InteractiveFly: GeneBrief

Autophagy-related 6: Biological Overview | References

Gene name - Autophagy-related 6

Synonyms - Beclin1, beclin,

Cytological map position - 95D10-95D10

Function - signaling

Keywords - a component of the Vps34 lipid kinase complex that is required for the formation of PI3P - functions in vesicle trafficking including autophagy and endocytosis - involved in stress, metabolic and immune signaling pathways - binds to Rab18 - promotes niche occupancy by bam mutant stem cells - Zonda, an Atg6 interactor, is critically required for starvation-induced autophagy - UVRAG interacts with Atg6 and together they promote autophagy - required for endolysosomal degradation and epithelial polarity during wing development

Symbol - Atg6

FlyBase ID: FBgn0264325

Genetic map position -

NCBI classification - APG6: Autophagy protein Apg6

Cellular location - cytoplasmic


NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine

Autophagy targets cytoplasmic materials for degradation, and influences cell health. Alterations in Atg6/Beclin-1, a key regulator of autophagy, are associated with multiple diseases. While the role of Atg6 in autophagy regulation is heavily studied, the role of Atg6 in organism health and disease progression remains poorly understood. This study discovered that loss of Atg6 in Drosophila results in various alterations to stress, metabolic and immune signaling pathways. The increased levels of circulating blood cells and tumor-like masses in atg6 mutants vary depending on tissue-specific function of Atg6, with contributions from intestine and hematopoietic cells. These phenotypes are suppressed by decreased function of macrophage and inflammatory response receptors crq and drpr. Thus, these findings provide a basis for understanding how Atg6 systemically regulates cell health within multiple organs, and highlight the importance of Atg6 in inflammation to organismal health (Shen, 2022).

Atg6/Beclin-1 has been implicated in the regulation of autophagy, endocytosis, apoptosis, and other cell processes. These diverse cellular roles suggest how Atg6/Beclin-1 loss may lead to health detriments and disease development. In mice, Beclin-1 loss enhanced tumorigenesis and invasive potential. Breast cancer cells deficient in Beclin-1 demonstrate enhanced tumorgenicity, potentially through modifications to cell membrane proteins like E-Cadherin. Like these mammalian models, Drosophila larvae lacking Atg6 possess cells with invasive properties in the form of blood cell masses. This study demonstrated the complex and tissue-specific roles of Atg6 by showing Atg6 rescue specifically in either blood or intestine cells differentially modulates atg6 deficiency phenotypes. Importantly, it was established that the macrophage receptors Crq and Drpr contribute to atg6 mutant blood cell tumor-like aggregation, providing strong evidence of Atg6 function as a suppressor of inflammation (Shen, 2022).

Studies of RNA levels in atg6 mutants revealed significant changes in multiple pathways. Stress response genes, including those in the Turandot and glutathione S-transferase family, were strongly upregulated with loss of atg6. These changes may reflect an increase in cellular stress, consistent with the metabolic roles of Atg6. Consistent with this possibility, a substantial difference in metabolite profiles were detected between atg6 mutants and controls in multiple tissue types. An increase was detected in specific metabolites in the atg6 mutant intestine and hemolymph, including phosphoserine and phosphoethanolamine. Interestingly, modulators of phosphoserine are known to affect cancer prognosis. In addition, previous work demonstrated that phosphoethanolamine accumulation protected breast cancer cells from glutamine deprivation and enhanced tumorigenicity. RNAs of genes associated with ribosomes were also increased with loss of Atg6, and increased ribosome biogenesis is a hallmark of many cancer types. Metabolic changes may provide an enhanced environment for tumor cells to establish and spread, which may explain why atg6 mutants are so prone to developing invasive blood cell masses. It is tempting to speculate if haploinsufficient beclin-1 mice, which also share a predisposition for developing spontaneous tumors, have a similar metabolic profile that promotes tumorous growth. Future analyses of the metabolic changes observed in atg6 loss-of-function mutant Drosophila in haplo-deficient beclin-1 mice will help address this question (Shen, 2022).

Alterations in stress response pathways and imbalances in amino acid content can adversely affect the health of the cell, potentially explaining the increased ROS in atg6 loss-of-function mutant intestine cells. It is certainly possible that with loss of Atg6 function, cells are unable to clear unstable cargo and waste through autophagy, leading to increased ROS. The mitophagy deficiency in intestine cells with decreased Atg6 function is consistent with this hypothesis, as a failure to clear dysfunctional mitochondria could result in increased mitochondrial ROS. However, it is unknown if this principle applies to non-mitochondrial autophagic cargoes. Furthermore, the possibility of a non-autophagy-related role of Atg6 in ROS production cannot be ruled out. Thus, these findings open new directions for how Atg6/Beclin-1 may function in tissue-specific manners to promote survival and stress-response during development, as well as suppress tumor formation (Shen, 2022).

Alterations in stress response markers, including GSTD1-GFP and TRE-GFP, in atg6 mutant intestine cells also support a role for Atg6 in suppression of stress. Furthermore, the upregulation of BomS1, BomS2, BomS3, Dso1, as well as genes involved in metabolism in atg6 mutants may be consistent with the role of Atg6 in inflammation and immunity. Loss of Atg6 could also affect nutrient-sensing pathways because of the established role of autophagy in catabolism, which could explain decreased activation of AKT and increased levels of phosphorylated AMPK. AKT normally inhibits Atg6 activity in response to growth factors. The nutrient-deprived and sepsis-like characteristics of Atg6 loss-of-function mutants are counter-intuitive with AKT activity. Likewise, activation of AMPK serves to upregulate Atg6 activity, which would be expected to be increased if Atg6 activity is missing and the animal is deprived of nutrients. Further investigation will determine if these phenotypes are due to either direct roles of Atg6 or if they reflect more global and indirect effects of Atg6 deficiency (Shen, 2022).

Previous work suggests that AKT activates SREBP. Therefore, it is interesting that SREBP activity is increased in atg6 mutants when AKT activity is decreased. It is possible that loss of Atg6 uncouples this relationship by alteration of lipid content. While speculative, decreased lipid droplets in cells may stimulate SREBP activity, which through a negative feedback loop could inhibit AKT activity. Further studies, including investigation of the role of mTOR in these pathways, are necessary to elucidate the relationship between SREBP and AKT in the absence of Atg6 (Shen, 2022).

Characterization of the blood cell masses in atg6 mutant larvae demonstrates that Atg6-deficient blood cells display invasive phenotypes, including MMP1 positivity and disruption of intestine smooth muscle. Furthermore, atg6 mutant phenotypes, including developmental arrest, blood cell aggregation, lymph gland enlargement, and increased circulating blood cell numbers, were suppressed to varying degrees by expression of Atg6 in either intestine or blood cells. However, rescue expression in some tissue subsets failed to suppress mutant lethality or blood cell phenotypes, supporting a hypothesis that altered Atg6 function in a subset of tissues could drive tumorigenesis and disrupt systemic homeostasis. Future studies should evaluate how Atg6 loss in one tissue affects metabolites in tissues that retain Atg6 function (Shen, 2022).

Significantly, the blood cell immune receptors Crq and Drpr contributed to blood cell aggregation in atg6 mutants. It remains unclear if similar inflammatory mechanisms account for both the blood cell differences and tumors in Beclin-1 deficient mice. Mmp1 plays a role in cancer invasiveness, likely as a collagenase that facilitates tumor cell invasion through the basement membrane . Although speculative, increased Mmp1 activity resulting from loss of Atg6/Beclin-1 and subsequent dysregulation of Crq and/or Drpr may explain the increase in invasive cell types in both Drosophila and mice. However, it cannot be excluded that increased Mmp1 activity is simply an indirect byproduct of atg6 loss (Shen, 2022).

Interestingly, Beclin-1 provides a protective role against sepsis (Sun, 2018). When compared to control animals, atg6 mutants display several molecular and metabolic hallmarks of sepsis. Upregulation of AICAR and AMP, as well as subsequent increase in pAMPK, is consistent with an increased response to septic shock. As in human patients with sepsis, atg6 mutants display an acute inflammatory response, immune cell activation, and increased oxidative stress as evidenced by JNK activation, upregulation of immune response genes, and increased oxidative stress. Likewise, MMP1 activation is believed to contribute to sepsis development. Taken together, these findings are consistent with a model whereby atg6 deficiency either results in increased susceptibility to sepsis or upregulates a sepsis-like response. These findings could explain the increased number of circulating blood cells, as increased circulating immune cells are characteristic of sepsis. In addition, the fact that Atg6 expression specifically in immune cell lineages rescues multiple mutant phenotypes suggests that Atg6 regulates this process, at least in part, through its role in the immune system. However, the possibility that loss of Atg6 contributes to this phenotype by enhancing blood cell proliferation through upregulation of cell proliferation regulators. Additional studies of Atg6 function in sepsis development, along with other diseases including cancer, will be required to better understand how Beclin-1 suppresses these human conditions (Shen, 2022).

The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. This work characterized a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compa a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. These mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. This study finds that GTP-bound Rab18 binds to p62 (refractory to sigma P) and Atg8a was not apparent in Rab3GAP2 mutant head lysates was surprising; but, it is possible that only a subset of neurons is defective for autophagy in these flies, which this study failed to detect. Although studies in mammalian WMS models are focusing on neuronal defects, the data suggest that it may be worth investigating other tissues of WMS patients as well (Takats, 2020).

For characterizing the role of the Rab3GAP-Rab18 module in autophagy, the genetically mosaic fat tissue of L3 larvae, a well-established system for autophagy analysis in Drosophila, was used. By analyzing Rab3GAP2 mutants and multiple independent RNAi lines, this study shows that the loss-of-function cells are less effective in autophagosome–lysosome fusion and are defective in autolysosome morphology and maturation. Additionally, it was found that the lack of Rab3GAP2 function causes striking perturbation of Rab7-positive late endosomes, autophagosomes, and (auto)lysosomes, but it does not affect Rab5-positive early endosomes. Thus, the data suggest that the Rab3GAP-Rab18 module can be considered as a general regulator of lysosome maturation. These results fit well with the findings of a recently published paper showing that Rab18 acts in concert with Rab7 during the lysosomal fusion events of autophagy and in the axonal transport of lysosomes. On the other hand, previous studies in C. elegans and cultured human cells suggested that the Rab3GAP subunits and Rab18 are rather involved in early steps of autophagy; however, it is important to note that these latter studies focused only on the amount and localization patterns of the autophagy markers Atg8 and p62, with no particular emphasis on ultrastructural analysis of the integrity of the lysosomal system. Electron microscopy observations concerning that numerous autophagosomes and autophagosome clusters are present in the cytoplasm of Rab3GAP2 mutant fat cells further suggest that loss of the Rab3GAP-Rab18 module causes major defects in (auto)lysosome function rather than in autophagosome formation. Of course, the possibility that the discrepancies between these studies arise due to a tissue-specific role of the Rab3GAP-Rab18 module in autophagy cannot be ruled out. The finding that Rab3GAP2 mutant adults obviously accumulate much more autophagy cargos in muscles than in their brain further corroborates this notion (Takats, 2020).

Since this study demonstrated that loss of Vps34 Complex I function results in phenotypes similar to those of inhibition of the Rab3GAP-Rab18 module and, furthermore, a physical interaction between the permanent Vps34 complex subunit Atg6 and the GTP-bound Rab18 was proven, it is proposed that Complex I is likely a novel Rab18 effector (Takats, 2020).

Vps34 Complex I is one of the most important regulators of autophagosome formation, and it also has a role in autophagosome maturation and fusion [(Diao, 2015). As matured, intact autophagosomes in Rab3GAP2 mutant cells were detected, it seems likely that Rab18 is critical for Vps34 Complex I activity following autophagosome formation. Localization of Rab3GAP subunits and Rab18 to autophagosomes also suggests that this module plays a role in later steps of autophagosome maturation: It facilitates their fusion with lysosomes and further enhances the maturation of the newly formed autolysosomes into enlarged degradative compartments (Takats, 2020).

The question of how could the autophagosome-localized Rab3GAP-Rab18 module affect vesicle maturation is yet to be answered. Based on the current results showing that Rab7 becomes dispersed in cells lacking the Rab3GAP-Rab18 module, it is suggested that the most important role of this module is to stabilize the Rab7-containing compartment. During their maturation, (auto)lysosomes undergo a series of membrane fusion events with endosomes, Golgi-derived vesicles, and autophagosomes. All these steps are mediated by Rab7 and its effectors such as the tethering factor HOPS complex or the adaptor protein PLEKHM1. As matured autophagosomes are also positive for Rab7, the autophagy-derived Rab7 proteins can be an important source for the lysosomal Rab7 pool. This scenario is even more likely in cell types such as fat cells, which show relatively low endocytic but high autophagic activity. Still, it cannot be ruled out that the Rab3GAP-Rab18 complex is also present on maturing endosomes or Golgi-derived transport vesicles that may also act as important Rab7 sources for maturing lysosomes. The precise contribution of these membrane transport pathways to maintaining the lysosomal Rab7 pool needs to be further investigated in the future (Takats, 2020).

This research highlights that the Rab3GAP-Rab18 module, in concert with the activity of the Vps34 Complex I, maintains the integrity of the Rab7-positive late endosomal/lysosomal compartment. Additionally, these findings that loss of Rab3GAP-Rab18 function perturbs autolysosome maturation and autophagic degradation shed light on a new possible cause of WMS development and open up potential novel therapeutic perspectives for this disease (Takats, 2020).

Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy
Alzheimer's disease (AD) is one the most common types of dementia. Plaques of amyloid beta and neurofibrillary tangles of tau are two major hallmarks of AD. Metabolism of these two proteins, in part, depends on autophagy pathways. Autophagy dysfunction and protein aggregation in AD may be involved in a vicious circle. The aim of this study was to investigate whether tau or amyloid beta 42 (Aβ42) could affect expression of autophagy genes, and whether they exert their effects in the same way or not. Misxpression levels of some autophagy genes, Hook, Atg6, Atg8, and Cathepsin D, were measured using quantitative PCR in transgenic Drosophila melanogaster expressing either Aβ42 or Tau R406W. Hook mRNA levels were downregulated in Aβ42-expressing flies both 5 and 25 days old, while they were increased in 25-day-old flies expressing Tau R406W. Both Atg6 and Atg8 were upregulated at day 5 and then downregulated in 25-day-old flies expressing either Aβ42 or Tau R406W. Cathepsin D expression levels were significantly increased in 5-day-old flies expressing Tau R406W, while there was no significant change in the expression levels of this gene in 5-day-old flies expressing Aβ42. Expression levels of Cathepsin D were significantly decreased in 25-day-old transgenic flies expressing Tau R406W or Aβ42. It is concluded that both Aβ42 and Tau R406W may affect autophagy through dysregulation of autophagy genes. Interestingly, it seems that these pathological proteins exert their toxic effects on autophagy through different pathways and independently (Haghi, 2020).

Autophagy promotes tumor-like stem cell niche occupancy

Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. This study used Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, it was found that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy (Zhao, 2018).

This study used Drosophila ovarian germline stem cells to study stem cell competition for niche occupancy. Significantly, it was found that autophagy promotes niche occupancy by bam mutant stem cells. Autophagy is required for proper cell cycle of bam mutant cells, and regulators of growth influence bam mutant stem cell niche occupancy. Previous reports indicate that autophagy is required for stem cell maintenance, proper differentiation, and homeostasis in different stem cell systems. By contrast, the data indicate that loss of autophagy does not have a negative impact on Drosophila ovarian germline stem cells, consistent with data indicating that autophagy is low in normal wild-type stem cells. Drosophila ovarian germline stem cells are the largest cells in germaria, and they have a high metabolic rate that is associated with the activation of BMP signaling, the expression of Myc that is a key regulator of cell growth and ribosome biogenesis, and a high level of rRNA transcription. Therefore, the catabolic autophagy pathway may be dispensable in Drosophila ovarian germline stem cells under normal conditions (Zhao, 2018).

Stem cell renewal is dependent on cell growth and division that is typically regulated by mTOR. In most cell contexts, autophagy is inhibited when mTOR-dependent cell growth is activated. Therefore, it is logical that autophagy levels are low in normal stem cells that are not stressed. By contrast, transformed cells, such as those with activated Ras, have been reported to possess elevated autophagy. Therefore, it seems reasonable that, like Ras transformed cells, bam mutant stem cells may depend on autophagy for cell division and ovarian growth. Interestingly, autophagy is required for the proliferation of fast-dividing germline progenitor cells in the C. elegans larval gonad. It is not clear why autophagy may be compatible with and required for tumor-like germline stem cell growth and division. One possibility is that autophagy is functioning to reduce cell stress associated with increased metabolic rate, protein, and organelle damage, but if this were the case, this would likely be reflected in increased cell death. Because non increase in cell death was observed, an alternative explanation is that autophagy is promoting bioenergetic homeostasis that is needed in tumor-like cells (Zhao, 2018).

Unlike previous studies of epithelial cell competition, the data indicate that loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influence the competition of tumor-like stem cells for niche occupancy. Stem cells possess properties that distinguish them from epithelial cells in the context of cell competition. First, adult stem cells are usually quiescent, while epithelial cell competition often takes place in fast-growing tissues. Second, stem cells compete for niche occupancy and there are no known specialized niches in epithelial cell competition systems. Third, loser stem cells are displaced from the niche and do not die, while loser epithelial cells die, enabling winners to expand during epithelial cell competition. In addition, tumor-like stem cells appear to divide faster than normal adult stem cells. These differences probably make tumor-like stem cells more sensitive to regulators of growth than epithelial cells during competition (Zhao, 2018).

Autophagy can either promote or suppress tumor growth, depending on cell and tissue context. Similar phenomena were also observed in different Drosophila tumor-like overgrowth models, where autophagy either enhanced or suppressed epithelial overgrowth phenotypes, depending on the oncogenic stimulus. The results indicate that autophagy promotes Drosophila ovarian germline tumor-like growth. Significantly, autophagy promotes niche occupancy by bam mutant tumor-like stem cells. Of note, the data indicate that the super-competition of bam mutant stem cells largely depends on their proliferative potential. In addition, the data contradict the current model that the niche stem cell adhesion factor E-cadherin plays a vital role, as no significant difference was observed in E-cadherin between wild-type and bam mutant stem cells. Furthermore, bam mutant stem cells that are out of the niche do not possess an E-cadherin connection with niche cap cells, but they are still more competitive than wild-type stem cells for niche occupancy, further challenging the current model that emphasizes a critical role of E-cadherin. In addition, although the super-competition of tumor-like stem cells for niche occupancy is proposed to be important for tumor initiation, it still cannot be excluded that autophagy also contributes to tumor progression in the tumor model system. Importantly, these studies indicate that specifically targeting autophagy in tumor-like stem cells could have potential for cancer therapy (Zhao, 2018).

Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila

Age-related changes of adult stem cell are crucial for tissue aging and age-related diseases. Thus, clarifying mechanisms to prevent adult stem cell aging is indispensable for healthy aging. Metformin, a drug for type 2 diabetes, has been highlighted for its anti-aging and anti-cancer effect. In Drosophila intestinal stem cell (ISC), the inhibitory effect of metformin on age-related phenotypes of has been previously reported. This study showed that knockdown of Atg6, a crucial autophagy-related factor, in ISC induces age-related phenotypes of ISC such as hyperproliferation, centrosome amplification, and DNA damage accumulation. Then, it was revealed that metformin inhibits ISC aging phenotypes in Atg6-dependent manner. Taken together, this study suggests that Atg6 is required for the inhibitory effect of metformin on ISC aging, providing an intervention mechanism of metformin on adult stem cell aging.

Zonda is a novel early component of the autophagy pathway in Drosophila

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. This study reports that a novel Drosophila immunophilin, named Zonda (CG5482), is critically required for starvation-induced autophagy. Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions, and soon after starvation nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13 and Atg17 but does not require Vps34, Vps15, Atg6 or Atg14. Zonda interacts physically with ATG1 through its kinase domain, as well as with ATG6 and Vps34. It is proposed that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation (Melani, 2017).

Autophagy, one of the main degradative pathways of the cell, begins with the formation of a membranous cistern called phagophore or isolation membrane that buds from a cup-shaped structure associated with the endoplasmic reticulum (ER) called omegasome. Thereafter, the phagophore expands and finally seals, giving rise to a double membrane organelle named autophagosome where cytoplasmic components including protein aggregates, ribosomes, and mitochondria are sequestered. Soon afterward, autophagosomes acquire degradative enzymes by successive fusion with late endosomes and lysosomes, thereby becoming an autophagolysosome where the engulfed material is degraded (Melani, 2017).

Autophagy, whose main stimulus is the stress generated by nutrient deprivation, is modulated by intracellular signaling pathways, mainly the target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) cascades, as well as by extracellular factors including hormones. Activation of the ULK1 complex (Atg1 complex in yeast and Drosophila) has been described as the first event in the autophagy cascade. This complex, formed by ULK1/2, FIP200/Atg17, Atg13, and Atg101, is constitutively assembled, and its kinase activity is negatively regulated by TOR signaling, which in turn depends on amino acid availability and the energy status of the cell. ULK1/Atg1 regulates the recruitment and activation of a second complex: the Vps34 lipid kinase complex, also called the autophagy nucleation complex, which is composed of the class 3 phosphatidylinositol 3-kinase Vps34 and the proteins PI3KR4 (Vps15), Beclin1 (BECN1)/Atg6, and Atg14 (Melani, 2017).

Vps34 mediates the synthesis of phosphatidylinositol 3-phosphate (PI3P). Local synthesis of this lipid defines the location of omegasome formation and, therefore the site of recruitment of several FYVE domain-containing proteins including DFCP1 and WIPI1, which in turn mediate phagophore elongation and autophagosome formation. Within the Vps34 complex, BECN1 is a direct target of ULK1/Atg1, and Vps34 kinase activity is believed to depend on the differential interaction of BECN1 with AMBRA1 or with the anti-apoptotic protein BCL-2. BCL-2 binding modulates the levels of BECN1 that become available to interact with Vps34 in the autophagy nucleation complex, thereby contributing to define if the cell will enter apoptosis or activate autophagy (Melani, 2017).

FK506-binding proteins (FKBPs) play a role in immunoregulation and participate in critical cellular functions that include protein trafficking and folding. Members of this family display peptidyl prolyl cis/trans isomerase (PPIase) activity, participating in de novo protein folding through the interconversion of intermediate folding states into the final tridimensional structure. This study has investigated a novel Drosophila gene-which has been named Zonda (Zda)-that encodes an immunophilin of the FKBP family, presumably homologous to mammalian FKBP8/FKBP38 (Bhujabal, 2017; Melani, 2017 and references therein).

By utilizing an in vivo approach, this study found that Zda is critically required for starvation-induced autophagy. Zda protein displays a cytoplasmic distribution in well-fed larvae and, shortly after the onset of starvation, nucleates in foci that colocalize with omegasome markers. Genetic manipulations revealed that components of the induction complex, Atg1, Atg13, and Atg17, but not components of the Vps34 complex, Vps34, Vps15, Atg6, or Atg14, are required for starvation-induced Zda nucleation. Moreover, Zda interacts physically with Atg1, Atg6, and Vps34 and is necessary for autophagic activation of Vps34 and omegasome formation, as revealed by DFCP1 foci formation following starvation. Zonda overexpression is sufficient to trigger a bona fide autophagic response, as evaluated by different autophagic markers. It is proposed that Zda is a novel component of the Drosophila autophagy machinery that forms part of the omegasome and is required for deposition of PI3P by the Vps34 complex and, hence, for the initiation of autophagosome biogenesis (Melani, 2017).

Previously, other immunophilins have been proposed both as positive or negative regulators of autophagy. In Drosophila, FKBP39 was found to be a negative regulator of developmentally triggered autophagy, possibly through the regulation of the transcription factor Foxo. Mammalian FKBP51 was described as a scaffold protein that recruits PHLPP, Akt, and Beclin1, leading to activation of autophagy. FKBP38 has been reported as a mitophagy receptor that interacts with LC3. Coexpression of FKBP38 along with LC3 can trigger Parkin-independent mitophagy (Melani, 2017).

Based on sequence homology, Zda is the likely orthologue of FKBP38. Not only do they share characteristics domains of FKBP proteins, but both proteins are the only members of their families to have a transmembrane domain on their C-terminal end. This study has shown that Zda is required for starvation-induced autophagy. Larval fat body cells in which Zda expression has been silenced fail to trigger autophagy, as assessed by several independent criteria: 1) inability of the cells to form autophagosomes and autolysosomes after starvation, as assessed by TEM and Atg8 nucleation; 2) their inability to increase the number and size of lysosomes, as evaluated by LysoTracker and GFP-Lamp markers; and 3) accumulation of Ref(2)P in these cells, which is indicative of impaired autophagic flux (Melani, 2017).

This study has found that, after nutrient deprivation, Zda can be detected in omegasomes, colocalizing with PI3P and DFCP1, from which early autophagic structures labeled with GFP-Atg5 and GFP-Atg8 bud off. Consistent with the notion that Zda is an early component of the autophagy cascade, genetic analysis revealed that starvation-induced Zda nucleation depends fully on components of the Atg1 induction complex but not on components of the Vps34 nucleation complex. Vps34 autophagic activation following starvation is regulated by the nutritional status of the cell downstream of Atg1. This study found that Zda interacts physically with the Atg1 kinase domain, as well as with components of the nucleation complex, including Atg6 and Vps34, suggesting that it may contribute to the activation of the latter complex by Atg1. This notion is consistent with the results of genetic experiments utilizing early autophagy markers, as they suggest that autophagy-dependent Vps34 activation and omegasome formation are dependent on Zda, this dependence being comparable to that on Atg1. Unlike Atg6, which was shown to be also required for Vps34 basal activity, Zda is clearly not necessary for early endosome formation but only for autophagic activation of Vps34. Thus, given the requirement of Zda for Vps34 autophagy-specific activation, and based on its localization at the omegasome, it is proposed that Zda contributes to define the location on the ER at which Vps34-dependent PI3P deposition and omegasome formation take place (Melani, 2017).

Induction of autophagy depends on the nutritional status of the cell and is subject to a contra-regulatory mechanism that occurs between mTOR and Atg1. Under nutrient-rich conditions, active mTOR phosphorylates and inactivates the Atg1 complex, and when nutrients are scarce, mTOR-dependent inactivation of Atg1 is released. Atg1 in turn reinforces down-regulation of mTOR through mechanisms that remain poorly defined. In line with this, Drosophila fat body cells that are mutant for atg1 grow bigger than control cells when subjected to prolonged nutrient deprivation, and conversely, Atg1 overexpression provokes cell size reduction and induces autophagosome formation. This study has shown that when overexpressed above certain levels, Zda can trigger a bona fide autophagic process, as assessed by several indicators, including TEM, Atg8 nucleation, and LysoTracker incorporation. This autophagic response fully depends on the activity of Vps34 and partially on Atg1. This suggests that Zonda operates upstream of Vps34 and in parallel to Atg1. Consistent with this, it was observed that under the same overexpression conditions, the TOR pathway is down-regulated and cell size is reduced similarly to what has been reported for Atg1. In line with these observations, adult flies that are homozygous for a Zda null mutation specifically in the head exhibit larger heads. Thus Zda mediates negative regulation of TOR, thereby exerting cell-­autonomous negative regulation of growth (Melani, 2017).

Given that immunophilins are known to work as chaperons or scaffolds, it is proposed that Zda might provide a platform where Atg1 and the Vps34 complex interact. Further research is required to define the mechanism by which Zda cooperates with Atg1 on the activation of the Vps34 nucleation complex that culminates in localized PI3P deposition for omegasome formation (Melani, 2017).

Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster

Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. This study used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila eye. Subsequently, their expression in D. melanogaster neurons was inhibited under the direction of a Dopa decarboxylase (Ddc) transgene, and the effects on longevity and motor function were examined. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, the roles of these genes were investigated in the well-studied alpha-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies (M'Angale, 2016).

The pro-apoptotic STK38 kinase is a new Beclin1 partner positively regulating autophagy
Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. This study describes the STK38 protein kinase (also termed NDR1 and Tricornered in Drosophila) as a conserved regulator of autophagy. STK38 was discovered as a novel binding partner of Beclin1 (Atg6; see Drosophila Atg6), a key regulator of autophagy. STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 (Pi3K59F in Drosophila) activity, as judged by PI3P formation. Furthermore, it was observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, STK38 was found to be stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, these data establish STK38 as a conserved regulator of autophagy in human cells and flies. Evidence is also provided demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions (Joffre, 2015).

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, has been identified as a new autophagy regulator; this gene is therefore referred to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved (Kim, 2015).

Atg6/UVRAG/Vps34-containing lipid kinase complex is required for receptor downregulation through endolysosomal degradation and epithelial polarity during Drosophila wing development

Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells was performed in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, these results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation (Lorincz, 2014).

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-kappaB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. This study generated Atg6 mutant Drosophila and demonstrated that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFkappaB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and the data indicate that this is due to defects in multiple membrane trafficking processes (Shravage, 2013).

The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation

Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII; see Phosphotidylinositol 3 kinase 59F) complex to promote pruning via the endocytic pathway. By studying several PI3P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).

Neuronal remodeling is an essential step of nervous system development in both vertebrates and invertebrates. One mechanism used to remodel neuronal circuits is by the elimination of long stretches of axons in a process known as axon pruning. With a few exceptions, the current dogma is that axon pruning of long stretches of axons occurs via local axon degeneration while axon pruning of short stretches occurs via retraction. While in some cases remodeling is directly affected by experience or neural activity, in cases of stereotypical pruning the identity of the axon that is destined to be prƒuned does not depend on experience or neural activity. Because of mechanistic similarities to Wallerian degeneration and dying back neurodegenerative diseases, understanding the molecular mechanisms of axon pruning should result in a broader insight into axon fragmentation and elimination during development and in disease (Issman-Zecharya, 2014).

The neuronal remodeling of the Drosophila mushroom body (MB) during development is a unique model system to study the molecular aspects of axon pruning. The stereotypic temporal and spatial occurrence of MB axon pruning combined with mosaic analyses provide a platform to perform genetic screens and molecular dissections of these processes in unprecedented resolution. The MB is comprised of three types of neurons that are sequentially born from four identical neuroblasts per hemisphere. Out of the three MB neuronal types, only the γ neurons undergo axon pruning, indicating that the process is cell-type specific. During the larval stage, γ neurons project a bifurcated axon to the dorsal and medial lobes. At the onset of metamorphosis, the dendrites of the γ neurons as well as specific parts of the axons are eliminated by localized fragmentation in a process that peaks at about 18 hr after puparium formation. Subsequently, γ neurons undergo developmental axon regrowth, which is distinct from initial axon outgrowth, to occupy the adult specific lobe (Issman-Zecharya, 2014).

Axon pruning of MB γ neurons depends on the cell-autonomous expression of the nuclear steroid hormone receptor, ecdysone receptor B1 (EcR-B1). The expression of EcR-B1 is regulated by at least three distinct pathways: the cohesin complex, the TGF-β pathway, and a network of nuclear receptors comprised of ftz-f1 and Hr39. While expression of EcR-B1 is required for pruning, it is not sufficient to drive ectopic pruning either in γ neurons or in other MB neurons that do not undergo remodeling. This raises two possible nonmutually exclusive scenarios: (1) additional molecules are required to initiate pruning and (2) an inhibitory signal needs to be attenuated in the MB for pruning to occur. Additionally, the ubiquitin pathway is also cell-autonomously required in γ neurons for pruning, but the target that must be ubiquitinated remains unknown. Thus, while understanding of the cellular sequence of events culminating in the elimination of specific axonal branches is quite detailed, understanding of the molecular mechanisms remains incomplete (Issman-Zecharya, 2014).

In a forward genetic screen, this study identified a cell-autonomous role for the UV radiation resistance-associated gene (UVRAG) in MB γ neuron pruning. UVRAG was originally identified based on its ability to confer UV resistance to nucleotide excision repair deficient cells. It was later shown to function as a tumor suppressor gene deleted in various types of cancers including colon and gastric carcinomas. UVRAG interacts with Atg6 (also known as Beclin1), another tumor suppressor gene, and together they promote autophagy in vitro. Their tumor suppression capabilities were first attributed to their autophagy-promoting function. However, a mutant form of UVRAG isolated from colon carcinomas promoted autophagy normally in cell culture. Both UVRAG and Atg6 are subunits in the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex, involved in autophagy and endocytosis. Recent studies have found that UVRAG mediates endocytosis in an Atg6-dependent manner suggesting that as part of the PI3K-cIII complex, both proteins regulate various aspects of vesicle trafficking. Two studies have recently identified new and seemingly unrelated functions for UVRAG in regulating DNA repair in response to UV-induced damage and ER to Golgi trafficking. Finally, an in vivo study has shown that UVRAG affects organ rotation in Drosophila by regulating Notch endocytosis in what seemed to be an Atg6-independent manner. A unifying understanding of the various aspects of UVRAG physiological function in vivo is still lacking. Likewise, although the PI3K-cIII complex has been extensively studied and implicated in autophagy, cytokinesis and endocytosis, its physiological roles during the normal course of development are not known (Issman-Zecharya, 2014).

This study reports that UVRAG and the PI3K-cIII complex mediate the endosome-lysosome degradation of Ptc to promote axon pruning. Furthermore, the results suggest that Ptc represses pruning via a Smo- and Hh-independent manner. This study provides evidence for the existence of a pruning inhibitory pathway originating at the membrane of MB neurons (Issman-Zecharya, 2014).

This study shows that the endosomal-lysosomal pathway is cell-autonomously required for developmental axon pruning of mushroom body (MB) γ neurons. Genetic loss-of-function experiments indicate that UVRAG, a tumor suppressor gene previously linked to both endocytosis and autophagy, promotes pruning as part of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex and that UVRAG is required in MB neurons for the formation of phosphatidylinositol 3-phosphate (PI3P). The ESCRT-0 complex, which is recruited to the PI3 moiety on endosomal membranes, is required for pruning, indicating that endosome to multivesicular body maturation is critical for the normal progression of axon pruning and suggesting that it involves receptor downregulation. Genetic loss-of-function and gain-of-function experiments suggest that downregulation of the Hedgehog receptor Patched (Ptc) by the endocytic machinery is instrumental in promoting pruning. Finally, the results suggest that Ptc inhibits pruning in a smo-independent and likely also hh-independent manner (Issman-Zecharya, 2014).

A recent study suggested that UVRAG is required for Notch endocytosis during organ rotation in Drosophila in an Atg6-independent manner. While the current study shows that Atg6 is required for pruning, these seemingly contradicting results can be easily explained by specific allele differences. The Atg600096 allele, used in the previous study, is a P element insertion about 100 bp upstream of the Atg6 gene that does not necessarily create a null allele. Indeed, this study could also not see any effect of this allele on axon pruning. This study used an Atg61 null allele created by homologous recombination resulting in a strong effect on pruning. Furthermore, the data clearly show that the entire PI3K-cIII complex is required for axon pruning (Issman-Zecharya, 2014).

The PI3K-cIII complex has been implicated in a wide variety of membrane trafficking processes ranging from autophagy to endocytosis to cytokinesis. How the PI3K-cIII is regulated to participate in these different processes and its physiological roles in vivo are not well understood. While its role in promoting autophagy is supported by several studies, deleting the catalytic unit, Vps34, in sensory neurons does not affect autophagy, but rather endocytosis. Whether this is a common feature of PI3K-cIII function in neurons remains to be further elucidated. One attractive hypothesis is that the PI3K-cIII function is determined by its complex composition. Indeed, it appears that in vitro, UVRAG and Atg14 are mutually exclusive subunits defining two distinct populations of the PI3K-cIII complex (Funderburk, 2010; Itakura, 2009). The current study is consistent with these findings, suggesting that UVRAG may define an endocytosis-specific PI3K-cIII complex at least in neurons. The full spectrum of the various PI3K-cIII complexes physiological roles in vivo remains to be further studied (Issman-Zecharya, 2014).

The PI3K-cIII complex phosphorylates PI to form PI3P on endosomal membranes. Indeed, this study found that UVRAG is essential for efficient PI3P formation and that PI3P is abundant throughout development. It is thus hypothesized that a PI3P binding protein mediates the effect of UVRAG and the PI3K-cIII complex on axon pruning. This study has identified Hrs, a subunit of the ESCRT-0 complex and a PI3P binding protein, as required for axon pruning. The role of ESCRT-0 in MVB maturation led to a hypothesis that the endolysosomal pathway is required to downregulate a signal that originates at the plasma membrane. While signaling can still occur in the early endosome, it is terminated at the MVB (Issman-Zecharya, 2014).

What is the identity of this transmembrane protein? Using genetic loss-of-function and gain-of-function experiments, it is suggested that Patched (Ptc) is at least one of the transmembrane proteins that is responsible for mediating the PI3K-cIII pruning defect. Strikingly, mutating ptc on the background of a Atg6 mutant significantly suppressed its pruning defect. Furthermore, overexpression of Ptc in WT brains resulted in a weak to mild pruning defect, depending on the Gal4 driver. Finally, overexpressing Ptc on the background of an endosomal defect significantly exacerbated the pruning defect. Together, these data suggest that Ptc mediates an inhibitory signal that needs to be attenuated for the normal progression of pruning. Interestingly, Ptc inactivation by endocytosis followed by lysosomal degradation was proposed before as a mechanism to activate the Hh pathway. What is the nature of this signal? Ptc is known to be the Hedgehog (Hh) receptor. Binding of Hh to Ptc relieves the Ptc-induced suppression of another transmembrane protein, Smoothened (Smo). Once derepressed, Smo initiates the intracellular Hh signal that culminates in the expression of specific nuclear transcription factors. Therefore this study tested the role of Smo and Hh in developmental axon pruning and, to surprisingly, demonstrated that both molecules seem to be irrelevant for pruning. Overexpressing Ptc mutant transgenes within MB neurons to identify the domains that are important for pruning inhibitions confirmed that Smo inhibition was not required to inhibit pruning. In contrast, the results suggest that the ligand binding domain is important. Because the results suggest that Hh is not required for pruning inhibition, it will be interesting to investigate in the future what other ligands might bind to Ptc. In this regard it is interesting to mention that a recent study has shown that Ptc is a lipoprotein receptor. The precise mechanism of Ptc action in MB neurons remains to be further elucidated in future studies (Issman-Zecharya, 2014).

This study has uncovered a role for the endocytic machinery in downregulating an inhibitory signal that is dependent on Ptc during MB axon pruning. A recently published study has shown that the Rab5/ESCRT endocytic pathways are required to downregulate neuroglian (Nrg) to promote dendrite pruning of sensory neurons in Drosophila. Both studies highlight that a combination of both promoting and inhibitory signals during developmental pruning is likely important to provide fail-safe mechanisms to regulate the process in a temporal, spatial, and cell-type specific resolution (Issman-Zecharya, 2014).

JNK protects Drosophila from oxidative stress by trancriptionally activating autophagy

JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, the role of autophagy was investigated as such a JNK-regulated protective mechanism. Oxidative stress was shown to induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms (Wu, 2009).

The data suggest that JNK signaling induces autophagy, at least in part, by transcriptional activation of ATG genes. Such a mechanism would be consistent with several previous reports indicating that conditions that stimulate autophagy, such as starvation and stress, also lead to increased expression levels of ATG genes. Furthermore, the deliberate expression of ATG1, ATG6 or ATG8a by itself is sufficient to drive cells into autophagy. It is therefore plausible that the JNK-induced increases in ATG gene expression levels observed in this study might drive and/or sustain autophagy in stressed organs. However, it is also clear that autophagy can be controlled by mechanisms other than gene expression. For instance, protein phosphorylation, lipidation, and processing events have been shown to regulate the process. Two recent studies conducted in mammalian cell lines indicate that JNK can induce autophagy by phosphorylating Bcl2, thereby relieving its inhibitory effect on Beclin 1, the ATG6 homolog (Pattingre, 2008; Wei, 2008). It thus emerges that JNK may impinge on autophagy at multiple regulatory levels. Such a scenario bears an interesting resemblance of the better-understood role of JNK in the regulation of apoptosis, a process that it can control by transcriptional, as well as non-transcriptional mechanisms. It is at present a matter of speculation how these different layers of regulation are integrated and how they may have evolved. In this regard it is interesting that the only anti-death Bcl2 family member in Drosophila, the buffy gene product, does not contain JNK phosphorylation sites, suggesting that Bcl2-dependent mechanisms do not contribute to JNK induced autophagy in Drosophila. It is possible that in flies JNK acts predominantly at the transcription level, and that the role of Bcl2 in this context has evolved later (Wu, 2009).

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, mutations were generated in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34-/- animals, but this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, this study showed that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. These results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation (Juhasz, 2008).

Functions of Atg6 orthologs in other species

Beclin-1 is involved in the regulation of antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis

Beclin-1, the mammalian ortholog of yeast Atg6, plays essential roles in the regulation of various processes, including autophagy, apoptosis, embryonic development and immune responses in vertebrates. However, the information about Beclin-1 in invertebrates especially in crustaceans is still very limited. In the present study, a novel Beclin-1 (designated as EsBeclin-1) was identified from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsBeclin-1 cDNA was of 1,275 bp, encoding a typical APG6 domain. The deduced amino acid sequence of EsBeclin-1 shared high similarity ranging from 42.9% to 63.6% with the previously identified Beclins. In the phylogenetic tree, EsBeclin-1 was firstly clustered with Drosophila melanogaster Atg6 and then assigned into the branch of invertebrate Beclin-1. The mRNA transcripts of EsBeclin-1 were highly expressed in hepatopancreas, hemocytes and gill. After lipopolysaccharide (LPS) and Aeromonas hydrophila stimulations, the relative mRNA expression of EsBeclin-1 in hemocytes was significantly increased from 3 to 24h with the peak level of 4.70-fold and 2.91-fold at 6h, respectively. EsBeclin-1 protein was diffusely distributed in the cytoplasm of crab hemocytes under normal conditions, whereas it displayed predominantly punctuate distribution after LPS stimulation. After EsBeclin-1 was interfered with specific EsBeclin-1-dsRNA, the mRNA transcripts of some antimicrobial peptides, including EsALF2, EsLYZ, EsCrus and EsCrus2 in crab hemocytes were significantly decreased at 6h post LPS stimulation. These results implicated that EsBeclin-1 played a role in regulating the antimicrobial peptides expressions in the immune responses of E. sinensis (Yang, 2019).

Beclin-1-Dependent Autophagy Protects the Heart During Sepsis

Cardiac dysfunction is a major component of sepsis-induced multiorgan failure in critical care units. Changes in cardiac autophagy and its role during sepsis pathogenesis have not been clearly defined. Targeted autophagy-based therapeutic approaches for sepsis are not yet developed. Beclin-1-dependent autophagy in the heart during sepsis and the potential therapeutic benefit of targeting this pathway were investigated in a mouse model of lipopolysaccharide (LPS)-induced sepsis. LPS induced a dose-dependent increase in autophagy at low doses, followed by a decline that was in conjunction with mammalian target of rapamycin activation at high doses. Cardiac-specific overexpression of Beclin-1 promoted autophagy, suppressed mammalian target of rapamycin signaling, improved cardiac function, and alleviated inflammation and fibrosis after LPS challenge. Haplosufficiency for beclin 1 resulted in opposite effects. Beclin-1 also protected mitochondria, reduced the release of mitochondrial danger-associated molecular patterns, and promoted mitophagy via PTEN-induced putative kinase 1-Parkin but not adaptor proteins in response to LPS. Injection of a cell-permeable Tat-Beclin-1 peptide to activate autophagy improved cardiac function, attenuated inflammation, and rescued the phenotypes caused by beclin 1 deficiency in LPS-challenged mice. These results suggest that Beclin-1 protects the heart during sepsis and that the targeted induction of Beclin-1 signaling may have important therapeutic potential (Sun, 2018).

USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 (see Drosophila Usp14) is known to inhibit UPS. This study shows that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1 (see Drosophila Atg6). Furthermore, activation of USP14 by Akt (see Drosophila Akt1)-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Data suggest that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. The study proposes that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases (Xu, 2016).

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. It was hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, this study evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. Unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells (Furuya, 2005).

Search PubMed for articles about Drosophila Atg6


Diao, J., Liu, R., Rong, Y., Zhao, M., Zhang, J., Lai, Y., Zhou, Q., Wilz, L. M., Li, J., Vivona, S., Pfuetzner, R. A., Brunger, A. T. and Zhong, Q. (2015). ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520(7548): 563-566. PubMed ID: 25686604

Furuya, N., Yu, J., Byfield, M., Pattingre, S. and Levine, B. (2005). The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function. Autophagy 1(1): 46-52. PubMed ID: 16874027

Haghi, M., Masoudi, R. and Najibi, S. M. (2020). Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy. Ups J Med Sci: 1-9. PubMed ID: 32657227

Issman-Zecharya, N. and Schuldiner, O. (2014). The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation. Dev Cell 31: 461-473. PubMed ID: 25458013

Joffre, C., et al. (2015). The pro-apoptotic STK38 kinase is a new Beclin1 partner positively regulating autophagy. Curr Biol 25: 2479-2492. PubMed ID: 26387716

Juhasz, G., Hill, J. H., Yan, Y., Sass, M., Baehrecke, E. H., Backer, J. M. and Neufeld, T. P. (2008). The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. J Cell Biol 181(4): 655-666. PubMed ID: 18474623

Kim, M., Semple, I., Kim, B., Kiers, A., Nam, S., Park, H. W., Park, H., Ro, S. H., Kim, J. S., Juhasz, G. and Lee, J. H. (2015). Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis. Autophagy 11(8): 1358-1372. PubMed ID: 26086452

Lorincz, P., Lakatos, Z., Maruzs, T., Szatmari, Z., Kis, V. and Sass, M. (2014). Atg6/UVRAG/Vps34-containing lipid kinase complex is required for receptor downregulation through endolysosomal degradation and epithelial polarity during Drosophila wing development. Biomed Res Int 2014: 851349. PubMed ID: 25006588

M'Angale, P. G. and Staveley, B. E. (2016). Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster. Genet Mol Res 15. PubMed ID: 27813607

Melani, M., Valko, A., Romero, N. M., Aguilera, M. O., Acevedo, J. M., Bhujabal, Z., Perez-Perri, J., de la Riva-Carrasco, R. V., Katz, M. J., Sorianello, E., D'Alessio, C., Juhasz, G., Johansen, T., Colombo, M. I. and Wappner, P. (2017). Zonda is a novel early component of the autophagy pathway in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28904211

Na, H. J., Pyo, J. H., Jeon, H. J., Park, J. S., Chung, H. Y. and Yoo, M. A. (2018). Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila. Biochem Biophys Res Commun 498(1): 18-24. PubMed ID: 29496445

Shravage, B. V., Hill, J. H., Powers, C. M., Wu, L. and Baehrecke, E. H. (2013). Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila. Development 140(6): 1321-1329. PubMed ID: 23406899

Shen, J. L., Doherty, J., Allen, E., Fortier, T. M. and Baehrecke, E. H. (2022). Atg6 promotes organismal health by suppression of cell stress and inflammation. Cell Death Differ. PubMed ID: 35523956

Sun, Y., Yao, X., Zhang, Q. J., Zhu, M., Liu, Z. P., Ci, B., Xie, Y., Carlson, D., Rothermel, B. A., Sun, Y., Levine, B., Hill, J. A., Wolf, S. E., Minei, J. P. and Zang, Q. S. (2018). Beclin-1-Dependent Autophagy Protects the Heart During Sepsis. Circulation 138(20): 2247-2262. PubMed ID: 29853517

Takats, S., Levay, L., Boda, A., Toth, S., Simon-Vecsei, Z., Rubics, A., Varga, A., Lippai, M., Lorincz, P., Glatz, G. and Juhasz, G. (2021). The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I. FEBS J 288(1): 190-211. PubMed ID: 32248620

Wu, H., Meng C. Wang, M. C. and Bohmann, D. (2009). JNK protects Drosophila from oxidative stress by trancriptionally activating autophagy. Mech. Dev. 126: 624-637. PubMed Citation: 19540338

Xu, D., Shan, B., Sun, H., Xiao, J., Zhu, K., Xie, X., Li, X., Liang, W., Lu, X., Qian, L. and Yuan, J. (2016). USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1. Genes Dev 30: 1718-1730. PubMed ID: 27542828

Yang, W., Liu, C., Xu, Q., Qu, C., Sun, J., Huang, S., Kong, N., Lv, X., Liu, Z., Wang, L. and Song, L. (2019). Beclin-1 is involved in the regulation of antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis. Fish Shellfish Immunol 89: 207-216. PubMed ID: 30936045

Zhao, S., Fortier, T. M. and Baehrecke, E. H. (2018). Autophagy promotes tumor-like stem cell niche occupancy. Curr Biol 28(19): 3056-3064. PubMed ID: 30270184

Biological Overview

date revised: 10 November 2022

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