RESUMEN
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ 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 Ca2+ 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 Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ 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, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Asunto(s)
Autofagosomas , Calcio , Animales , Autofagosomas/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismoRESUMEN
Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.
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Autofagia/fisiología , Ácidos Grasos/metabolismo , Fagosomas/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligasas/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Fagosomas/fisiología , Fosfolípidos/biosíntesis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Macroautophagy is an intracellular degradation system that delivers diverse cytoplasmic materials to lysosomes via autophagosomes. Recent advances have enabled identification of several selective autophagy substrates and receptors, greatly expanding our understanding of the cellular functions of autophagy. In this review, we describe the diverse cellular functions of macroautophagy, including its essential contribution to metabolic adaptation and cellular homeostasis. We also discuss emerging findings on the mechanisms and functions of various types of selective autophagy.
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Autofagosomas/metabolismo , Autofagia/genética , Retículo Endoplásmico/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Animales , Autofagosomas/enzimología , Autofagosomas/microbiología , Autofagia/fisiología , Retículo Endoplásmico/fisiología , Homeostasis/genética , Homeostasis/fisiología , Humanos , Lisosomas/patología , Mitocondrias/patología , Nutrientes/deficiencia , Nutrientes/metabolismo , Peroxisomas/metabolismo , Peroxisomas/fisiologíaRESUMEN
Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.
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Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas SNARE/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Proteínas Relacionadas con la Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Citoesqueleto/química , Citoesqueleto/metabolismo , Endocitosis , Humanos , Lisosomas/metabolismo , Mamíferos , Modelos Moleculares , Fagosomas/metabolismo , Proteínas SNARE/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/genéticaRESUMEN
Cargo sequestration is a fundamental step of selective autophagy in which cells generate a double-membrane structure termed an "autophagosome" on the surface of cargoes. NDP52, TAX1BP1, and p62 bind FIP200, which recruits the ULK1/2 complex to initiate autophagosome formation on cargoes. How OPTN initiates autophagosome formation during selective autophagy remains unknown despite its importance in neurodegeneration. Here, we uncover an unconventional path of PINK1/Parkin mitophagy initiation by OPTN that does not begin with FIP200 binding or require the ULK1/2 kinases. Using gene-edited cell lines and in vitro reconstitutions, we show that OPTN utilizes the kinase TBK1, which binds directly to the class III phosphatidylinositol 3-kinase complex I to initiate mitophagy. During NDP52 mitophagy initiation, TBK1 is functionally redundant with ULK1/2, classifying TBK1's role as a selective autophagy-initiating kinase. Overall, this work reveals that OPTN mitophagy initiation is mechanistically distinct and highlights the mechanistic plasticity of selective autophagy pathways.
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Mitofagia , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Autofagosomas/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , AutofagiaRESUMEN
The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.
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Daño del ADN , Lamina Tipo A , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Fosforilación , Nucleotidiltransferasas/genética , ADN/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
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.
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Autofagosomas , Autofagia , Microscopía por Crioelectrón , Bioensayo , LípidosRESUMEN
The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Cisteína Endopeptidasas/metabolismo , Metabolismo de los Lípidos , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Inteligencia Artificial , Autofagosomas/genética , Autofagosomas/ultraestructura , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Cisteína Endopeptidasas/genética , Células HEK293 , Células HeLa , Humanos , Imagenología Tridimensional , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Mitocondrias/ultraestructura , Mitofagia , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Metabolismo Energético , Galectinas/metabolismo , Lisosomas/enzimología , Ubiquitina/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Adolescente , Adulto , Animales , Autofagia/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Activación Enzimática , Femenino , Galectinas/genética , Células HEK293 , Células HeLa , Humanos , Hipoglucemiantes/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/microbiología , Lisosomas/patología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Metformina/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium tuberculosis/patogenicidad , Transducción de Señal , Células THP-1 , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Adulto JovenRESUMEN
Autophagy is one of the major highly inducible degradation processes in response to plant developmental and environmental signals. In response to different stimuli, cellular materials, including proteins and organelles, can be sequestered into a double membrane autophagosome structure either selectively or non-selectively. The formation of an autophagosome as well as its delivery into the vacuole involves complex and dynamic membrane processes. The identification and characterization of the conserved autophagy-related (ATG) proteins and their related regulators have greatly advanced our understanding of the molecular mechanism underlying autophagosome biogenesis and function in plant cells. Autophagosome biogenesis is tightly regulated by the coordination of multiple ATG and non-ATG proteins, and selective cargo recruitment. This review updates our current knowledge of autophagosome biogenesis, with special emphasis on the core molecular machinery that drives autophagosome formation, and autophagosome-organelle interactions under abiotic stress conditions.
RESUMEN
mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.
Asunto(s)
Autofagosomas/enzimología , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Glucógeno Sintasa Quinasa 3/metabolismo , Metabolismo de los Lípidos , Hígado/enzimología , Lisina Acetiltransferasa 5/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Transactivadores/metabolismo , Acetilación , Animales , Autofagosomas/patología , Proteínas Relacionadas con la Autofagia/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Glucógeno Sintasa Quinasa 3/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Gotas Lipídicas/metabolismo , Hígado/patología , Lisina Acetiltransferasa 5/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas de la Membrana , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión a Fosfato/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Transducción de Señal , Transactivadores/genética , Proteínas Supresoras de TumorRESUMEN
Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPα KO brain. Significantly, all neuronal classes in CSPα KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPα KO cortex and reveals insights into synapse maintenance and neurodegeneration.
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Proteínas del Choque Térmico HSP40 , Proteínas de la Membrana , Ratones Noqueados , Neuronas , Sinapsis , Transcriptoma , Animales , Sinapsis/metabolismo , Ratones , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Neuronas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Neuroglía/metabolismoRESUMEN
Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.
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Autofagosomas , Fosfolípidos , Autofagosomas/metabolismo , Fosfolípidos/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Colina/metabolismo , Citidina Difosfato/metabolismoRESUMEN
One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12-ATG5-ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.
RESUMEN
Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of Salmonella, range from 2 to 5 µm in length and 0.5 to 1.5 µm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic Salmonella to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged Salmonella-containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of Salmonella, or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.
Asunto(s)
Autofagosomas , Macroautofagia , Humanos , Autofagosomas/metabolismo , Autofagia , Retículo Endoplásmico/metabolismo , Células HeLaRESUMEN
Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.
Asunto(s)
Autofagia , Proteínas de Saccharomyces cerevisiae , Animales , Humanos , Proteínas R-SNARE/metabolismo , Fosforilación , Autofagia/genética , Autofagosomas/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Fusión de Membrana/fisiología , Saccharomyces cerevisiae/metabolismo , Lisosomas/metabolismo , Mamíferos/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.
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Autofagosomas/enzimología , Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Proteínas de la Membrana/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Animales Modificados Genéticamente , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia , Células COS , Sistemas CRISPR-Cas , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/metabolismo , Chlorocebus aethiops , Genotipo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de la Membrana/genética , Proteínas Musculares/metabolismo , Fenotipo , Fosfatos de Fosfatidilinositol/metabolismo , Proteolípidos/metabolismo , Interferencia de ARN , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , TransfecciónRESUMEN
Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
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Autofagosomas , Macroautofagia , Vacuolas , Autofagosomas/metabolismo , Membrana Celular , Retículo Endoplásmico/metabolismo , Saccharomyces cerevisiae , Vacuolas/metabolismoRESUMEN
BACKGROUND: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. RESULTS: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. CONCLUSIONS: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.
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Autofagosomas , Glioblastoma , Humanos , Autofagosomas/metabolismo , Glioblastoma/metabolismo , Autofagia , Proteómica , Lisosomas/metabolismo , Estrés del Retículo EndoplásmicoRESUMEN
Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.