens and appear to amplify pathogen degradation as well as Toll-like receptor responses and antigen presentation. The findings that HIV-1 downregulates autophagy and impedes immune functions of DCs represent a pathogenesis mechanism that can be pharmacologically countered with therapeutic and prophylactic implications.
Autophagy initiation is strictly dependent on phosphatidylinositol 3-phosphate (PI3P) synthesis. PI3P production is under tight control of PI3Kinase, hVps34, in complex with Beclin-1. Mammalian cells express several PI3P phosphatases that belong to the myotubularin family. Even though some of them have been linked to serious human diseases, their cellular function is largely unknown. Two recent studies indicate that PI3P metabolism involved in autophagy initiation is further regulated by the PI3P phosphatases Jumpy and MTMR3. Additional pools of PI3P, upstream of mTOR and on the endocytic pathway, may modulate autophagy indirectly, suggesting that other PI3P phosphatases might be involved in this process. This review sums up our knowledge on PI3P phosphatases and discusses the recent progress on their role in autophagy.
autophagy; myotubularin; PI3P; phosphatase; Jumpy; MTMR14