Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/microm2, whereas isolated lysosomes had about 2000 particles/microm2). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/microm2), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearence of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi- or multilamellar autophagic vacuoles appeared to engage in such fusions.     

Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes

Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth. After transfer of methanol-grown cells into media containing glucose - a substrate that fully represses alcohol oxidase synthesis - the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein. The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer. This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded. Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment. Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes. The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis. Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment. The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment. The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins. The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle. Peroxisomes of methanol-grown H. polymorpha were degraded individually. In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome. However, fusions of autophagic vacuoles in one cell were frequently observed. After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.     

Endoplasmic reticulum and Golgi complex: Contributions to, and turnover by, autophagy

The degradation of cytoplasmic contents, especially organelles [mitochondria, peroxisomes, endoplasmic reticulum (ER), Golgi complex (GC)], cannot be accomplished solely by the cytosolic degradation machinery, of which the most prominent component is the proteasome. However, it is possible that such organelles (or portions thereof) can be degraded by the cell's autophagic machinery. In this manner, organelles can be either specifically or non-specifically targeted to the vacuole/lysosome for degradation. These processes can be triggered in response to different environmental cues. Here, we focus on two particular organelles, the ER and the GC, and their relationship with the autophagic process. Firstly, we briefly consider how these two organelles contribute to the synthesis and delivery of hydrolytic enzymes involved in autophagy as well as how they may potentially contribute to their own degradation by addressing the origin of the autophagic membrane. Secondly, we summarize the evidence for the turnover of these two organelles by autophagic processes in different organisms.     

Bacterial pathogens and the autophagic response

The host cell recognition and removal of invading pathogens are crucial for the control of microbial infections. However, several microorganisms have developed mechanisms that allow them to survive and replicate intracellularly. Autophagy is an ubiquitous physiological pathway in eukaryotic cells, which maintains the cellular homeostasis and acts as a cell quality control mechanism to eliminate aged organelles and unnecessary structures. In addition, autophagy has an important role as a housekeeper since cells that have to get rid of invading pathogens use this pathway to assist this eradication. In this review we will summarize some strategies employed by bacterial pathogens to modulate autophagy to their own benefit and, on the other hand, the role of autophagy as a protective process of the host cell. In addition, we will discuss here recent studies that show the association of LC3 to a pathogen-containing compartment without a classical autophagic sequestering process (i.e. formation of a double membrane structure).     

Autophagosome formation: core machinery and adaptations

Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.     

Lipids in autophagy: constituents, signaling molecules and cargo with relevance to disease

The balance between protein and lipid biosynthesis and their eventual degradation is a critical component of cellular health. Autophagy, the catabolic process by which cytoplasmic material becomes degraded in lysosomes, can be induced by various physiological stimuli to maintain cellular homeostasis. Autophagy was for a long time considered a non-selective bulk process, but recent data have shown that unwanted components such as aberrant protein aggregates, dysfunctional organelles and invading pathogens can be selectively eliminated by autophagy. Recently, also intracellular lipid droplets were described as specific autophagic cargo, indicating that autophagy plays a role in lipid metabolism and storage (Singh et al., 2009 [1]). Moreover, over the past several years, it has become increasingly evident that lipids and lipid-modifying enzymes play important roles in the autophagy process itself, both at the level of regulation of autophagy and as membrane constituents required for formation of autophagic vesicles. In this review, we will discuss the interplay between lipids and autophagy, as well as the role of lipid-binding proteins in autophagy. We also comment on the possible implications of this mutual interaction in the context of disease. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Curvature‐sensitive trans‐assembly of human Atg8‐family proteins in autophagy‐related membrane tethering

In macroautophagy, de novo formation of the double membrane‐bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8‐family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8‐mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE‐16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE‐16 retained the capacities to trigger efficient membrane tethering at the protein‐to‐lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8‐mediated membrane‐tethering reactions require trans‐assembly between the membrane‐anchored forms of LC3B and GATE‐16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B‐ and GATE‐16‐mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE‐16 for highly curved small vesicles (e.g., 50 nm in diameter), although GATE‐16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g., 200 and 400 nm in diameter). Our findings establish curvature‐sensitive trans‐assembly of human Atg8‐family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.     

Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling

Reactive oxygen and nitrogen species change cellular responses through diverse mechanisms that are now being defined. At low levels, they are signalling molecules, and at high levels, they damage organelles, particularly the mitochondria. Oxidative damage and the associated mitochondrial dysfunction may result in energy depletion, accumulation of cytotoxic mediators and cell death. Understanding the interface between stress adaptation and cell death then is important for understanding redox biology and disease pathogenesis. Recent studies have found that one major sensor of redox signalling at this switch in cellular responses is autophagy. Autophagic activities are mediated by a complex molecular machinery including more than 30 Atg (AuTophaGy-related) proteins and 50 lysosomal hydrolases. Autophagosomes form membrane structures, sequester damaged, oxidized or dysfunctional intracellular components and organelles, and direct them to the lysosomes for degradation. This autophagic process is the sole known mechanism for mitochondrial turnover. It has been speculated that dysfunction of autophagy may result in abnormal mitochondrial function and oxidative or nitrative stress. Emerging investigations have provided new understanding of how autophagy of mitochondria (also known as mitophagy) is controlled, and the impact of autophagic dysfunction on cellular oxidative stress. The present review highlights recent studies on redox signalling in the regulation of autophagy, in the context of the basic mechanisms of mitophagy. Furthermore, we discuss the impact of autophagy on mitochondrial function and accumulation of reactive species. This is particularly relevant to degenerative diseases in which oxidative stress occurs over time, and dysfunction in both the mitochondrial and autophagic pathways play a role.     

Lysosomes: fusion and function

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.     

Methods for monitoring autophagy

Autophagy is an intracellular bulk degradation system that is found ubiquitously in eukaryotes. Autophagy is responsible for the degradation of most long-lived proteins and some organelles. Cytoplasmic constituents, including organelles, are sequestered into double-membraned autophagosomes, which subsequently fuse with lysosomes where their contents are degraded. This system has been implicated in various physiological processes including protein and organelle turnover, the starvation response, cellular differentiation, cell death, and pathogenesis. However, methods for monitoring autophagy have been very limited and unsatisfactory. The most standard method is conventional electron microscopy. In addition, some biochemical methods have been utilized to measure autophagic activity. Recently, the molecular basis of autophagosome formation has been extensively studied using yeast cells; these studies have provided useful marker proteins for autophagosomes. Importantly, most of these proteins are conserved in mammals. Using these probes, we can now specifically monitor autophagic activity.     

Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand..

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Deconstructing Pompe Disease by Analyzing Single Muscle Fibers: “To See a World in a Grain of Sand…”

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogen-filled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.     

Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation

Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPgammaS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.     

Interplay between autophagy and apoptosis in TrkA-induced cell death

Autophagy is a self-eating process to eradicate damaged proteins or organelles in cells. This process begins with formation of a double-membrane structure, called an autophagosome, which can sequester soluble proteins and organelles eventually degraded by lysosomal proteases after fusion with the lysosome. Autophagy was initially identified as a cell survival mechanism under stress conditions such as nutrient deprivation. More recently, it is also considered as type-II programmed cell death. In our recent report, we observed that overexpression of TrkA caused massive cell death via both apoptosis and autophagy. Overexpression of TrkA abated catalase activity and subsequently resulted in the production of a large amount of reactive oxygen species in cells. These consequences led to autophagic cell death. The autophagic cell death in TrkA-overexpressing cells was validated by GFP-LC3 dot formation, production of autophagosomes or acidic vacuoles, LC3 lipidation, and depletion of autopahgy-related genes. In addition, we also observed some evidence for apoptosis in TrkA-expressing cells. Many cells expressing TrkA exhibited annexin V-positive staining, activation of caspase-7 and BAX. Moreover, TrkA activated the JNK pathway, leading to phosphorylation of H2AX. In this report, we suggest that two cell death mechanisms occur simultaneously and interlink with each other. The JNK-calpain pathway might be a central process to mediate the two processes in TrkA-overexpressing cells, although further study still remains to prove the interplay between autophagy and apoptosis.     

Spatiotemporal dynamics of autophagy receptors in selective mitophagy

Damaged mitochondria are turned over through a process of selective autophagy termed mitophagy. In mitophagy, unhealthy mitochondria are recognized and ubiquitinated by Parkinson disease-linked proteins PINK1 and PARK2. The subsequent recruitment of ubiquitin-binding autophagy receptors leads in turn to the sequestration of the damaged organelles into LC3-positive phagophores, precursors to autophagosomes. The precise identity of these receptors and how they are regulated has been the focus of considerable attention. Our recent work uses live-cell imaging to explore the dynamics and regulation of autophagy receptor recruitment. Utilizing multiple paradigms to induce mitochondrial damage, we identified the rapid, 2-step recruitment of autophagy receptors OPTN, CALCOCO2/NDP52, and TAX1BP1. All 3 receptors are recruited to damaged mitochondria with similar kinetics; however, only OPTN is necessary for efficient formation of a phagophore sequestering damaged mitochondria from the cytosol. OPTN is co-recruited to damaged mitochondria along with its upstream kinase TBK1. Depletion of OPTN or TBK1, or expression of amyotrophic lateral sclerosis (ALS)-linked mutations in either protein, interfere with efficient autophagic engulfment of depolarized mitochondria. These observations suggest that insufficient autophagy of damaged mitochondria may contribute to neurodegenerative disease.     

NBR1 co-operates with p62 in selective autophagy of ubiquitinated targets

Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation. Although the exact mechanism underlying selection of autophagic substrates is not known, ubiquitination is a candidate signal for autophagic degradation of misfolded and aggregated proteins. p62/SQSTM1 was the first protein shown to bind both target-associated ubiquitin (Ub) and LC3 conjugated to the phagophore membrane, thereby effectively acting as an autophagic receptor for ubiquitinated targets. Importantly, p62 not only mediates selective degradation but also promotes aggregation of ubiquitinated proteins that can be harmful in some cell types. Is p62 the only autophagic receptor for selective autophagy? Looking for proteins that interact with ATG8 family proteins, we identified NBR1 (neighbor of BRCA1 gene 1) as an additional LC3- and Ub-binding protein. NBR1 is degraded by autophagy depending on its LC3-interacting region (LIR) but does not strictly require p62 for this process. Like p62, NBR1 accumulates and aggregates when autophagy is inhibited and is a part of pathological inclusions. We propose that NBR1 together with p62 promotes autophagic degradation of ubiquitinated targets and simultaneously regulates their aggregation when autophagy becomes limited.     

Chasing the elusive mammalian microautophagy

Different mechanisms for delivery of intracellular components (proteins and organelles) to lysosomes and late endosomes for degradation co-exist in almost all cells and set the basis for distinct autophagic pathways. Cargo can be sequestered inside double-membrane vesicles (or autophagosomes) and reach the lysosomal compartment upon fusion of these vesicles to lysosomes through macroautophagy. In a different type of autophagy, known as chaperone-mediated autophagy (CMA), single individual soluble proteins can be targeted one by one to the lysosomal membrane and translocated into the lumen for degradation. Direct sequestration of proteins and organelles by invaginations at the lysosomal membrane that pinch off into the lumen has also been proposed. This process, known as microautophagy, remains poorly understood in mammalian cells. In our recent work, we demonstrate the occurrence of both "in bulk" and "selective" internalization of cytosolic components in late endosomes and identify some of the molecular players of this process that we have named endosomalmicroautophagy (e-MI) due to its resemblance to microautophagy.     

Mutation at the cargo-receptor binding site of Atg8 also affects its general autophagy regulation function

Autophagy is a highly conserved degradation pathway for intracellular macromolecules and organelles. Among those characterized autophagy regulators, the ubiquitin-like protein Atg8 is found to be a membrane modifier that both regulates biogenesis of transport vesicles and interacts with the cargo receptor Atg19 for selective autophagic transport of the vacuolar enzyme prApe1 in budding yeast. The role of Atg8 in the enlargement of vesicle membrane during autophagosome biogenesis has been well documented, but how Atg8 coordinates vesicle formation and sorting of selective cargo is largely unknown. Identification of the cargo-receptor binding site of Atg8 would provide information to solve this issue. Here we characterized Atg8 mutants that were defective in interaction with the prApe1 receptor Atg19 and found that the vesicle formation function of these Atg8 mutants was also compromised to different extents. Atg8 mutants with single-residue substitution at the Atg19-binding site were defective in lipid conjugation and/or subcellular localization. Additional Atg8 mutants were found defective in autophagosome formation without affecting their interaction with Atg19, suggesting partially overlapping of the cargo-sorting site and its domains critical for autophagy control. Our observation paves the road for a more comprehensive understanding on how Atg8 coordinates cargo sorting and vesicle formation in selective autophagic pathways.