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David C. Rubinsztein - One of the best experts on this subject based on the ideXlab platform.
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a location location location mutation impairs dnm2 mediated release of nascent Autophagosomes from recycling endosomes
Autophagy, 2020Co-Authors: Claudia Puri, David C. RubinszteinAbstract:Elucidation of the membranes contributing to Autophagosomes has been a critical question in the field, and an area of active research. Recently, we showed that key events in Autophagosome formation...
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lc3 ii tagging and western blotting for monitoring autophagic activity in mammalian cells
Methods of Molecular Biology, 2016Co-Authors: Anne Streeter, Fiona M. Menzies, David C. RubinszteinAbstract:Abstract The Autophagosome-associated protein LC3-II is commonly used as a marker of autophagic activity within cells, but its levels are affected by both formation and degradation of Autophagosomes. This can make the significance of altered LC3-II levels ambiguous. Here we describe the method of Bafilomycin A1 blotting, in which the degradation of Autophagosomes is prevented in cultured cells, allowing the causes of altered LC3-II levels to be determined.
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atg16l1 meets atg9 in recycling endosomes additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis
Autophagy, 2014Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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ATG16L1 meets ATG9 in recycling endosomes: additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis.
Autophagy, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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Diverse Autophagosome Membrane Sources Coalesce in Recycling Endosomes
Cell, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagic protein degradation is mediated by Autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of Autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during Autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of Autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with Autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.
Claudia Puri - One of the best experts on this subject based on the ideXlab platform.
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a location location location mutation impairs dnm2 mediated release of nascent Autophagosomes from recycling endosomes
Autophagy, 2020Co-Authors: Claudia Puri, David C. RubinszteinAbstract:Elucidation of the membranes contributing to Autophagosomes has been a critical question in the field, and an area of active research. Recently, we showed that key events in Autophagosome formation...
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A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
Developmental Cell, 2020Co-Authors: Claudia Puri, Marco M. Manni, Mariella Vicinanza, Christine Hilcenko, Ye Zhu, Gautam Runwal, Eleanna Stamatakou, Fiona M. Menzies, Kamel MamchaouiAbstract:Summary Autophagy involves engulfment of cytoplasmic contents by double-membraned Autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of Autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of Autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from Autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This “scission” step is, thus, critical for Autophagosome formation, is defective in a human disease, and influences the way we consider how Autophagosomes are formed.
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atg16l1 meets atg9 in recycling endosomes additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis
Autophagy, 2014Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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ATG16L1 meets ATG9 in recycling endosomes: additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis.
Autophagy, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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Diverse Autophagosome Membrane Sources Coalesce in Recycling Endosomes
Cell, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagic protein degradation is mediated by Autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of Autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during Autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of Autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with Autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.
Qing Zhong - One of the best experts on this subject based on the ideXlab platform.
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ATG14 controls SNARE-mediated Autophagosome fusion with a lysosome
Autophagy, 2015Co-Authors: Rong Liu, Xiaoyong Zhi, Qing ZhongAbstract:Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between Autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on Autophagosomes and promotes STX17-SNAP29-VAMP8-mediated Autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and Autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for Autophagosome fusion with a lysosome, but is dispensable for Autophagosome biogenesis. These data support a key role of ATG14 in controlling Autophagosome fusion with a lysosome.
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A tethering coherent protein in Autophagosome maturation
Autophagy, 2012Co-Authors: Dandan Chen, Qing ZhongAbstract:Autophagy is a cellular pathway that degrades damaged organelles, cytosol and microorganisms, thereby maintaining human health by preventing various diseases including cancers, neurodegenerative disorders and diabetes. In autophagy, Autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. The proper Autophagosome-lysosome fusion is pivotal for efficient autophagy activity. However, the molecular mechanism that specifically directs the fusion process is not clear. Our study reported that lysosome-localized TECPR1 (TECtonin β-Propeller Repeat containing 1) binds the Autophagosome-localized ATG12–ATG5 conjugate and recruits it to autolysosomes. TECPR1 also binds PtdIns3P in an ATG12–ATG5-dependent manner. Consequently, depletion of TECPR1 leads to a severe defect in Autophagosome maturation. We propose that the interaction between TECPR1 and ATG12–ATG5 initiates the fusion between the Autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in Autophagosome maturation.
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a mammalian Autophagosome maturation mechanism mediated by tecpr1 and the atg12 atg5 conjugate
Molecular Cell, 2012Co-Authors: Dandan Chen, Yiting Lu, Xiaojun Ding, She Chen, Qing ZhongAbstract:Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, Autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying Autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns[3]P) to promote Autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate, and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of Autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, Autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1-deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5, and PtdIns(3)P provide the fusion specificity between Autophagosomes and lysosomes and that the assembly of this complex initiates the Autophagosome maturation process.
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a mammalian Autophagosome maturation mechanism mediated by tecpr1 and the atg12 atg5 conjugate
Molecular Cell, 2012Co-Authors: Dandan Chen, Yiting Lu, Xiaojun Ding, She Chen, Qing ZhongAbstract:Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, Autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying Autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns[3]P) to promote Autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate, and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of Autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, Autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1-deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5, and PtdIns(3)P provide the fusion specificity between Autophagosomes and lysosomes and that the assembly of this complex initiates the Autophagosome maturation process.
Noboru Mizushima - One of the best experts on this subject based on the ideXlab platform.
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modeling membrane morphological change during Autophagosome formation
arXiv: Biological Physics, 2020Co-Authors: Yuji Sakai, Ikuko Koyamahonda, Masashi Tachikawa, Roland L Knorr, Noboru MizushimaAbstract:Autophagy is an intracellular degradation process that is mediated by de novo formation of Autophagosomes. Autophagosome formation involves dynamic morphological changes; a disk-shaped membrane cisterna grows, bends to become a cup-shaped structure, and finally develops into a spherical Autophagosome. We have constructed a theoretical model that integrates the membrane morphological change and entropic partitioning of putative curvature generators, which we have used to investigate the Autophagosome formation process quantitatively. We show that the membrane curvature and the distribution of the curvature generators stabilize disk- and cup-shaped intermediate structures during Autophagosome formation, which is quantitatively consistent with in vivo observations. These results suggest that various autophagy proteins with membrane curvature-sensing properties control morphological change by stabilizing these intermediate structures. Our model provides a framework for understanding Autophagosome formation.
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autophagosomal ykt6 is required for fusion with lysosomes independently of syntaxin 17
Journal of Cell Biology, 2018Co-Authors: Takahide Matsui, Peidu Jiang, Saori Nakano, Yuriko Sakamaki, Hayashi Yamamoto, Noboru MizushimaAbstract:Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using Autophagosomes. To achieve degradation, lysosomes must fuse with closed Autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to Autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that Autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited Autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to Autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate Autophagosome–lysosome fusion.
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accumulation of undegraded Autophagosomes by expression of dominant negative stx17 syntaxin 17 mutants
Autophagy, 2017Co-Authors: Masaaki Uematsu, Hayashi Yamamoto, Yuriko Sakamaki, Taki Nishimura, Noboru MizushimaAbstract:Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the Autophagosome. Purification of Autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the Autophagosome. Here, we report a simple method to accumulate undigested Autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of Autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested Autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent Autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent Autophagosomes.
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syntaxin 17 the autophagosomal snare
Autophagy, 2013Co-Authors: Eisuke Itakura, Noboru MizushimaAbstract:The phagophore (also called isolation membrane) elongates and encloses a portion of cytoplasm, resulting in formation of the Autophagosome. After completion of Autophagosome formation, the outer autophagosomal membrane becomes ready to fuse with the lysosome for degradation of enclosed cytoplasmic materials. However, the molecular mechanism for how the fusion of completed Autophagosomes with the lysosome is regulated has not been fully understood. We discovered syntaxin 17 (STX17) as an autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). STX17 has a hairpin-type structure mediated by two transmembrane domains, each containing glycine zipper motifs. This unique transmembrane structure contributes to its specific localization to completed Autophagosomes but not to phagophores. STX17 interacts with SNAP29 and the lysosomal SNARE VAMP8, and all of these proteins are required for Autophagosome–lysosome fusion. The late recruitment of STX17 to completed Autophagosomes could prevent premature fusion of the lysosome with unclosed phagophores.
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the hairpin type tail anchored snare syntaxin 17 targets to Autophagosomes for fusion with endosomes lysosomes
Cell, 2012Co-Authors: Eisuke Itakura, Chieko Kishiitakura, Noboru MizushimaAbstract:The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying Autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed Autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of Autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed Autophagosome.
Kevin Moreau - One of the best experts on this subject based on the ideXlab platform.
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atg16l1 meets atg9 in recycling endosomes additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis
Autophagy, 2014Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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ATG16L1 meets ATG9 in recycling endosomes: additional roles for the plasma membrane and endocytosis in Autophagosome biogenesis.
Autophagy, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the Autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent Autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to Autophagosomes.
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Diverse Autophagosome Membrane Sources Coalesce in Recycling Endosomes
Cell, 2013Co-Authors: Claudia Puri, Kevin Moreau, Maurizio Renna, Carla F Bento, David C. RubinszteinAbstract:Autophagic protein degradation is mediated by Autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of Autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during Autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of Autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with Autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.
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arf6 promotes Autophagosome formation via effects on phosphatidylinositol 4 5 bisphosphate and phospholipase d
Journal of Cell Biology, 2012Co-Authors: Kevin Moreau, Claudia Puri, Brinda Ravikumar, David C. RubinszteinAbstract:Macroautophagy (in this paper referred to as autophagy) and the ubiquitin–proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane–bounded vesicles called Autophagosomes. The membrane source for Autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of Autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in Autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP2) and in inducing phospholipase D (PLD) activity. PIP2 and PLD may themselves promote Autophagosome biogenesis by influencing endocytic uptake of plasma membrane into Autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.
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Autophagosome Precursor Maturation Requires Homotypic Fusion
Cell, 2011Co-Authors: Kevin Moreau, Claudia Puri, Brinda Ravikumar, Maurizio Renna, David C. RubinszteinAbstract:Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned Autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed Autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent Autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and Autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive Autophagosomes.
