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Journal of Molecular Biology Apr 2020Macroautophagy is a conserved catabolic process observed in all eukaryotic cells, during which selected cellular components are transported to and broken down within... (Review)
Review
Macroautophagy is a conserved catabolic process observed in all eukaryotic cells, during which selected cellular components are transported to and broken down within lysosomes. The process starts with the capture of unnecessary material into autophagosomes, which is followed by autophagosome-lysosome fusion to generate autolysosomes that degrade the cargo. In the past quarter-century, our knowledge about autophagosome formation almost exponentially increased, while the later steps were much less studied. This fortunately changed in the past few years, with more and more publications focusing on the fate of the completed autophagosome. In this review, we aspire to summarize the current knowledge about the molecular mechanisms of autophagosome-lysosome fusion.
Topics: Animals; Autophagosomes; Autophagy; Humans; Lysosomes; Neurodegenerative Diseases; SNARE Proteins
PubMed: 31682838
DOI: 10.1016/j.jmb.2019.10.028 -
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 Reviews. Molecular Cell Biology Aug 2020Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to... (Review)
Review
Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. Autophagosome biogenesis responds to a plethora of signals and involves unique and dynamic membrane processes. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer. Thus, not surprisingly, the elucidation of the molecular mechanisms governing autophagosome biogenesis has attracted considerable interest. Key molecules and organelles involved in autophagosome biogenesis, including autophagy-related (ATG) proteins and the endoplasmic reticulum, have been discovered, and their roles and relationships have been investigated intensely. However, several fundamental questions, such as what supplies membranes/lipids to build the autophagosome and how the membrane nucleates, expands, bends into a spherical shape and finally closes, have proven difficult to address. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying these processes on a molecular level. We now know that autophagosome biogenesis is a highly complex process, in which multiple proteins and lipids from various membrane sources, supported by the formation of membrane contact sites, cooperate with biophysical phenomena, including membrane shaping and liquid-liquid phase separation, to ensure seamless segregation of the autophagic cargo. Together, these studies pave the way to obtaining a holistic view of autophagosome biogenesis.
Topics: Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Cell Membrane; Endoplasmic Reticulum; Humans; Lysosomes; Macroautophagy; Protein Transport
PubMed: 32372019
DOI: 10.1038/s41580-020-0241-0 -
Autophagy 2018Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered...
Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A (BafA), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.
Topics: Animals; Autophagosomes; Autophagy; Cell Line, Tumor; Chloroquine; Endocytosis; Endosomes; ErbB Receptors; Female; Golgi Apparatus; Humans; Hydroxychloroquine; Lysosomes; Macrolides; Membrane Fusion; Mice, Inbred C57BL; Proteolysis; Sequestosome-1 Protein
PubMed: 29940786
DOI: 10.1080/15548627.2018.1474314 -
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 -
Cell Oct 2022The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca transients on the...
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca transients. Multi-modal SIM analysis revealed that Ca transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Topics: Animals; Autophagosomes; Calcium; Endoplasmic Reticulum; Autophagy-Related Proteins; Autophagy; Cell Cycle Proteins
PubMed: 36198318
DOI: 10.1016/j.cell.2022.09.001 -
The Journal of Cell Biology Jun 2020Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an... (Review)
Review
Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an autophagosome). This process has remained enigmatic since its initial discovery >50 yr ago, but our understanding of the mechanisms involved in autophagosome biogenesis has increased substantially during the last 20 yr. Several key questions do remain open, however, including, What determines the site of autophagosome nucleation? What is the origin and lipid composition of the autophagosome membrane? How is cargo sequestration regulated under nonselective and selective types of autophagy? This review provides key insight into the core molecular mechanisms underlying autophagosome biogenesis, with a specific emphasis on membrane modeling events, and highlights recent conceptual advances in the field.
Topics: Autophagosomes; Autophagy; Autophagy-Related Proteins; Biological Transport, Active; Endoplasmic Reticulum; Humans; Lipid Metabolism; Lipids; Membranes; Signal Transduction
PubMed: 32357219
DOI: 10.1083/jcb.202002085 -
Autophagy May 2021The fusion of autophagosomes and endosomes/lysosomes, also called autophagosome maturation, ensures the degradation of autophagic cargoes. It is an important regulatory...
The fusion of autophagosomes and endosomes/lysosomes, also called autophagosome maturation, ensures the degradation of autophagic cargoes. It is an important regulatory step of the macroautophagy/autophagy process. STX17 is the key autophagosomal SNARE protein that mediates autophagosome maturation. Here, we report that the acetylation of STX17 regulates its SNARE activity and autophagic degradation. The histone acetyltransferase CREBBP/CBP and the deacetylase HDAC2 specifically regulate the acetylation of STX17. In response to cell starvation and MTORC1 inhibition, the inactivation of CREBBP leads to the deacetylation of STX17 at its SNARE domain. This deacetylation promotes the interaction between STX17 and SNAP29 and the formation of the STX17-SNAP29-VAMP8 SNARE complex with no effect on the recruitment of STX17 to autophagosomal membranes. Deacetylation of STX17 also enhances the interaction between STX17 and the tethering complex HOPS, thereby further promoting autophagosome-lysosome fusion. Our study suggests a mechanism by which acetylation regulates the late-stage of autophagy, and possibly other STX17-related intracellular membrane fusion events. ACTB: actin beta; CREBBP/CBP: CREB binding protein; Ctrl: control; GFP: green fluorescent protein; GST: glutathione S-transferase; HDAC: histone deacetylase; HOPS: homotypic fusion and protein sorting complex; KO: knockout; LAMP1/2: lysosomal associated membrane protein 1/2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MS: mass spectrometry; MTORC1: mechanistic target of rapamycin kinase complex 1; NAM: nicotinamide; PtdIns3K: phosphatidylinositol 3-kinase; RFP: red fluorescent protein; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TSA: trichostatin A; TSC1/2: TSC complex subunit 1/2; VAMP8: vesicle associated membrane protein 8; WT: wild type.
Topics: Autophagosomes; Autophagy; Endosomes; Fibroblasts; Humans; Lysosomes; Macroautophagy; Membrane Fusion; Qa-SNARE Proteins
PubMed: 32264736
DOI: 10.1080/15548627.2020.1752471 -
Autophagy Jan 2023SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of...
SCFD1 (sec1 family domain containing 1) was recently shown to function in autophagosome-lysosome fusion, and multiple studies have demonstrated the regulatory impacts of acetylation (a post-translational modification) on macroautophagy/autophagy. Here, we demonstrate that both acetylation- and phosphorylation-dependent mechanisms control SCFD1's function in autophagosome-lysosome fusion. After detecting a decrease in the extent of SCFD1 acetylation under autophagy-stimulated conditions, we found that KAT2B/PCAF catalyzes the acetylation of residues K126 and K515 of SCFD1; we also showed that these two residues are deacetylated by SIRT4. Importantly, we showed that AMPK-controlled SCFD1 phosphorylation strongly disrupts the capacity of SCFD1 to interact with KAT2B, thus ensuring that the SCFD1 acetylation level remains low. Finally, we demonstrated that SCFD1 acetylation inhibits autophagic flux, specifically by blocking STX17-SNAP29-VAMP8 SNARE complex formation. Thus, our study reveals a mechanism through which phosphorylation and acetylation modifications of SCFD1 mediate SNARE complex formation to regulate autophagosome maturation.ACLY: ATP citrate lyase; CREB: cAMP responsive element binding protein; EBSS: nutrient-deprivation medium; EP300: E1A binding protein p300; KAT5/TIP60: lysine acetyltransferase 5; HOPS: homotypic fusion and protein sorting; MS: mass spectroscopy; SCFD1: sec1 family domain containing 1; SM: Sec1/Munc18; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UVRAG: UV radiation resistance associated.
Topics: Autophagy; Autophagosomes; Macroautophagy; Acetylation; Lysosomes; Protein Processing, Post-Translational; SNARE Proteins; Membrane Fusion
PubMed: 35465820
DOI: 10.1080/15548627.2022.2064624 -
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