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Cells Feb 2023Autophagy-the lysosomal degradation of cytoplasm-plays a central role in cellular homeostasis and protects cells from potentially harmful agents that may accumulate in... (Review)
Review
Autophagy-the lysosomal degradation of cytoplasm-plays a central role in cellular homeostasis and protects cells from potentially harmful agents that may accumulate in the cytoplasm, including pathogens, protein aggregates, and dysfunctional organelles. This process is initiated by the formation of a phagophore membrane, which wraps around a portion of cytoplasm or cargo and closes to form a double-membrane autophagosome. Upon the fusion of the autophagosome with a lysosome, the sequestered material is degraded by lysosomal hydrolases in the resulting autolysosome. Several alternative membrane sources of autophagosomes have been proposed, including the plasma membrane, endosomes, mitochondria, endoplasmic reticulum, lipid droplets, hybrid organelles, and de novo synthesis. Here, we review recent progress in our understanding of how the autophagosome is formed and highlight the proposed role of vesicles that contain the lipid scramblase ATG9 as potential seeds for phagophore biogenesis. We also discuss how the phagophore is sealed by the action of the endosomal sorting complex required for transport (ESCRT) proteins.
Topics: Autophagosomes; Macroautophagy; Autophagy; Endosomes; Cell Membrane
PubMed: 36831335
DOI: 10.3390/cells12040668 -
Nature Communications Mar 2023Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their...
Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.
Topics: Lipogenesis; Autophagosomes; Adipocytes; Fatty Acids; Autophagy; Lysosomes
PubMed: 36914626
DOI: 10.1038/s41467-023-37016-8 -
Autophagy Aug 2021TMEM41B and VMP1, two endoplasmic reticulum (ER)-resident transmembrane proteins, play important roles in regulating the formation of lipid droplets (LDs), autophagy... (Review)
Review
TMEM41B and VMP1, two endoplasmic reticulum (ER)-resident transmembrane proteins, play important roles in regulating the formation of lipid droplets (LDs), autophagy initiation, and viral infection. However, the biochemical functions of TMEM41B and VMP1 are unclear. A lipids distribution screen suggested TMEM41B and VMP1 are critical to the normal distribution of cholesterol and phosphatidylserine. Biochemical analyses unveiled that TMEM41B and VMP1 have scramblase activity. These findings shed light on the mechanism by which TMEM41B and VMP1 regulate LD formation, lipids distribution, macroautophagy, and viral infection.
Topics: Animals; Autophagosomes; Autophagy; Humans; Macroautophagy; Membrane Proteins; Phospholipid Transfer Proteins
PubMed: 34074213
DOI: 10.1080/15548627.2021.1937898 -
Methods in Molecular Biology (Clifton,... 2019Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis....
Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy. Autophagy induction can be detected using Western blotting of LC3 (marker protein for autophagosomes) in which LC3-II levels represent the quantity of autophagosomes formed on induction to a particular stimulus. This can also be confirmed by puncta formation assay using confocal microscopy. Further, the autophagic flux can be examined using bafilomycin A1 as inhibitor of autophagosome-lysosome fusion and acidification of lysosomal compartments, thereby leading to accumulation of autophagosomes which is represented by high LC3-II levels. The autophagolysosomal degradation or proteolysis which is the last step of autophagy can be analyzed by DQ-BSA assay.
Topics: Animals; Autophagosomes; Autophagy; Fluorescence; Lysosomes; Mice; Microscopy, Confocal; Proteolysis; RAW 264.7 Cells
PubMed: 30242567
DOI: 10.1007/7651_2018_190 -
Autophagy Dec 2023Macroautophagy/autophagy and lipid droplet (LD) biology are intricately linked, with autophagosome-dependent degradation of LDs in response to different signals. LDs...
Macroautophagy/autophagy and lipid droplet (LD) biology are intricately linked, with autophagosome-dependent degradation of LDs in response to different signals. LDs play crucial roles in forming autophagosomes possibly by providing essential lipids and serving as a supportive autophagosome assembly platform at the endoplasmic reticulum (ER)-LD interface. LDs and autophagosomes share common proteins, such as VPS13, ATG2, ZFYVE1/DFCP1, and ATG14, but their dual functions remain poorly understood. In our recent study, we found that prolonged starvation leads to ATG3 localizing to large LDs and lipidating LC3B, revealing a non-canonical autophagic role on LDs. In vitro, ATG3 associates with purified and artificial LDs, and conjugated Atg8-family proteins. In long-term starved cells, only LC3B is found on the specific large LDs, positioned near LC3B-positive membranes that undergo lysosome-mediated acidification. This implies that LD-lipidated LC3B acts as a tethering factor, connecting phagophores to LDs and promoting degradation. Our data also support the notion that certain LD surfaces may function as lipidation stations for LC3B, which may move to nearby sites of autophagosome formation. Overall, our study unveils an unknown non-canonical implication of LDs in autophagy processes. ATG: autophagy-related enzyme, ATP: adenosine triphosphate, E2 enzyme: ubiquitin-conjugating enzyme, ER: endoplasmic reticulum, LD: lipid droplet, LIR motif: LC3-interacting region, MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta, PE: phosphatidylethanolamine, PLIN1: perilipin 1, PNPLA2/ATGL: patatin-like phospholipase domain containing 2, SQSTM1/p62: sequestosome 1, VSP13: vacuolar protein sorting 13, ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1.
Topics: Autophagy; Lipid Droplets; Autophagosomes; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins
PubMed: 37599471
DOI: 10.1080/15548627.2023.2249390 -
Current Biology : CB Jul 2019In 1955, the biologist and Nobel Prize laureate Christian de Duve discovered that cells possess specialized organelles filled with hydrolytic enzymes and he called these...
In 1955, the biologist and Nobel Prize laureate Christian de Duve discovered that cells possess specialized organelles filled with hydrolytic enzymes and he called these organelles lysosomes. At the same time, electron microscopy studies by Novikoff and colleagues showed that intracellular dense bodies, which later turned out to be lysosomes, contain cytoplasmic components. Together, these groundbreaking observations revealed that cells can deliver cytoplasmic components to lysosomes for degradation. The hallmark of this degradative process, which de Duve called autophagy, is the formation of double-membrane-limited vesicles. Further morphological characterization of these vesicles (autophagosomes) revealed that they mainly contain bulk cytoplasm. Although this suggested that autophagy leads to a non-selective degradation of cytoplasmic material, de Duve anticipated that a regulated and selective type of this pathway must also exist. Today we know that, under normal conditions, macroautophagy is a highly selective pathway that sequesters damaged or superfluous material from the cytoplasm through the formation of double-membrane-limited autophagosomes. Upon fusion with lysosomes, the content of autophagosomes is degraded and the resulting building blocks are released into the cytoplasm. However, in response to cytotoxic stress or starvation, cells start to produce autophagosomes that capture bulk cytoplasm non-selectively. This stress response is essential for cells to survive adverse environmental conditions, whereas the selective sequestration of cargo is important to maintain cellular homeostasis.
Topics: Autophagosomes; Autophagy; Cytosol; Lysosomes; Macroautophagy
PubMed: 31336079
DOI: 10.1016/j.cub.2019.06.014 -
Molecular Cell May 2021Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8...
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Cysteine Endopeptidases; Female; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Influenza A virus; Macrolides; Male; Mice; Microtubule-Associated Proteins; Monensin; Phagocytosis; Phosphatidylethanolamines; Phosphatidylserines; Protein Processing, Post-Translational; RAW 264.7 Cells; Signal Transduction
PubMed: 33909989
DOI: 10.1016/j.molcel.2021.03.020 -
Advances in Experimental Medicine and... 2021Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5...
Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.
Topics: Animals; Autophagosomes; Autophagy; Endosomal Sorting Complexes Required for Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34260020
DOI: 10.1007/978-981-16-2830-6_3 -
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 -
Journal of Molecular Biology Apr 2020We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the... (Review)
Review
We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well coordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of nonselective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.
Topics: Animals; Autophagosomes; Autophagy; Humans; Mitophagy; Neurodegenerative Diseases; Neurons
PubMed: 31705882
DOI: 10.1016/j.jmb.2019.10.027