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Molecular Cell Nov 2022ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas...
ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.
Topics: Autophagosomes; Cryoelectron Microscopy; Autophagy; Biological Assay; Lipids
PubMed: 36347259
DOI: 10.1016/j.molcel.2022.10.017 -
The Biochemical Journal May 2021Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during... (Review)
Review
Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during a sequential maturation process of autophagosomes before their ultimate fusion with lysosomes for cargo degradation. This process is highly regulated with multiple protein machineries, such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs, are involved to facilitate autophagic flux to proceed. In neurons, autophagosomes are robustly generated in axonal terminals and then rapidly fuse with late endosomes to form amphisomes. This fusion event allows newly generated autophagosomes to gain retrograde transport motility and move toward the soma, where proteolytically active lysosomes are predominantly located. Amphisomes are not only the products of autophagosome maturation but also the intersection of the autophagy and endo-lysosomal pathways. Importantly, amphisomes can also participate in non-canonical functions, such as retrograde neurotrophic signaling or autophagy-based unconventional secretion by fusion with the plasma membrane. In this review, we provide an updated overview of the recent discoveries and advancements on the molecular and cellular mechanisms underlying amphisome biogenesis and the emerging roles of amphisomes. We discuss recent developments towards the understanding of amphisome regulation as well as the implications in the context of major neurodegenerative diseases, with a comparative focus on Alzheimer's disease and Parkinson's disease.
Topics: Animals; Autophagosomes; Autophagy; Endosomes; Humans; Neurodegenerative Diseases; Neurons
PubMed: 34047789
DOI: 10.1042/BCJ20200917 -
The Journal of Cell Biology Jul 2023As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the...
As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.
Topics: Animals; Autophagosomes; Membrane Proteins; Autophagy; Protein Transport; Autophagy-Related Proteins; Lipids; Mammals
PubMed: 37115958
DOI: 10.1083/jcb.202208088 -
Autophagy Jan 2024Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is...
Omega-shaped domains of the endoplasmic reticulum, known as omegasomes, have been suggested to contribute to autophagosome biogenesis, although their exact function is not known. Omegasomes are characterized by the presence of the double FYVE domain containing protein ZFYVE1/DFCP1, but it has remained a paradox that depletion of ZFYVE1 does not prevent bulk macroautophagy/autophagy. We recently showed that ZFYVE1 contains an N-terminal ATPase domain which dimerizes upon ATP binding. Mutations in the ATPase domain that inhibit ATP binding or hydrolysis do not prevent omegasome expansion and maturation. However, omegasome constriction is inhibited by these mutations, which results in an increased lifetime and thereby higher number of omegasomes. Interestingly, whereas knockout or mutations do not significantly affect bulk autophagy, selective autophagy of mitochondria, protein aggregates and micronuclei is inhibited. We propose that ATP binding and hydrolysis control the di- or multimerization state of ZFYVE1 which could provide the mechanochemical energy to drive large omegasome constriction and autophagosome completion.
Topics: Autophagy; Autophagosomes; Macroautophagy; Adenosine Triphosphatases; Adenosine Triphosphate
PubMed: 37722386
DOI: 10.1080/15548627.2023.2255967 -
Developmental Cell Dec 2023The sequence of morphological intermediates that leads to mammalian autophagosome formation and closure is a crucial yet poorly understood issue. Previous studies have...
The sequence of morphological intermediates that leads to mammalian autophagosome formation and closure is a crucial yet poorly understood issue. Previous studies have shown that yeast autophagosomes evolve from cup-shaped phagophores with only one closure point, and mammalian studies have inferred that mammalian phagophores also have single openings. Our superresolution microscopy studies in different human cell lines in conditions of basal and nutrient-deprivation-induced autophagy identified autophagosome precursors with multifocal origins that evolved into unexpected finger-like phagophores with multiple openings before becoming more spherical structures. Compatible phagophore structures were observed with whole-mount and conventional electron microscopy. This sequence of events was visualized using advanced SIM superresolution live microscopy. The finger-shaped phagophore apertures remained open when ESCRT function was compromised. The efficient closure of autophagic structures is important for their release from the recycling endosome. This has important implications for understanding how autophagosomes form and capture various cargoes.
Topics: Animals; Humans; Autophagosomes; Autophagy; Endosomes; Cell Line; Phagocytosis; Mammals
PubMed: 37683632
DOI: 10.1016/j.devcel.2023.08.016 -
Cells Aug 2021Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...].
Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...].
Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Autophagy-Related Proteins; Humans; Signal Transduction
PubMed: 34440756
DOI: 10.3390/cells10081987 -
Cells Nov 2022Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or... (Review)
Review
Eukaryotes utilize different communication strategies to coordinate processes between different cellular compartments either indirectly, through vesicular transport, or directly, via membrane contact sites (MCSs). MCSs have been implicated in lipid metabolism, calcium signaling and the regulation of organelle biogenesis in various cell types. Several studies have shown that MCSs play a crucial role in the regulation of macroautophagy, an intracellular catabolic transport route that is characterized by the delivery of cargoes (proteins, protein complexes or aggregates, organelles and pathogens) to yeast and plant vacuoles or mammalian lysosomes, for their degradation and recycling into basic metabolites. Macroautophagy is characterized by the de novo formation of double-membrane vesicles called autophagosomes, and their biogenesis requires an enormous amount of lipids. MCSs appear to have a central role in this supply, as well as in the organization of the autophagy-related (ATG) machinery. In this review, we will summarize the evidence for the participation of specific MCSs in autophagosome formation, with a focus on the budding yeast and mammalian systems.
Topics: Animals; Endoplasmic Reticulum; Autophagosomes; Autophagy; Lysosomes; Saccharomyces cerevisiae; Mammals
PubMed: 36497073
DOI: 10.3390/cells11233813 -
Neuron Mar 2022Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of...
Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN. We found that nucleoid-associated proteins are enriched compared with other mitochondrial components. In the axon, autophagic engulfment of nucleoid-enriched mitochondrial fragments requires the mitochondrial fission machinery Drp1. We proposed that localized Drp1-dependent fission of nucleoid-enriched fragments in proximity to the sites of autophagosome biogenesis enhances their capture. The resulting efficient autophagic turnover of nucleoids may prevent accumulation of mitochondrial DNA in the neuron, thus mitigating activation of proinflammatory pathways that contribute to neurodegeneration.
Topics: Animals; Autophagosomes; Autophagy; Brain; Mice; Neurodegenerative Diseases; Neurons
PubMed: 35051374
DOI: 10.1016/j.neuron.2021.12.029 -
Disease Models & Mechanisms Mar 2021Epidemiological research has shown that elevated serum urate concentration is a risk factor for the development of kidney disease; however, the mechanisms underlying...
Epidemiological research has shown that elevated serum urate concentration is a risk factor for the development of kidney disease; however, the mechanisms underlying this process have not yet been elucidated. To examine the role of urate in the kidney, we used Wistar rats to functionally disrupt expression of urate oxidase (UOX) by using the CRISPR/Cas9 system. In comparison to wild-type (WT) rats, serum urate levels spontaneously and persistently increased in -KO rats, without showing a significant decrease in survival rate. Architecture and function of the kidneys in -KO rats were impaired. Injury to the kidney resulted in increased interstitial fibrosis, macrophage infiltration, increased expression of NLRP3 and IL-1β, and activation of multiple cell-signaling pathways associated with autophagy, such as AMPK, p38 MAPK, ERK and JNK pathways. Inhibition of autophagy with the PI3K inhibitor 3-MA abrogated the development of kidney damage and attenuated renal fibrosis, macrophage infiltration, and expression of NLRP3 and IL-1β in injured kidneys. In conclusion, the -KO rat is a great model to study hyperuricemia-related diseases. Hyperuricemia-induced autophagy and NLRP3-dependent inflammation are critically involved in the development of renal damage and, therefore, highlight the inhibition of autophagy and inflammation in search of therapeutic strategies to treat uric acid nephropathy.
Topics: Animals; Autophagosomes; Autophagy; Base Sequence; Gene Knockout Techniques; Hyperuricemia; Inflammation; Kidney; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Rats, Wistar; Urate Oxidase; Rats
PubMed: 33648977
DOI: 10.1242/dmm.048041 -
Cell Research Feb 2022Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system...
Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system remodels has been poorly understood. Here we identify a new type of membrane contact formed between the ER-Golgi intermediate compartment (ERGIC) and the ER-exit site (ERES) in the ER-Golgi system, which is essential for promoting autophagosome biogenesis induced by different stress stimuli. The ERGIC-ERES contact is established by the interaction between TMED9 and SEC12 which generates a short distance opposition (as close as 2-5 nm) between the two compartments. The tight membrane contact allows the ERES-located SEC12 to transactivate COPII assembly on the ERGIC. In addition, a portion of SEC12 also relocates to the ERGIC. Through both mechanisms, the ERGIC-ERES contact promotes formation of the ERGIC-derived COPII vesicle, a membrane precursor of the autophagosome. The ERGIC-ERES contact is physically and functionally different from the TFG-mediated ERGIC-ERES adjunction involved in secretory protein transport, and therefore defines a unique endomembrane structure generated upon stress conditions for autophagic membrane formation.
Topics: Autophagosomes; Autophagy; Endoplasmic Reticulum; Golgi Apparatus; Protein Transport
PubMed: 34561617
DOI: 10.1038/s41422-021-00563-0