RESUMO
Autophagosome formation requires PROPPIN/WIPI proteins and monophosphorylated phosphoinositides, such as phosphatidylinositol-3-phosphate (PtdIns3P) or PtdIns5P. This process occurs in association with mammalian endosomes, where the PROPPIN WIPI1 has additional, undefined roles in vesicular traffic. To explore whether these functions are interconnected, we dissected routes and subreactions of endosomal trafficking requiring WIPI1. WIPI1 specifically acts in the formation and fission of tubulo-vesicular endosomal transport carriers. This activity supports the PtdIns(3,5)P2-dependent transport of endosomal cargo toward the plasma membrane, Golgi, and lysosomes, suggesting a general role of WIPI1 in endosomal protein exit. Three features differentiate the endosomal and macroautophagic/autophagic activities of WIPI1: phosphoinositide binding site II, the requirement for PtdIns(3,5)P2, and bilayer deformation through a conserved amphipathic α-helix. Their inactivation preserves autophagy but leads to a strong enlargement of endosomes, which accumulate micrometer-long endosomal membrane tubules carrying cargo proteins. WIPI1 thus supports autophagy and protein exit from endosomes by different modes of action. We propose that the type of phosphoinositides occupying its two lipid binding sites, the most unusual feature of PROPPIN/WIPI family proteins, switches between these effector functions.Abbreviations: EGF: epidermal growth factorEGFR: epidermal growth factor receptorKD: knockdownKO: knockoutPtdIns3P: phosphatidylinositol-3-phosphatePtdIns5P: phosphatidylinositol-5-phosphatePtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphateTF: transferrinTFRC: transferrin receptorWT: wildtype.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas Relacionadas à Autofagia/fisiologia , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular , Endocitose , Edição de Genes , Humanos , Proteínas de Membrana/fisiologia , Microscopia Confocal , Corpos Multivesiculares/fisiologia , Mutagênese Sítio-DirigidaRESUMO
Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.
Assuntos
Neoplasias da Mama/genética , Exossomos/patologia , GTP Fosfo-Hidrolases/metabolismo , Metástase Neoplásica/genética , Animais , Neoplasias da Mama/secundário , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Corpos Multivesiculares/fisiologia , Peixe-ZebraRESUMO
Endogenous toxicity caused by systemic inflammation as well as by acute liver failure triggers a wide range of dysfunctional disorders in the brain ranging from delirium and acute psychosis to coma. Astrocytes, the main homeostatic cells of the central nervous system (CNS), play a key role in pathophysiology of neurotoxic insults. We examined the cecal ligation and puncture (CLP) and acetaminophen-induced liver failure (AILF) of Wistar rats, and analysed ultrastructure of astrocytes in the brain cortex and subcortical white matter of sensorimotor zone with transmission electron microscopy. Both models showed significant similarities in reactive changes of astroglial endosomal machinery. In survived animals (with relative prevalence in the CLP-model), at 24 h after intervention we found an increase in number of multivesicular bodies (MVBs) in astroglial perikarya and astroglial processes. In particular, the number of MVBs substantially (3 times of control values) increased in the perivascular astroglial endfeet. Increased number of MVBs in astrocytes was associated with the lesser degree of intracellular oedema and with signs of compensated oedematous tissue changes. In deceased animals, up to 24 h after intervention, single MVBs were localised mainly in astroglial perikarya, and their number was not significantly changed compared to control. Activation of astroglial endosomal-exosomal machinery in both models reflects the uniform pattern of reactive changes of astroglia in these two systemic conditions and may represent activation of astroglial defence in sepsis-associated encephalopathy (SAE) and acute hepatic encephalopathy (AHE). Our data highlight the special role of astroglial adaptive activity in the counterbalancing of an impaired brain homeostasis under action of endogenous toxins. Accumulation of MVBs in astrocytic processes indicates the activation of their intercellular and gliovascular interactions through endo- and exocytosis in SAE and AHE.
Assuntos
Astrócitos/fisiologia , Corpos Multivesiculares/fisiologia , Síndromes Neurotóxicas/fisiopatologia , Doença Aguda , Animais , Astrócitos/metabolismo , Encéfalo/fisiopatologia , Encefalopatias/metabolismo , Encefalopatias/fisiopatologia , Sistema Nervoso Central/citologia , Masculino , Corpos Multivesiculares/metabolismo , Neurônios/metabolismo , Síndromes Neurotóxicas/metabolismo , Ratos , Ratos Wistar , Córtex Sensório-Motor/fisiopatologia , Sepse/fisiopatologia , Encefalopatia Associada a Sepse/fisiopatologiaRESUMO
NEW FINDINGS: What is the central question of this study? What is the impact of acute aerobic and aerobic + resistance (concurrent) exercise on the regulation of multivesicular body formation in human skeletal muscle? What is the main finding and its importance? Gene expression for proteins associated with multivesicular body biogenesis was increased in response to concurrent exercise, and gene expression of microRNA processing (genetic information) was increased in response to aerobic and concurrent exercise. A greater understanding of the processing of multivesicular bodies in response to acute exercise may lead to novel treatments focused on intercellular communication pathways. ABSTRACT: Regular aerobic exercise (AEx) and resistance exercise (REx) promote many beneficial adaptations. Skeletal muscle participates in intercellular communication in part through the release of myokines and extracellular vesicles including exosomes (EXOs), the latter containing mRNA, microRNA (miRNA), lipids and proteins. Exercise-induced regulation of skeletal muscle multivesicular body (MVB) biogenesis leading to EXO formation and release is poorly understood. We hypothesized that acute exercise would increase skeletal muscle MVB biogenesis and EXO release pathways with a greater response to aerobic + resistance exercise (A+REx) than to AEx alone. Twelve sedentary, healthy male subjects exercised on a cycle ergometer for 45 min (AEx) followed by single leg, knee extensor, resistance exercise (A+REx). Vastus lateralis biopsies were obtained at rest and 1 h post-exercise. Key components of the MVB biogenesis, EXO biogenesis and release, and miRNA processing pathways were analysed. Clathrin and Alix mRNA (MVB biogenesis) were increased by A+REx, while DICER and exportin mRNA (miRNA processing) were increased by AEx and A+REx. There were positive relationships between MVBs and miRNA processing genes following both AEx and A+REx consistent with coordinated regulation of these interrelated processes (Alix mRNA increased with Drosha, exportin and Dicer mRNA). Acute exercise increases the regulation of components of MVB and EXO pathways as well as miRNA processing components. A greater understanding of the production and packaging of skeletal muscle MVBs, EXOs and mature miRNA could lead to novel treatments focused on intercellular communication.
Assuntos
Exercício Físico/fisiologia , Exossomos/metabolismo , Exossomos/fisiologia , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/fisiologia , Transdução de Sinais/fisiologia , Adolescente , Adulto , Humanos , Masculino , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Músculo Quadríceps/fisiologia , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Vírus da Hepatite E/fisiologia , Corpos Multivesiculares/fisiologia , Corpos Multivesiculares/virologia , Liberação de Vírus , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Transporte Proteico , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Autophagy plays a key role in cellular homeostasis since it allows optimal cellular functioning and provides energy under conditions of stress. Initial is revealed that alterations of macroautophagy disturb adipogenic differentiation in cultured cells, and in mice, leading to a drastic reduction of adipose tissue depots. Nevertheless, more recent studies indicate that autophagy participates in the control of adipose tissue biology in a more complex manner. The protein TP53INP2 activates the formation of autophagosomes by binding to ATG8 proteins such as LC3 or GATE16, and its genetic elimination reduces but does not cancel this activity. TP53INP2 deficiency increases adipogenic differentiation and induces a gain in adiposity in the mouse. At the cellular level, TP53INP2 promotes the sequestration of the regulatory protein GSK3ß in multivesicular bodies (MVBs) by a process that involves autophagic activity and the participation of the endosomal sorting complexes required for transport (ESCRT) machinery. Through this mechanism, TP53INP2 stabilizes and activates ß-catenin, which in turn triggers the inhibition of adipogenesis. In summary, autophagic pathways provide a whole set of mechanisms that may regulate in an opposite way the biology of adipose tissue, and consequently, have a variable impact on the whole body adiposity. This concept may be extensible to other cell types.
Assuntos
Tecido Adiposo/fisiologia , Autofagia/fisiologia , Adipogenia/fisiologia , Adiposidade/fisiologia , Animais , Diferenciação Celular/fisiologia , Humanos , Corpos Multivesiculares/fisiologiaRESUMO
Inter-cellular and inter-kingdom signaling systems of various levels of complexity regulate pathogenic and mutualistic interactions between bacteria, parasites, and fungi and animal and plant hosts. Inter-kingdom interactions between mutualistic bacteria such as rhizobia and legumes during nodulation and between fungi and plants during mycorrhizal associations, are characterized by the extensive exchange of molecular signals, which allow nitrogen and phosphate assimilation, respectively. A novel aspect of this signaling exchange is the existence of specific structures, the exosomes, that carry important molecules that shape the plant-pathogen interactions. Exosomes contain a wide array of molecules, such as lipids, proteins, messenger RNA, and microRNAs, that play important roles in cell-to-cell communication in animal and plant cells by affecting gene expression and other physiological activity in distant cells within the same organism (e.g., during cancer metastases and neuron injuries). In plant cells, it has been recently reported that exosomes go beyond organism boundaries and inhibit a pathogenic interaction in plants. Plant produce and send exosomes loaded with specific small miRNA which inhibit the pathogen infection, but the pathogen can also produce exosomes carrying pro-pathogenic proteins and microRNAs. Therefore, exosomes are the important bridge regulating the signal exchange. Exosomes are small membrane-bound vesicles derived from multivesicular bodies (MVBs), which carries selected cargos from the cytoplasm (protein, lipids, and microRNAs) and under certain circumstances, they fuse with the plasma membrane, releasing the small vesicles as cargo-carrying exosomes into the extracellular space during intercellular and inter-kingdom communication. Animal and plant proteomic studies have demonstrated that tetraspanin proteins are an integral part of exosome membranes, positioning tetraspanins as essential components for endosome organization, with key roles in membrane fusion, cell trafficking, and membrane recognition. We discuss the similarities and differences between animal tetraspanins and plant tetraspanins formed during plant-microbe interactions and their potential role in mutualistic communication.
Assuntos
Comunicação Celular/fisiologia , Exossomos , Simbiose/fisiologia , Tetraspaninas , Animais , Transporte Biológico , Exossomos/fisiologia , Humanos , MicroRNAs/metabolismo , Corpos Multivesiculares/fisiologia , Micorrizas/genética , Micorrizas/metabolismo , Células Vegetais/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Tetraspaninas/química , Tetraspaninas/genética , Tetraspaninas/fisiologiaRESUMO
Exosomes and ectosomes, two distinct types of extracellular vesicles generated by all types of cell, play key roles in intercellular communication. The formation of these vesicles depends on local microdomains assembled in endocytic membranes for exosomes and in the plasma membrane for ectosomes. These microdomains govern the accumulation of proteins and various types of RNA associated with their cytosolic surface, followed by membrane budding inward for exosome precursors and outward for ectosomes. A fraction of endocytic cisternae filled with vesicles - multivesicular bodies - are later destined to undergo regulated exocytosis, leading to the extracellular release of exosomes. In contrast, the regulated release of ectosomes follows promptly after their generation. These two types of vesicle differ in size - 50-150 nm for exosomes and 100-500 nm for ectosomes - and in the mechanisms of assembly, composition, and regulation of release, albeit only partially. For both exosomes and ectosomes, the surface and luminal cargoes are heterogeneous when comparing vesicles released by different cell types or by single cells in different functional states. Upon release, the two types of vesicle navigate through extracellular fluid for varying times and distances. Subsequently, they interact with recognized target cells and undergo fusion with endocytic or plasma membranes, followed by integration of vesicle membranes into their fusion membranes and discharge of luminal cargoes into the cytosol, resulting in changes to cellular physiology. After fusion, exosome/ectosome components can be reassembled in new vesicles that are then recycled to other cells, activating effector networks. Extracellular vesicles also play critical roles in brain and heart diseases and in cancer, and are useful as biomarkers and in the development of innovative therapeutic approaches.
Assuntos
Comunicação Celular/fisiologia , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Animais , Membrana Celular/metabolismo , Exocitose , Exossomos/fisiologia , Vesículas Extracelulares/fisiologia , Humanos , Fusão de Membrana/fisiologia , Corpos Multivesiculares/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.
Assuntos
Membrana Celular/fisiologia , Fusão de Membrana/fisiologia , Corpos Multivesiculares/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Comunicação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Células HCT116 , Células HeLa , Histamina/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Fusão de Membrana/efeitos dos fármacos , Corpos Multivesiculares/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Histamínicos H1/efeitos dos fármacos , Análise de Célula Única , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
Loss-of-function mutations in human endosomal Na+(K+)/H+ exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function.
Assuntos
Corpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Transporte Biológico , Endocitose/fisiologia , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Corpos Multivesiculares/fisiologia , Antiportadores de Potássio-Hidrogênio/metabolismo , Transporte Proteico , Proteólise , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment ß (C99), generated by cleavage of APP by ß-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.-González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Autofagossomos/fisiologia , Lisossomos/fisiologia , Corpos Multivesiculares/fisiologia , Precursor de Proteína beta-Amiloide/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Humanos , Naftiridinas/farmacologia , Neuroglia , RNA Interferente Pequeno , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The endoplasmic reticulum (ER) plays a pivotal role in maintaining cellular homeostasis. However, numerous environmental and genetic factors give rise to ER stress by inducing an accumulation of unfolded proteins. Under ER stress conditions, cells initiate the unfolded protein response (UPR). Here, we demonstrate a novel aspect of the UPR by electron microscopy and immunostaining analyses, whereby multivesicular body (MVB) formation was enhanced after ER stress. This MVB formation was influenced by inhibition of ER stress transducers inositol required enzyme 1 (IRE1) and PKR-like ER kinase (PERK). Furthermore, exosome release was also increased during ER stress. However, in IRE1 or PERK deficient cells, exosome release was not upregulated, indicating that IRE1- and PERK-mediated pathways are involved in ER stress-dependent exosome release.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Exossomos/metabolismo , Corpos Multivesiculares/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Células HeLa , Humanos , Transdução de SinaisRESUMO
Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells.
Assuntos
Envelhecimento/fisiologia , Membrana Celular/fisiologia , Exossomos/fisiologia , Lisossomos/fisiologia , Doenças Neurodegenerativas/metabolismo , Transporte Proteico/fisiologia , Autofagia/fisiologia , Humanos , Corpos Multivesiculares/fisiologiaRESUMO
Eukaryotes possess an elaborate endomembrane system with endoplasmic reticulum, nucleus, Golgi, lysosomes, peroxisomes, autophagosomes, and dynamic vesicle traffic. Theories addressing the evolutionary origin of eukaryotic endomembranes have overlooked the outer membrane vesicles (OMVs) that bacteria, archaea, and mitochondria secrete into their surroundings. We propose that the eukaryotic endomembrane system originated from bacterial OMVs released by the mitochondrial ancestor within the cytosol of its archaeal host at eukaryote origin. Confined within the host's cytosol, OMVs accumulated naturally, fusing either with each other or with the host's plasma membrane. This matched the host's archaeal secretory pathway for cotranslational protein insertion with outward bound mitochondrial-derived vesicles consisting of bacterial lipids, forging a primordial, secretory endoplasmic reticulum as the cornerstone of the eukaryotic endomembrane system. VIDEO ABSTRACT.
Assuntos
Evolução Biológica , Retículo Endoplasmático/fisiologia , Eucariotos/fisiologia , Complexo de Golgi/fisiologia , Membranas Mitocondriais/fisiologia , Corpos Multivesiculares/fisiologia , Archaea/fisiologia , Bactérias , Células Eucarióticas/citologia , Mitocôndrias/fisiologia , Membrana Nuclear/fisiologiaRESUMO
This brief overview of endocytic trafficking is written in honor of Renate Fuchs, who retires this year. In the mid-1980s, Renate pioneered studies on the ion-conducting properties of the recently discovered early and late endosomes and the mechanisms governing endosomal acidification. As described in this review, after uptake through one of many mechanistically distinct endocytic pathways, internalized proteins merge into a common early/sorting endosome. From there they again diverge along distinct sorting pathways, back to the cell surface, on to the trans-Golgi network or across polarized cells. Other transmembrane receptors are packaged into intraluminal vesicles of late endosomes/multivesicular bodies that eventually fuse with and deliver their content to lysosomes for degradation. Endosomal acidification, in part, determines sorting along this pathway. We describe other sorting machinery and mechanisms, as well as the rab proteins and phosphatidylinositol lipids that serve to dynamically define membrane compartments along the endocytic pathway.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Corpos Multivesiculares/fisiologia , Animais , Clatrina/fisiologia , HumanosRESUMO
Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.
Assuntos
Compartimento Celular/fisiologia , Endossomos/metabolismo , Corpos de Inclusão Viral/metabolismo , Marburgvirus/crescimento & desenvolvimento , Corpos Multivesiculares/fisiologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/virologia , Doença do Vírus de Marburg/virologia , Nucleocapsídeo/biossíntese , Nucleocapsídeo/metabolismo , Nucleoproteínas/metabolismo , Transporte Proteico , Pseudópodes/metabolismo , Fatores de Transcrição/metabolismo , Liberação de Vírus/fisiologia , Replicação Viral/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
UNLABELLED: Autophagy is a central mechanism by which hepatocytes catabolize lipid droplets (LDs). Currently, the regulatory mechanisms that control this important process are poorly defined. The small guanosine triphosphatase (GTPase) Rab7 has been implicated in the late endocytic pathway and is known to associate with LDs, although its role in LD breakdown has not been tested. In this study, we demonstrate that Rab7 is indispensable for LD breakdown ("lipophagy") in hepatocytes subjected to nutrient deprivation. Importantly, Rab7 is dramatically activated in cells placed under nutrient stress; this activation is required for the trafficking of both multivesicular bodies and lysosomes to the LD surface during lipophagy, resulting in the formation of a lipophagic "synapse." Depletion of Rab7 leads to gross morphological changes of multivesicular bodies, lysosomes, and autophagosomes, consequently leading to attenuation of hepatocellular lipophagy. CONCLUSION: These findings provide additional support for the role of autophagy in hepatocellular LD catabolism while implicating the small GTPase Rab7 as a key regulatory component of this essential process.
Assuntos
Autofagia , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Humanos , Lisossomos/fisiologia , Corpos Multivesiculares/fisiologia , proteínas de unión al GTP Rab7RESUMO
During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismo , Vacúolos/fisiologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Ebola virus is an enveloped virus with filamentous structure and causes a severe hemorrhagic fever in human and nonhuman primates. Host cell entry is the first essential step in the viral life cycle, which has been extensively studied as one of the therapeutic targets. A virus factor of cell entry is a surface glycoprotein (GP), which is an only essential viral protein in the step, as well as the unique particle structure. The virus also interacts with a lot of host factors to successfully enter host cells. Ebola virus at first binds to cell surface proteins and internalizes into cells, followed by trafficking through endosomal vesicles to intracellular acidic compartments. There, host proteases process GPs, which can interact with an intracellular receptor. Then, under an appropriate circumstance, viral and endosomal membranes are fused, which is enhanced by major structural changes of GPs, to complete host cell entry. Recently the basic research of Ebola virus infection mechanism has markedly progressed, largely contributed by identification of host factors and detailed structural analyses of GPs. This article highlights the mechanism of Ebola virus host cell entry, including recent findings.
Assuntos
Células/virologia , Ebolavirus/fisiologia , Ebolavirus/patogenicidade , Interações Hospedeiro-Patógeno , Glicoproteínas de Membrana/fisiologia , Proteínas Virais/fisiologia , Internalização do Vírus , Animais , Fenômenos Fisiológicos Celulares , Ebolavirus/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Corpos Multivesiculares/fisiologia , Corpos Multivesiculares/virologia , Peptídeo Hidrolases/fisiologia , Receptores Virais/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Ligação Viral , Replicação ViralRESUMO
The endosomal sorting complex required for transport (ESCRT) protein complexes function at the endosome in the formation of intraluminal vesicles (ILVs) containing cargo proteins destined for the vacuolar/lysosomal lumen. The early ESCRTs (ESCRT-0 and -I) are likely involved in cargo sorting, whereas ESCRT-III and Vps4 function to sever the neck of the forming ILVs. ESCRT-II links these functions by initiating ESCRT-III formation in an ESCRT-I-regulated manner. We identify a constitutively active mutant of ESCRT-II that partially suppresses the phenotype of an ESCRT-I or ESCRT-0 deletion strain, suggesting that these early ESCRTs are not essential and have redundant functions. However, the ESCRT-III/Vps4 system alone is not sufficient for ILV formation but requires cargo sorting mediated by one of the early ESCRTs.