A novel Drosophila model system of chloroquine myopathy reveals how glycogen is targeted to the lysosome and what the significance of this process is for muscle cells.
Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.
Lysosomes are organelles that work as a disposal system for the cell. It is known that lysosomes can degrade glycogen and that defects in this function trigger the accumulation of vesicles containing glycogen in animals that lead to vacuolar myopathies—diseases that result in muscle weakness. However, it remains unclear how and why glycogen is degraded through this system, and what significance it has for the pathology of such diseases. Here, we addressed these questions by establishing a fruitfly model system to study glycogen autophagy in skeletal muscles. By feeding the flies chloroquine (CQ), we induce a vacuolar myopathy associated with massive accumulation of glycogen-filled vesicles, and assay the role of autophagy and glycogen metabolic enzymes in this process. We show that CQ-induced glycogen autophagy is completely dependent on the core conserved autophagy genes and that this autophagy is triggered by nutrient deprivation in a Tor-dependent manner. Interestingly, while glycogen autophagy and enzymatic glycogen breakdown can compensate for each other, concurrent inhibition of both systems blocks glycogen breakdown. Finally, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of glycogen synthase—the main enzyme involved in converting glucose to glycogen—in regulating autophagy through its interaction with the autophagosome.
Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.
Atg1; Atg13; autophagy; Drosophila; Atg17/FIP200; lysosome; Ref(2)P/p62; TOR
Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
Autophagy is a catabolic process involved in maintaining cellular homeostasis in eukaryotic cells, while coping with their changing environmental conditions. Mechanistically, it is also a process of considerable complexity involving multiple protein factors and implying numerous protein-protein and protein-membrane interactions. The cellular material to be degraded by autophagy is contained in a membrane-bound compartment called the autophagosome. We have characterised the formation of autophagosomes in the protozoan parasite Toxoplasma gondii by following the relocalisation of autophagosome-bound TgAtg8. Thus, exploiting GFP-TgAtg8 as a marker, we showed that it is a process that is regulated and can be induced artificially by amino acid starvation. Autophagic vesicles were also observed in normally dividing intracellular parasites. Depleting Toxoplasma of the TgAtg3 autophagy protein led to an impairment of TgAtg8 conjugation to the autophagosomal membrane and, at the cellular level, to a fragmentation of the single mitochondrion of the parasite and to a severe growth arrest. We have thus found that TgAtg3-dependent autophagy is essential for normal intracellular development of T. gondii tachyzoites.
RACK1 is a versatile scaffold protein in mammals, regulating diverse developmental processes. Unlike in non-plant organisms where RACK1 is encoded by a single gene, Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively. Previous studies indicated that the loss-of-function alleles of RACK1A displayed multiple defects in plant development. However, the functions of RACK1B and RACK1C remain elusive. Further, the relationships between three RACK1 homologous genes are unknown.
We isolated mutant alleles with loss-of-function mutations in RACK1B and RACK1C, and examined the impact of these mutations on plant development. We found that unlike in RACK1A, loss-of-function mutations in RACK1B or RACK1C do not confer apparent defects in plant development, including rosette leaf production and root development. Analyses of rack1a, rack1b and rack1c double and triple mutants, however, revealed that rack1b and rack1c can enhance the rack1a mutant's developmental defects, and an extreme developmental defect and lethality were observed in rack1a rack1b rack1c triple mutant. Complementation studies indicated that RACK1B and RACK1C are in principle functionally equivalent to RACK1A. Gene expression studies indicated that three RACK1 genes display similar expression patterns but are expressed at different levels. Further, RACK1 genes positively regulate each other's expression.
These results suggested that RACK1 genes are critical regulators of plant development and that RACK1 genes function in an unequally redundant manner. Both the difference in RACK1 gene expression level and the cross-regulation are likely the molecular determinants of their unequal genetic redundancy.
Receptor for Activated C Kinase 1 (RACK1) is viewed as a versatile scaffold protein in mammals. The protein sequence of RACK1 is highly conserved in eukaryotes. However, the function of RACK1 in plants remains poorly understood. Accumulating evidence suggested that RACK1 may be involved in hormone responses, but the precise role of RACK1 in any hormone signalling pathway remains elusive. Molecular and genetic evidence that Arabidopsis RACK1 is a negative regulator of ABA responses is provided here. It is shown that three RACK1 genes act redundantly to regulate ABA responses in seed germination, cotyledon greening and root growth, because rack1a single and double mutants are hypersensitive to ABA in each of these processes. On the other hand, plants overexpressing RACK1A displayed ABA insensitivity. Consistent with their proposed roles in seed germination and early seedling development, all three RACK1 genes were expressed in imbibed, germinating and germinated seeds. It was found that the ABA-responsive marker genes, RD29B and RAB18, were up-regulated in rack1a mutants. Furthermore, the expression of all three RACK1 genes themselves was down-regulated by ABA. Consistent with the view that RACK1 negatively regulates ABA responses, rack1a mutants lose water significantly more slowly from the rosettes and are hypersensitive to high concentrations of NaCl during seed germination. In addition, the expression of some putative RACK1-interacting, ABA-, or abiotic stress-regulated genes was mis-regulated in rack1a rack1b double mutants in response to ABA. Taken together, these findings provide compelling evidence that RACK1 is a critical, negative regulator of ABA responses.
ABA; drought; early seedling development; RACK1; salt; seed germination
B cell lymphoma 2 (Bcl-2) proteins are the central regulators of apoptosis. The two bcl-2 genes in Drosophila modulate the response to stress-induced cell death, but not developmental cell death. Because null mutants are viable, Drosophila provides an optimum model system to investigate alternate functions of Bcl-2 proteins. In this report, we explore the role of one bcl-2 gene in nutrient stress responses.
We report that starvation of Drosophila larvae lacking the bcl-2 gene, buffy, decreases survival rate by more than twofold relative to wild-type larvae. The buffy null mutant reacted to starvation with the expected responses such as inhibition of target of rapamycin (Tor) signaling, autophagy initiation and mobilization of stored lipids. However, the autophagic response to starvation initiated faster in larvae lacking buffy and was inhibited by ectopic buffy. We demonstrate that unusually high basal Tor signaling, indicated by more phosphorylated S6K, was detected in the buffy mutant and that removal of a genomic copy of S6K, but not inactivation of Tor by rapamycin, reverted the precocious autophagy phenotype. Instead, Tor inactivation also required loss of a positive nutrient signal to trigger autophagy and loss of both was sufficient to activate autophagy in the buffy mutant even in the presence of enforced phosphoinositide 3-kinase (PI3K) signaling. Prior to starvation, the fed buffy mutant stored less lipid and glycogen, had high lactate levels and maintained a reduced pool of cellular ATP. These observations, together with the inability of buffy mutant larvae to adapt to nutrient restriction, indicate altered energy metabolism in the absence of buffy.
All animals in their natural habitats are faced with periods of reduced nutrient availability. This study demonstrates that buffy is required for adaptation to both starvation and nutrient restriction. Thus, Buffy is a Bcl-2 protein that plays an important non-apoptotic role to promote survival of the whole organism in a stressful situation.
autophagy; Bcl-2; metabolism; non-apoptotic; nutrient restriction; S6K; starvation; Tor
In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17Δ mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13Δ mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17C24R, showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.
In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.
Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Δ cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1–Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.
To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, due in part to the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1.
We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth, and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself.
Our results reveal a central role for Atg1 in mounting a coordinated autophagic response, and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.
autophagy; cell growth; programmed cell death; Target of Rapamycin (TOR); Drosophila
The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes.
We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development.
DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.
Dictyostelium discoideum; G protein signaling; RACK1; WD40 repeat protein; Phosphoinositides; Phosphorylation; Dimerization
Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy.
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
Autophagy is a eukaryotic cellular process that transports cytoplasmic contents into lysosomes/vacuoles for degradation. It has been linked to multiple human diseases, including cancer and neurodegenerative disorders. The molecular machinery of autophagy was first identified and has been best characterized in the budding yeast Saccharomyces cerevisiae, but little is known about the autophagy machinery in another important unicellular model organism, the fission yeast Schizosaccharomyces pombe. In this study, we performed an unbiased and comprehensive screening of the fission yeast autophagy genes by profiling the mating phenotypes of nearly 3000 deletion strains. Following up on the screening results, we systematically characterized both previously known and newly identified fission yeast autophagy factors by examining their localization and the phenotype of their mutants. Our analysis increased the number of experimentally defined fission yeast autophagy factors from 14 to 23, including two novel factors that act in ways different from all previously known autophagy proteins. Together, our data reveal unexpected evolutionary divergence of autophagy mechanisms and establish a new model system for unraveling the molecular details of the autophagy process.
The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gβ homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and β1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible β1 integrin-associated kinase activity and association of Crk with p130CAS were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G1 or G1/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21Cip1/WAF1 and p27Kip1, and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.
Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.
autophagy; Drosophila; oogenesis; starvation; insulin/TOR
Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.
The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.
The receptor for activated C kinase 1 (RACK1) is one member of the most important WD repeat–containing family of proteins found in all eukaryotes and is involved in multiple signaling pathways. However, compared with the progress in the area of mammalian RACK1, our understanding of the functions and molecular mechanisms of RACK1 in the regulation of plant growth and development is still in its infancy. In the present study, we investigated the roles of rice RACK1A gene (OsRACK1A) in controlling seed germination and its molecular mechanisms by generating a series of transgenic rice lines, of which OsRACK1A was either over-expressed or under-expressed. Our results showed that OsRACK1A positively regulated seed germination and negatively regulated the responses of seed germination to both exogenous ABA and H2O2. Inhibition of ABA biosynthesis had no enhancing effect on germination, whereas inhibition of ABA catabolism significantly suppressed germination. ABA inhibition on seed germination was almost fully recovered by exogenous H2O2 treatment. Quantitative analyses showed that endogenous ABA levels were significantly higher and H2O2 levels significantly lower in OsRACK1A-down regulated transgenic lines as compared with those in wildtype or OsRACK1A-up regulated lines. Quantitative real-time PCR analyses showed that the transcript levels of OsRbohs and amylase genes, RAmy1A and RAmy3D, were significantly lower in OsRACK1A-down regulated transgenic lines. It is concluded that OsRACK1A positively regulates seed germination by controlling endogenous levels of ABA and H2O2 and their interaction.
Aberrant Aur-A signaling is associated with tumor malignant behaviors. However, its involvement in tumor metabolic stress is not fully elucidated. In the present study, prolonged nutrient deprivation was conducted into breast cancer cells to mimic metabolic stress in tumors. In these cells, autophagy was induced, leading to caspase-independent cell death, which was blocked by either targeted knockdown of autophagic gene ATG5 or autophagy inhibitor 3-Methyladenine (3-MA). Aur-A overexpression mediated resistance to autophagic cell death and promoted breast cancer cells survival when exposed to metabolic stress. Moreover, we provided evidence that Aur-A suppressed autophagy in a kinase-dependent manner. Furthermore, we revealed that Aur-A overexpression enhanced the mammalian target of rapamycin (mTOR) activity under metabolic stress by inhibiting glycogen synthase kinase 3β (GSK3β). Inhibition of mTOR activity by rapamycin sensitized Aur-A-overexpressed breast cancer cells to metabolic stress-induced cell death. Consistently, we presented an inverse correlation between Aur-A expression (high) and autophagic levels (low) in clinical breast cancer samples. In conclusion, our data provided a novel insight into the cyto-protective role of Aur-A against metabolic stress by suppressing autophagic cell death, which might help to develop alternative cell death avenues for breast cancer therapy.
aurora kinase; metabolic stress; autophagy; cell death; breast cancer
Background: ATG4 is a cysteine peptidase crucial for macroautophagy.
Results: Gene deletion mutants show that the two ATG4s of Leishmania perform distinct roles, although there is some redundancy.
Conclusion: ATG4s are not individually essential but macroautophagy, a process important in the virulence of the parasite, requires one.
Significance: Highlights the distinct roles of ATG4 isoforms and their importance for autophagy and parasite infectivity.
Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.
Autophagy; Cysteine Protease; Leishmania; Parasite; Parasite Metabolism; Peptidases; Protease; ATG4
Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12–5-16L1 complex. The molecular mechanisms of Atg12–5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12–5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12–5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.
•WIPI2 binds Atg16L1 directly and recruits the Atg12–5-16L1 complex•WIPI2 binding to Atg16L1 is required for LC3 lipidation and autophagosome formation•WIPI2-Atg16L1 function requires PI3P binding by WIPI2 and is independent of FIP200•Autophagosomal engulfment of Salmonella requires the WIPI2-Atg16L1 complex
Starvation-induced autophagy requires PtdIns(3)P locally produced on ER-derived membranes. Dooley et al. demonstrate that the PtdIns(3)P effector WIPI2b binds Atg16L1 to recruit Atg12–5-16L1 to PtdIns(3)P-positive omegasomes, resulting in LC3 lipidation and starvation-induced autophagy. These findings suggest that WIPI2b senses increases in PtdIns(3)P and directs LC3 conjugation to developing autophagosomes.
Atg9 is a transmembrane protein essential for autophagy which cycles between the Golgi network, late endosomes and LC3-positive autophagosomes in mammalian cells during starvation through a mechanism that is dependent on ULK1 and requires the activity of the class III phosphatidylinositol-3-kinase (PI3KC3). In this study, we demonstrate that the N-BAR-containing protein, Bif-1, is required for Atg9 trafficking and the fission of Golgi membranes during the induction of autophagy. Upon starvation, Atg9-positive membranes undergo continuous tubulation and fragmentation to produce cytoplasmic punctate structures that are positive for Rab5, Atg16L and LC3. Loss of Bif-1 or inhibition of the PI3KC3 complex II suppresses starvation-induced fission of Golgi membranes and peripheral cytoplasmic redistribution of Atg9. Moreover, Bif-1 mutants, which lack the functional regions of the N-BAR domain that are responsible for membrane binding and/or bending activity, fail to restore the fission of Golgi membranes as well as the formation of Atg9 foci and autophagosomes in Bif-1-deficient cells starved of nutrients. Taken together, these findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.
Bif-1; Atg9 trafficking; Golgi fragmentation; membrane curvature; Beclin 1; UVRAG; class III PI3-kinase; autophagosome; starvation
Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.
Beclin 1 was not only the first-described mammalian autophagy protein, but is one of the most widely-characterized players in autophagy regulation. It is implicated in multiple human disease conditions. As a core component of the essential lipid kinase complex (PI3K-III), beclin 1 has largely been characterized to date in the context of autophagy through its recruitment of additional autophagy proteins for the assembly of the PI3K-III complexes involved in the nucleation of the autophagosome. Little is known, however, about how beclin 1 regulates specific functions of PI3K-III in other membrane trafficking pathways. Furthermore, although beclin 1 has been linked to multiple neurodegenerative diseases, the function of beclin 1 in the brain remains uncharacterized. Herein, we used genetic animal models and mutant cell lines to demonstrate that beclin 1 participates in multiple organelle trafficking pathways. Beclin 1 deficiency in neurons causes severe neurodegeneration, concomitant with aberrant late endosome formation and impaired phospholipid localization. Our mechanistic study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy. Our study clarifies the physiological function of beclin 1, which leads for further understanding of its role in tumorigenesis, infectious disease and neurodegenerative disease.
Blocking autophagy protects the apoptosis inhibitor dBruce from destruction and promotes nurse cell survival in developing egg chambers.
Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.