Monitoring autophagy in wheat living cells by visualization of fluorescence protein-tagged ATG8

Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Apg2p functions in autophagosome formation on the perivacuolar structure

Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole. We have identified and characterized a novel protein Apg2p essential for autophagy in yeast. Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation. Apg2p localizes to some membranous structure distinct from any known organelle. Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p. This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol 3-kinase complex and Apg9p. Apg2p(G83E), encoded by an apg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm. Overexpression of the mutant Apg2p lessens the defect in autophagy. These results suggest that the dot structure is physiologically important. Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome.     

Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process

Autophagy in plant cells is induced by nutrient starvation. Initially, double membrane-bound organelles, termed autophagosomes, enclose a portion of cytoplasm, and then fuse with a vacuole or lysosome to give an autolysosome. Autolysosomes can be visualized by incubating cells in the presence of a membrane-permeable cysteine protease inhibitor. The inhibitor presumably decreases proteolytic degradation of the autolysosome contents that are composed of portions of cytoplasm enclosed by the membrane originating from the inner membrane of autophagosomes, and allows them to accumulate. The origin of membranes that give rise to autophagosomes and autolysosomes is unknown. Here we use an acidotropic fluorescent dye, LysoTracker Red, to label autolysosomes specifically. We demonstrate that autolysosome membranes are marked by the presence of alpha-tonoplast intrinsic protein (alpha-TIP) but not by gamma-TIP or delta-TIP. The identification of a TIP specifically associated with membranes derived from an autophagic process may help our understanding of how plant cells generate and maintain functionally distinct types of vacuoles.     

Atg11 directs autophagosome cargoes to the PAS along actin cables

For more than 40 years, autophagy has been almost exclusively studied as a cellular response that allows adaptation to starvation situations. In nutrient-deprived conditions, cytoplasmic components and organelles are randomly sequestered into double-membrane vesicles called autophagosomes, creating the notion that this pathway is a nonselective process (reviewed in Refs 1, 2). Recent results, however, have demonstrated that under certain circumstances, cargoes such as protein complexes, organelles and bacteria can be selectively and exclusively incorporated into double-membrane vesicles.(1) We have recently shown that actin plays an essential role in two selective types of autophagy in the yeast Saccharomyces cerevisiae, the cytoplasm to vacuole targeting (Cvt) pathway and pexophagy, raising the possibility that the structures formed by polymers of this protein helps autophagosomes in recognizing the cargoes that must be delivered to the vacuole.(3) In this addendum, we discuss the possible central role of Atg11 as a molecule connecting cargoes, actin and pre-utophagosomal structure (PAS) elements.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.     

Yeast autophagosomes: de novo formation of a membrane structure

Autophagy - the degradation of organelles and cytoplasmic material - occurs through dynamic rearrangements of cellular membrane structures. Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation. The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed de novo many times. The autophagosome is formed through the nucleation, assembly and elongation of membrane structures. The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.     

Induction of Autophagy by Amino Acid Starvation in Fish Cells

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.

Addendum to: Monastyrska I, He C, Geng J, Hoppe D, Li Z, Klionsky DJ.Arp2 links autophagic machinery with the actin cytoskeleton. Mol Biol Cell 2008; 19:1962-75.     

Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation.

Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.     

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.     

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

Delivery of cytoplasmic proteins to autophagosomes

Autophagy represents a signaling-dependent regulated process that allows the degradation of some cellular proteins in autophagosomes, and plays a critical role in the management of cellular homeostasis under various stress conditions. In recent years, selective degradation of cytoplasmic proteins during stress has attracted considerable scientific interest. Here we examined the ability of resveratrol to induce autophagy in a variety of human cancer cell lines. We found that resveratrol-induced autophagy is accompanied by colocalization of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) with the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) in autophagosomes. In addition, we found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a previously shown PELP1-interacting protein, is co-recruited to autophagosomes in the presence of resveratrol. Although autophagy has been assumed to be a bulk and non-selective degradation process, in recent years, evidence of selective degradation of cytosolic proteins and organelles by autophagy is mounting. These observations suggest that the interaction of the target protein(s) with the delivery protein or proteins such as HRS facilitates the transport of certain cytoplasmic proteins to autophagosomes for their selective degradation, and thus, could influence the cytoplasmic as well as nuclear functions of nuclear receptor coregulators. Since PELP1 and, perhaps, other nuclear receptor coregulators are widely dysregulated in human cancers, these findings highlight the significance of the autophagic selective degradation of PELP1 following resveratrol (or other phytoestrogens) treatment in developing future strategies to use resveratrol under cancer prevention and therapeutic settings.     

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.     

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.     

Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Maturation of Autophagic Vacuoles in Mammalian Cells

The autophagic process was first described in mammalian cells several decades ago. After their formation as double-membraned vacuoles containing cytoplasmic material, autophagic vacuoles or autophagosomes undergo a stepwise maturation including fusion with both endosomal and lysosomal vesicles. However, the molecular mechanisms regulating these fusion steps have begun to emerge only recently. The list of newly discovered molecules that regulate the maturation of autophagosomes to degradative autolysosomes includes the AAA ATPase SKD1, the small GTP binding protein Rab7, and possibly also the Alzheimer-linked presenilin 1. This review combines previous data on the endo/lysosomal fusion steps during autophagic vacuole maturation with recent findings on the molecules regulating these fusion steps. Interestingly, autophagic vacuole maturation appears to be blocked in certain human diseases including neuronal ceroid lipofuscinosis and Danon disease. This suggests that autophagy has important housekeeping or protective functions, because a block in autophagic maturation causes a disease.