Steps Of Autophagosome Formation Research Articles

Structure of the WIPI3/ATG16L1 Complex Reveals the Molecular Basis for the Recruitment of the ATG12~ATG5-ATG16L1 Complex by WIPI3.

Macroautophagy deploys a wealth of autophagy-related proteins to synthesize the double-membrane autophagosome, in order to engulf cytosolic components for lysosome-dependent degradation. The recruitment of the ATG12~ATG5-ATG16L1 complex by WIPI family proteins is a crucial step in autophagosome formation. Nevertheless, the molecular mechanism by which WIPI3 facilitates the recruitment of the ATG12~ATG5-ATG16L1 complex remains largely unknown. Here, we uncover that WIPI3 can directly interact with the coiled-coil domain of ATG16L1. By determining the crystal structure of WIPI3 in complex with ATG16L1 coiled-coil, we elucidate the molecular basis underpinning the specific recruitment of the ATG12~ATG5-ATG16L1 complex by WIPI3. Moreover, we demonstrate that WIPI2 and WIPI3 are competitive for interacting with ATG16L1 coiled-coil, and ATG16L1 and ATG2 are mutually exclusive in binding to WIPI3. In all, our findings provide mechanistic insights into the WIPI3/ATG16L1 interaction, and are valuable for further understanding the activation mechanism of the ATG12~ATG5-ATG16L1 complex as well as the working mode of WIPI3 in autophagy.

Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

A novel autophagy inhibitor, bTBT, disturbs autophagosome formation

Macroautophagy (hereafter, autophagy) is a form of intracellular degradation in which autophagosome formation is systematically coordinated by multiple processes involving numerous autophagy-related gene (ATG) proteins. Autophagy-modulating compounds are valuable for understanding the molecular mechanism of autophagy and its clinical application. Although several autophagy inhibitors have been identified, their inhibitory steps during autophagosome formation by the inhibitors are limited. Herein, we identified a novel autophagy inhibitor, bis-tributyltin (bTBT), which inhibits a unique step in autophagosome formation. In mammalian cells, bTBT treatment suppresses LC3 flux and accumulates most of ATG proteins, including LC3 and early ATG proteins (ULK1, ATG16L1, and WIPI2), in punctate structures. On the other hand, LAMP1, a lysosomal marker, did not co-localize with accumulated LC3 after bTBT treatment, indicating bTBT inhibits a late step of autophagosome formation. Stx17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that mediates autophagosome–lysosome fusion, is usually recruited to LC3-positive structures after the dissociation of early ATG proteins. However, bTBT accumulates Stx17 and WIPI2 positive large autophagic structures and maintains the autophagic structures for much longer. In conclusion, we identified a novel type of autophagy inhibitor, bTBT, which disturbs autophagosome formation.

The role of FIP200 in Phagophore formation during Autophagy

The Moeller Molecular Modeling Team in conjunction with Dr. Jiajie Diao at the University of Cincinnati Medical College used 3D modeling and printing technology to study the vital role of the focal adhesion kinase family interacting protein of 200 kD (FIP200) in the autophagy process. The cellular process of autophagy is enabled by formation of autophagosomes, a double membraned structure that isolates substrates designated by the cell to be eliminated by autophagy. FIP200 is a 200 kD subunit of the ULK1 complex which facilitates the formation of phagophores, the first step in autophagosome formation. An ULK1 protein kinase is necessary for the activation of the ULK1 complex during autophagosome formation. The positively charged “claw domain” area at the C‐terminal region of FIP200 interacts with p62, a receptor protein, which delivers ubiquitinated cargo proteins bound to LC3B, a central regulator for autophagosome formation. After the binding of these proteins, the autophagosome forms around the ubiquitinated cargo and continues to the next step of the autophagy process.

Andrographolide derivative as antagonist of vitamin D receptor to induce lipidation of microtubule associate protein 1 light chain 3 (LC3)

Lipidation of microtubule associated protein 1 light chain 3 (LC3) is the critical step in autophagosome formation, numerous efforts have been made to design and develop small molecules that trigger LC3 lipidation to activate autophagy. In this study, we discovered a series of andrographolide derivatives as potent antagonists of vitamin D receptor (VDR) by luciferase reporter assay. Structure-activity-relationship study revealed that andrographolide derivative ZAV-12 specifically inhibited VDR signaling but not NF-κB or STAT3 activation. Western blot analysis indicates that ZAV-12 markedly triggered lipidation of LC3 in MPP+-induced Parkinsonism in vitro in an mTOR-independent manner. The ZAV-12 triggered lipidation was mediated through SREBP2 activation instead of changing expression levels of lipid synthesis genes. Furthermore, ZAV-12 treatment increased the ratio of LC3-II/LC3-I and oligomerization of A53T α-synuclein (SNCA) in SNCA triggered neurotoxicity. Taken together, these results demonstrate the therapeutic potential of VDR antagonist as novel drug candidate for neurodegenerative diseases.

Reconstituting autophagosome nucleation

Cell Biology To stay healthy, our cells must constantly dispose of harmful material. Autophagy, or self-eating, is an important mechanism to ensure the clearance of bulky material. Such material is enwrapped by cellular membranes to form autophagosomes, the contents of which are then degraded. The formation of autophagosomes is a complicated process involving a large number of factors. How they act together in this process is still enigmatic. Sawa-Makarska et al. recapitulated the initial steps of autophagosome formation using purified autophagy factors from yeast. This approach elucidated some of the organizational principles of the autophagy machinery during the assembly of autophagosomes. Science , this issue p. [eaaz7714][1] [1]: /lookup/doi/10.1126/science.aaz7714

Emerging Role of the Nucleolar Stress Response in Autophagy.

Autophagy represents a conserved self-digestion program, which allows regulated degradation of cellular material. Autophagy is activated by cellular stress, serum starvation and nutrient deprivation. Several autophagic pathways have been uncovered, which either non-selectively or selectively target the cellular cargo for lysosomal degradation. Autophagy engages the coordinated action of various key regulators involved in the steps of autophagosome formation, cargo targeting and lysosomal fusion. While non-selective (macro)autophagy is required for removal of bulk material or recycling of nutrients, selective autophagy mediates specific targeting of damaged organelles or protein aggregates. By proper action of the autophagic machinery, cellular homeostasis is maintained. In contrast, failure of this fundamental process is accompanied by severe pathophysiological conditions. Hallmarks of neuropathological disorders are for instance accumulated, mis-folded protein aggregates and damaged mitochondria. The nucleolus has been recognized as central hub in the cellular stress response. It represents a sub-nuclear organelle essential for ribosome biogenesis and also functions as stress sensor by mediating cell cycle arrest or apoptosis. Thus, proper nucleolar function is mandatory for cell growth and survival. Here, I highlight the emerging role of nucleolar factors in the regulation of autophagy. Moreover, I discuss the nucleolar stress response as a novel signaling pathway in the context of autophagy, health and disease.

Two distinct mechanisms target the autophagy-related E3 complex to the pre-autophagosomal structure.

In autophagy, Atg proteins organize the pre-autophagosomal structure (PAS) to initiate autophagosome formation. Previous studies in yeast revealed that the autophagy-related E3 complex Atg12-Atg5-Atg16 is recruited to the PAS via Atg16 interaction with Atg21, which binds phosphatidylinositol 3-phosphate (PI3P) produced at the PAS, to stimulate conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine. Here, we discover a novel mechanism for the PAS targeting of Atg12-Atg5-Atg16, which is mediated by the interaction of Atg12 with the Atg1 kinase complex that serves as a scaffold for PAS organization. While autophagy is partially defective without one of these mechanisms, cells lacking both completely lose the PAS localization of Atg12-Atg5-Atg16 and show no autophagic activity. As with the PI3P-dependent mechanism, Atg12-Atg5-Atg16 recruited via the Atg12-dependent mechanism stimulates Atg8 lipidation, but also has the specific function of facilitating PAS scaffold assembly. Thus, this study significantly advances our understanding of the nucleation step in autophagosome formation.

Role of VMP1-USP9x interaction in the initial steps of autophagosome formation

Role of VMP1-USP9x interaction in the initial steps of autophagosome formation

Mir505–3p regulates axonal development via inhibiting the autophagy pathway by targeting Atg12

ABSTRACTIn addition to the canonical role in protein homeostasis, autophagy has recently been found to be involved in axonal dystrophy and neurodegeneration. Whether autophagy may also be involved in neural development remains largely unclear. Here we report that Mir505–3p is a crucial regulator for axonal elongation and branching in vitro and in vivo, through modulating autophagy in neurons. We identify that the key target gene of Mir505–3p in neurons is Atg12, encoding ATG12 (autophagy-related 12) which is an essential component of the autophagy machinery during the initiation and expansion steps of autophagosome formation. Importantly, axonal development is compromised in brains of mir505 knockout mice, in which autophagy signaling and formation of autophagosomes are consistently enhanced. These results define Mir505–3p-ATG12 as a vital signaling cascade for axonal development via the autophagy pathway, further suggesting the critical role of autophagy in neural development.

Regulation of autophagic flux by CHIP.

Autophagy is a major degradation system which processes substrates through the steps of autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. Aberrant autophagic flux is present in many pathological conditions including neurodegeneration and tumors. CHIP/STUB1, an E3 ligase, plays an important role in neurodegeneration. In this study, we identified the regulation of autophagic flux by CHIP (carboxy-terminus of Hsc70-interacting protein). Knockdown of CHIP induced autophagosome formation through increasing the PTEN protein level and decreasing the AKT/mTOR activity as well as decreasing phosphorylation of ULK1 on Ser757. However, degradation of the autophagic substrate p62 was disturbed by knockdown of CHIP, suggesting an abnormality of autophagic flux. Furthermore, knockdown of CHIP increased the susceptibility of cells to autophagic cell death induced by bafilomycin A1. Thus, our data suggest that CHIP plays roles in the regulation of autophagic flux.

Interaction of Bcl-2 with the Autophagy-related GABAA Receptor-associated Protein (GABARAP): BIOPHYSICAL CHARACTERIZATION AND FUNCTIONAL IMPLICATIONS

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

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

Abstract SY31-02: Autophagy, cancer cell survival, and mitochondrial homeostasis

Abstract Autophagy is a tightly regulated lysosomal self-digestion process that serves as an important adaptive mechanism during stress or starvation. My laboratory recently discovered that autophagy is strongly induced in epithelial cells deprived of extracellular (ECM) contact, which protects them from apoptosis. In further work, we have uncovered that autophagy is robustly induced in epithelial cells transformed by mutationally active oncogenes, raising the hypothesis that autophagy functions as a survival mechanism in cancerous cells deprived of proper ECM contact. We are currently dissecting whether detachment-induced autophagy contributes to oncogenic cell survival and transformation in vitro, and promotes the dissemination and metastasis of tumor cells in vivo. In addition, we are uncovering novel cellular functions for ATGs, the proteins that regulate autophagy. ATG12 is an ubiquitin-like modifier required for autophagy, which to date, has been proposed to possess a single conjugation target, another autophagy regulator called ATG5. Now, we identify ATG3 as a novel substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Mutation of this single lysine abolishes ATG12-ATG3 complex formation. To uncover the biological functions of ATG12-ATG3, we reconstituted ATG3-deficient cells with wild type ATG3 (ATG3WT) or a mutant version unable to conjugate to ATG12 (ATG3KR). Remarkably, disrupting ATG12-3 complex formation has no observable effect on autophagy. In fact, during nutrient starvation and rapamycin- mediated mTORC1 inhibition, ATG3WT and ATG3KR-expressing cells exhibit equal rates of both LC3 lipidation (LC3-II) and turnover in the lysosome, as well as the robust proteolytic degradation of autophagy substrates like p62/SQSTM. Instead, the most obvious consequence of disrupting ATG12-3 is an expansion in mitochondrial mass due to autophagy- and mitophagy-independent mechanisms. Lack of ATG12 conjugation to ATG3 also produces an increase in mitochondrial fragmentation, arising from a defect in mitochondrial fusion. Furthermore, cells lacking ATG12-3 exhibit resistance to the intrinsic apoptotic pathway. Most importantly, these unique functions of the ATG12-3 complex can be completely separated from the established roles of either individual ATG on autophagosome formation. Overall, these results reveal unexpected novel roles for ATG12-3 in mitochondrial homeostasis and cell death, and for the first time, implicate the ATG12 conjugation system in new cellular functions distinct from the early steps of autophagosome formation. Citation Format: Jayanta Debnath. Autophagy, cancer cell survival, and mitochondrial homeostasis [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY31-02

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H 2O 2 stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H 2O 2, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.

The effects of dynein inhibition on the autophagic pathway in glioma cells

Autophagy has multiple physiological functions, including protein degradation, organelle turnover and the response of cancer cells to chemotherapy. Because autophagy is implicated in a number of diseases, a better understanding of the molecular mechanisms of autophagy is needed for therapeutic purposes, including rational design of drugs. Autophagy is a process that occurs in several steps as follows: formation of phagophores, formation of mature autophagosomes, targeting and trafficking of autophagosomes to lysosomes, formation of autolysosomes by fusion between autophagosomes and lysosomes, and finally, degradation of the autophagic bodies within the lysosomes. It has been suggested that autophagosome formation is driven by molecular motor machineries, and, once formed, autophagosomes need to reach lysosomes, enriched perinuclearly around the microtubule-organizing centre. While it is recognized that all these steps require the cytoskeletal network, little is known about the mechanisms involved. Here we assessed the role of cytoplasmic dynein in the autophagic process of human glioma cells to determine the part played by dynein in autophagy. We observed that chemical interference with dynein function led to an accumulation of autophagosomes, suggesting impaired autophagosome-lysosome fusion. In contrast, we found that overexpression of dynamitin, which disrupts the dynein complex, reduced the number of autophagosomes, suggesting the requirement of the dynein-dynactin interaction in the early membrane trafficking step in autophagosome formation. These results suggest that dynein plays a variety of crucial roles during the autophagic process in glioma cells.

Autophagy and the functional roles of Atg5 and beclin-1 in the anti-tumor effects of 3β androstene 17α diol neuro-steroid on malignant glioma cells

In this study, we demonstrate that the anti-tumor activity of the neuro-steroid, 3β androstene 17α diol (17α-AED) on malignant glioma cells is mediated by the induction of autophagy. 17α-AED can inhibit the proliferation an induce cell death of multiple, unrelated gliomas with an IC 50 between 8 and 25 μM. 17α-AED treatment induced the formation of autophagosomes and acidic vesicular organelles in human malignant gliomas which was blocked by bafilomycin A1 or 3-methyladenine. Cleavage of microtubule-associated protein-light chain 3 (LC3), an essential step in autophagosome formation, was detected in human malignant glioma cells exposed to 17α-AED. In 17α-AED treated T98G glioma cells there was an increase in the autophagy related proteins Atg5 and beclin-1. Silencing of ATG5 or beclin-1 with small interfering RNA significantly reduced the incidence of autophagy in 17α-AED treated malignant gliomas and attenuated the cytotoxic effects of the neuro-steroid indicating that the induction of autophagy mediates the anti-glioma activity of 17α-AED rather than serving as a cyto-protective response. These results demonstrate that 17α-AED possesses significant anti-glioma activity when used at pharmacologically relevant concentrations in vitro and the cytotoxic effects are resultant from the induction of autophagy.

Apg2p Functions in Autophagosome Formation on the Perivacuolar Structure

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