RESUMEN
50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.
RESUMEN
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.
Asunto(s)
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
Organelles are continuously turned over as part of cellular homeostasis and adaptation. Most organelles, even including the nucleus, are degraded by lysosomes via different pathways, such as macroautophagy, microautophagy, organelle-derived vesicle degradation, and crinophagy. In some specific cases-for example, in lens fiber cells-organelles are degraded by cytosolic phospholipases. To view this SnapShot, open or download the PDF.
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Autofagia , Cristalino , Citosol , Cristalino/metabolismo , Lisosomas , Orgánulos/metabolismoRESUMEN
The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Liposomas/metabolismo , Mamíferos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Macroautophagy and microautophagy are highly conserved eukaryotic cellular processes that degrade cytoplasmic material in lysosomes. Both pathways involve characteristic membrane dynamics regulated by autophagy-related proteins and other molecules, some of which are shared between the two pathways. Over the past few years, the application of new technologies, such as cryo-electron microscopy, coevolution-based structural prediction and in vitro reconstitution, has revealed the functions of individual autophagy gene products, especially in autophagy induction, membrane reorganization and cargo recognition. Concomitantly, mutations in autophagy genes have been linked to human disorders, particularly neurodegenerative diseases, emphasizing the potential pathogenic implications of autophagy defects. Accumulating genome data have also illuminated the evolution of autophagy genes within eukaryotes as well as their transition from possible ancestral elements in prokaryotes.
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Autofagia , Lisosomas , Humanos , Microscopía por Crioelectrón , Autofagia/genética , Lisosomas/metabolismo , Proteínas/metabolismo , Eucariontes , BiologíaRESUMEN
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.
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Endosomas/metabolismo , Lisosomas/metabolismo , Fagosomas/metabolismo , Proteínas Qa-SNARE/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Autofagia , Línea Celular , Citosol/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/química , Alineación de SecuenciaRESUMEN
The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.
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Cristalino/citología , Cristalino/enzimología , Orgánulos/metabolismo , Fosfolipasas A2 Calcio-Independiente/metabolismo , Fosfolipasas A/metabolismo , Proteínas de Pez Cebra/metabolismo , Aciltransferasas/metabolismo , Animales , Catarata/metabolismo , Línea Celular , Femenino , Factores de Transcripción del Choque Térmico/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Pez Cebra/metabolismoRESUMEN
Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.
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Autofagosomas/metabolismo , Autofagia , Compartimento Celular , Citosol/metabolismo , Humectabilidad , Adhesividad , Autofagosomas/química , Línea Celular , Citosol/química , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Proteína Sequestosoma-1/metabolismo , Tensión SuperficialRESUMEN
Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.
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Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Retículo Endoplásmico/genética , Proteínas Intrínsecamente Desordenadas/genética , Secuencia de Aminoácidos/genética , Proteínas Relacionadas con la Autofagia/química , Proteínas de Ciclo Celular/genética , Retículo Endoplásmico/química , Estrés del Retículo Endoplásmico/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , ProteolisisRESUMEN
Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the lysosome. However, the purpose of autophagy is not the simple elimination of materials, but instead, autophagy serves as a dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis. Here we provide a multidisciplinary review of our current understanding of autophagy's role in metabolic adaptation, intracellular quality control, and renovation during development and differentiation. We also explore how recent mouse models in combination with advances in human genetics are providing key insights into how the impairment or activation of autophagy contributes to pathogenesis of diverse diseases, from neurodegenerative diseases such as Parkinson disease to inflammatory disorders such as Crohn disease.
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Autofagia , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Enfermedades Neurodegenerativas/patología , Fagosomas/metabolismo , Células Vegetales/metabolismoRESUMEN
ß-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21 and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including ß-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3 and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.
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Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/genética , Mamíferos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismoRESUMEN
Selective autophagy relies on adaptor proteins to bind and transport cargos (or substrates) to the lysosome or vacuole, yet the mechanisms for cargo recognition are not well understood. In this issue, Wang et al (2021) showed that in the fission yeast, Nbr1, a homolog of a mammalian selective autophagy adaptor, recognizes vacuolar hydrolases Ams1 and Ape4 through both versatile and cargo-specific interactions with the Nbr1 ZZ1 domain.
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Schizosaccharomyces , Vacuolas , Proteínas Adaptadoras Transductoras de Señales , Animales , Autofagia , Citosol , Schizosaccharomyces/genéticaRESUMEN
Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.
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Autofagia , Susceptibilidad a Enfermedades , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Autofagia/inmunología , Biomarcadores , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Homeostasis , Interacciones Huésped-Patógeno , Humanos , Especificidad de Órganos , Transducción de SeñalRESUMEN
Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.
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Autofagia , Técnicas Citológicas , Animales , Humanos , Fagosomas/metabolismoRESUMEN
Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.
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Autofagia/fisiología , Complejos Multiproteicos/metabolismo , Fagosomas/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Fagosomas/ultraestructura , Fosfatidilinositol 3-Quinasas/metabolismo , Levaduras/citología , Levaduras/metabolismoRESUMEN
Autophagy is a lysosome-dependent intracellular degradation system required for various physiological processes and can be dysregulated in human disease. To understand its biological significance and underlying mechanisms, measuring autophagic activity (i.e., autophagic flux) is critical. However, navigating which assays to use, and when, is complicated and at times the results are often interpreted inappropriately. This review will summarize both advantages and disadvantages of currently available methods to monitor autophagy. In addition, we discuss how these assays should be used in high-throughput screens to identify autophagy-modulating drugs and genes and the general features needed for biomarkers to assess autophagy in humans.
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Autofagia , Bioensayo , Autofagia/efectos de los fármacos , Autofagia/genética , Biomarcadores/análisis , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Lisosomas/metabolismoRESUMEN
Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases. We show that the expression of the active form of Rim101 increased the size of wildtype cells. Furthermore, the size of wildtype cells increased in response to external alkalization. Microscopic observation revealed that this cell size increase was associated with changes in both vacuolar and cytoplasmic volume. We also found that these volume changes were dependent on Rim21 and Rim101. In addition, a mutant lacking Vph1, a component of V-ATPase that is transcriptionally regulated by Rim101, was also smaller than wildtype cells, with no increase in size in response to alkalization. We demonstrate that the loss of Vph1 suppressed the Rim101-induced increase in cell size under physiological pH conditions. Taken together, our results suggest that the cell size of budding yeast is regulated by the Rim101-V-ATPase axis under physiological conditions as well as in response to alkaline stresses.
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Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Concentración de Iones de Hidrógeno , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Eliminación de SecuenciaRESUMEN
The endoplasmic reticulum (ER) is a central hub for the biogenesis of various organelles and lipid-containing structures. Recent studies suggest that vacuole membrane protein 1 (VMP1) and transmembrane protein 41B (TMEM41B), multispanning ER membrane proteins, regulate the formation of many of these ER-derived structures, including autophagosomes, lipid droplets, lipoproteins, and double-membrane structures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. VMP1 and TMEM41B possess a DedA domain that is widely distributed not only in eukaryotes but also in prokaryotes and predicted to adopt a characteristic structure containing two reentrant loops. Furthermore, recent studies show that both proteins have lipid scrambling activity. Based on these findings, the potential roles of VMP1 and TMEM41B in the dynamic remodeling of ER membranes and the biogenesis of ER-derived structures are discussed.
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Autofagia , Proteínas de la Membrana/genética , Retículo Endoplásmico/genética , HumanosRESUMEN
Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3-interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR-ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)-phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER-phagy. Structural analysis shows that phosphorylation of these serine residues increases binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER-phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR-ATG8 interactions, even in the absence of auxiliary interactions.
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Quinasa de la Caseína II , Proteínas Asociadas a Microtúbulos , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Proteínas Portadoras/metabolismo , Quinasa de la Caseína II/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Serina/metabolismoRESUMEN
Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.