Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes.

Autophagy power expands: fuse those cells!

The lysosomal degradation pathway of autophagy depends on a set of evolutionarily conserved autophagy-related molecules (ATGs) bestowed with the power to direct membrane trafficking and biology. In this issue of EMBO Journal, Kakanj P et al reveal a surprising role for the autophagy machinery in cell fusion (Kakanj et al, 2022). Autophagy is physiologically required for cell syncytium formation through dismantling the lateral plasma membrane during wound healing, and unchecked autophagy can drive cell fusion in epithelial tissues without compromising epithelial integrity.

Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.

During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.

PHLPP1 regulates CFTR activity and lumen expansion through AMPK.

Complex organ development depends on single lumen formation and its expansion during tubulogenesis. This can be achieved by correct mitotic spindle orientation during cell division, combined with luminal fluid filling that generates hydrostatic pressure. Using a human 3D cell culture model, we have identified two regulators of these processes. We find that pleckstrin homology leucine-rich repeat protein phosphatase (PHLPP) 2 regulates mitotic spindle orientation, and thereby midbody positioning and maintenance of a single lumen. Silencing the sole PHLPP family phosphatase in Drosophila melanogaster, phlpp, resulted in defective spindle orientation in Drosophila neuroblasts. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) is the main channel regulating fluid transport in this system, stimulated by phosphorylation by protein kinase A and inhibited by the AMP-activated protein kinase AMPK. During lumen expansion, CFTR remains open through the action of PHLPP1, which stops activated AMPK from inhibiting ion transport through CFTR. In the absence of PHLPP1, the restraint on AMPK activity is lost and this tips the balance in the favour of channel closing, resulting in the lack of lumen expansion and accumulation of mucus.

Microenvironmental autophagy promotes tumour growth.

As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. Here we demonstrate, using a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor and interleukin-6-like signalling from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. We show that dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy.

Autoimmunity gene IRGM suppresses cGAS-STING and RIG-I-MAVS signaling to control interferon response.

Activation of the type 1 interferon response is extensively connected to the pathogenesis of autoimmune diseases. Loss of function of Immunity Related GTPase M (IRGM) has also been associated to several autoimmune diseases, but its mechanism of action is unknown. Here, we found that IRGM is a master negative regulator of the interferon response. Several nucleic acid-sensing pathways leading to interferon-stimulated gene expression are highly activated in IRGM knockout mice and human cells. Mechanistically, we show that IRGM interacts with nucleic acid sensor proteins, including cGAS and RIG-I, and mediates their p62-dependent autophagic degradation to restrain interferon signaling. Further, IRGM deficiency results in defective mitophagy leading to the accumulation of defunct leaky mitochondria that release cytosolic DAMPs and mtROS. Hence, IRGM deficiency increases not only the levels of the sensors, but also those of the stimuli that trigger the activation of the cGAS-STING and RIG-I-MAVS signaling axes, leading to robust induction of IFN responses. Taken together, this study defines the molecular mechanisms by which IRGM maintains interferon homeostasis and protects from autoimmune diseases.

Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo.

BACKGROUND: Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, a labeled metabolite can be enzymatically changed in cellular metabolism, complicating the use of this experimental strategy to understand how a labeled metabolite moves between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (IRMS). RESULTS: We exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. IRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 µg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues. CONCLUSION: Measuring tumor growth in a D. melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.

RasV12; scrib-/- Tumors: A Cooperative Oncogenesis Model Fueled by Tumor/Host Interactions.

The phenomenon of how oncogenes and tumor-suppressor mutations can synergize to promote tumor fitness and cancer progression can be studied in relatively simple animal model systems such as Drosophila melanogaster. Almost two decades after the landmark discovery of cooperative oncogenesis between oncogenic RasV12 and the loss of the tumor suppressor scribble in flies, this and other tumor models have provided new concepts and findings in cancer biology that has remarkable parallels and relevance to human cancer. Here we review findings using the RasV12; scrib-/- tumor model and how it has contributed to our understanding of how these initial simple genetic insults cooperate within the tumor cell to set in motion the malignant transformation program leading to tumor growth through cell growth, cell survival and proliferation, dismantling of cell-cell interactions, degradation of basement membrane and spreading to other organs. Recent findings have demonstrated that cooperativity goes beyond cell intrinsic mechanisms as the tumor interacts with the immediate cells of the microenvironment, the immune system and systemic organs to eventually facilitate malignant progression.

Autophagy and Tumorigenesis in Drosophila.

The resurgence of Drosophila as a recognized model for carcinogenesis has contributed greatly to our conceptual advance and mechanistic understanding of tumor growth in vivo. With its powerful genetics, Drosophila has emerged as a prime model organism to study cell biology and physiological functions of autophagy. This has enabled exploration of the contributions of autophagy in several tumor models. Here we review the literature of autophagy related to tumorigenesis in Drosophila. Functional analysis of core autophagy components does not provide proof for a classical tumor suppression role for autophagy alone. Autophagy both serve to suppress or support tumor growth. These effects are context-specific, depending on cell type and oncogenic or tumor suppressive lesion. Future delineation of how autophagy impinges on tumorigenesis will demand to untangle in detail, the regulation and flux of autophagy in the respective tumor models. The downstream tumor-regulative roles of autophagy through organelle homeostasis, metabolism, selective autophagy or alternative mechanisms remain largely unexplored.

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB.

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element in the p62 promoter. Second, p62 accumulation, occurring when autophagy is impaired, leads to increased p62 binding to the NRF2 inhibitor KEAP1, resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. Here, we show that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via an LC3-interacting region motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional antioxidant response element that is directly bound by the NRF2 orthologue, CncC, which can induce ref(2)P expression along with the oxidative stress-associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region motif; nor does ectopically expressed Ref(2)P or autophagy deficiency activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress-sensing NRF2/CncC transcription factors and autophagy and suggest that NRF2/CncC may regulate autophagic activity in other organisms too.

Two-tiered control of epithelial growth and autophagy by the insulin receptor and the ret-like receptor, stitcher.

Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. Here, we reveal that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration.

Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

Removal of hypersignaling endosomes by simaphagy.

Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination.Abbreviations: AKT: AKT serine/threonine kinase; APEX2: apurinic/apyrimidinic endodoexyribonuclease 2; ctrl: control; EEA1: early endosome antigen 1; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HGS/HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; IF: immunofluorescence; ILV: intralumenal vesicle; KO: knockout; LIR: LC3-interacting region; LLOMe: L-leucyl-L-leucine methyl ester (hydrochloride); MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NBR1: NBR1 autophagy cargo receptor; PAG10: Protein A-conjugated 10-nm gold; RB1CC1/FIP200: RB1 inducible coiled-coil 1; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TUB: Tubulin; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VCL: Vinculin; VPS37: VPS37 subunit of ESCRT-I; WB: western blot; WT: wild-type.

Computed tomography with segmentation and quantification of individual organs in a D. melanogaster tumor model.

Drosophila melanogaster tumor models are growing in popularity, driven by the high degree of genetic as well as functional conservation to humans. The most common method to measure the effects of a tumor on distant organs of a human cancer patient is to use computed tomography (CT), often used in diagnosing cachexia, a debilitating cancer-induced syndrome most visibly characterized by loss of muscle mass. Successful application of high resolution micro-CT scanning of D. melanogaster was recently reported and we here present the segmentation of all visible larval organs at several stages of tumor development. We previously showed the strong expected reduction in muscle mass as the tumor develops, and we here report a surprisingly strong reduction also in gut and Malpighian tubules (kidney) volume. Time-point of tumor development was found to have a stronger correlation to cachectic organ volume loss than tumor volume, giving support to the previously proposed idea that tumor size does not directly determine degree of cachexia.

Protein sorting into multivesicular endosomes.

Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.

RNA-Binding RING E3-Ligase DZIP3/hRUL138 Stabilizes Cyclin D1 to Drive Cell-Cycle and Cancer Progression.

DZIP3/hRUL138 is a poorly characterized RNA-binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G1 arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3' untranslated region of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G1 phase of the cell cycle, where it is needed for cell-cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 uses a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell-cycle and cancer progression. SIGNIFICANCE: These findings show that DZIP3 is a novel driver of cell-cycle and cancer progression via its control of Cyclin D1 mRNA and protein stability in a cell-cycle phase-dependent manner. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/315/F1.large.jpg.

ESCRTs and Fab1 regulate distinct steps of autophagy.

Eukaryotes use autophagy to turn over organelles, protein aggregates, and cytoplasmic constituents. The impairment of autophagy causes developmental defects, starvation sensitivity, the accumulation of protein aggregates, neuronal degradation, and cell death [1, 2]. Double-membraned autophagosomes sequester cytoplasm and fuse with endosomes or lysosomes in higher eukaryotes [3], but the importance of the endocytic pathway for autophagy and associated disease is not known. Here, we show that regulators of endosomal biogenesis and functions play a critical role in autophagy in Drosophila melanogaster. Genetic and ultrastructural analysis showed that subunits of endosomal sorting complex required for transport (ESCRT)-I, -II and -III, as well as their regulatory ATPase Vps4 and the endosomal PtdIns(3)P 5-kinase Fab1, all are required for autophagy. Although the loss of ESCRT or Vps4 function caused the accumulation of autophagosomes, probably because of inhibited fusion with the endolysosomal system, Fab1 activity was necessary for the maturation of autolysosomes. Importantly, reduced ESCRT functions aggravated polyglutamine-induced neurotoxicity in a model for Huntington's disease. Thus, this study links ESCRT function with autophagy and aggregate-induced neurodegeneration, thereby providing a plausible explanation for the fact that ESCRT mutations are involved in inherited neurodegenerative disease in humans [4].