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1.
Science ; 367(6484): 1366-1371, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193326

RESUMO

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Fosfatos de Fosfatidilinositol/metabolismo , Rede trans-Golgi/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Dinaminas/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Microdomínios da Membrana , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Interferência de RNA
2.
Nat Commun ; 11(1): 507, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980612

RESUMO

The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aß) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aß burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Animais , Núcleo Celular/metabolismo , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/patologia , Simulação por Computador , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Feminino , Proteína HMGB1/líquido cefalorraquidiano , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Camundongos Transgênicos , Necrose , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Imagem com Lapso de Tempo
3.
Proc Natl Acad Sci U S A ; 117(2): 1069-1080, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882451

RESUMO

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.


Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteólise , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Microscopia Crioeletrônica , Citosol/metabolismo , Endopeptidases , Imagem Óptica , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Proteína com Valosina/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-30890442

RESUMO

The origin of the autophagosomal membrane started to be debated by scientists working in the field within one year of the modern definition of autophagy in 1963. There is now converging evidence from older and newer studies that the endoplasmic reticulum is involved in formation of autophagosomes. Thus, it is possible to trace from early morphological work - done without the benefit of molecular descriptions - to recent studies - dissecting how specific proteins nucleate autophagosome biogenesis - a long series of experimental findings that are beginning to answer the 55-year old question with some confidence. The view that has emerged is that specialised regions of the endoplasmic reticulum, in dynamic cross talk with most intracellular organelles via membrane contact sites, provide a platform for autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Autofagossomos/ultraestrutura , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Proteínas de Membrana/metabolismo
5.
Fluids Barriers CNS ; 16(1): 39, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31849332

RESUMO

BACKGROUND: A growing body of evidence suggests that the accumulation of amyloid-ß and tau (HPτ) in the brain of patients with the dementia subtype idiopathic normal pressure hydrocephalus (iNPH) is associated with delayed extravascular clearance of metabolic waste. Whether also clearance of intracellular debris is affected in these patients needs to be examined. Hypothetically, defective extra- and intra-cellular clearance of metabolites may be instrumental in the neurodegeneration and dementia characterizing iNPH. This study explores whether iNPH is associated with altered mitochondria phenotype in neurons and astrocytes. METHODS: Cortical brain biopsies of 9 reference (REF) individuals and 30 iNPH patients were analyzed for subcellular distribution and morphology of mitochondria using transmission electron microscopy. In neuronal soma of REF and iNPH patients, we identified normal, pathological and clustered mitochondria, mitochondria-endoplasmic reticulum contact sites and autophagic vacuoles. We also differentiated normal and pathological mitochondria in pre- and post-synaptic nerve terminals, as well as in astrocytic endfoot processes towards vessels. RESULTS: We found a high prevalence of pathological mitochondria in neuronal soma and pre- and post-synaptic terminals, as well as increased mitochondrial clustering, and altered number of mitochondria-endoplasmic reticulum contact sites in iNPH. Non-fused autophagic vacuoles were more abundant in neuronal soma of iNPH patients, suggestive of cellular clearance failure. Moreover, the length of postsynaptic densities was reduced in iNPH, potentially related to reduced synaptic activity. In astrocytic endfoot processes, we also found increased number, area and area fraction of pathological mitochondria in iNPH patients. The proportion of pathological mitochondria correlated significantly with increasing degree of astrogliosis and reduced perivascular expression of aquaporin-4 (AQP4), assessed by light microscopy immunohistochemistry. CONCLUSION: Our results provide evidence of mitochondrial pathology and signs of impaired cellular clearance in iNPH patients. The results indicate that iNPH is a neurodegenerative disease with close similarity to Alzheimer's disease.


Assuntos
Astrócitos/patologia , Encéfalo/patologia , Sistema Glinfático/patologia , Hidrocefalia de Pressão Normal/patologia , Mitocôndrias/patologia , Neurônios/patologia , Astrócitos/ultraestrutura , Autofagia , Encéfalo/ultraestrutura , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Sistema Glinfático/ultraestrutura , Humanos , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Sinapses/patologia , Sinapses/ultraestrutura
6.
Nat Commun ; 10(1): 5132, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723129

RESUMO

The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.


Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagossomos/metabolismo , Membrana Celular/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Arabidopsis/ultraestrutura , Autofagossomos/ultraestrutura , Autofagia , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Filogenia , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nat Cell Biol ; 21(10): 1234-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570833

RESUMO

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Nat Methods ; 16(12): 1226-1232, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570887

RESUMO

We present ilastik, an easy-to-use interactive tool that brings machine-learning-based (bio)image analysis to end users without substantial computational expertise. It contains pre-defined workflows for image segmentation, object classification, counting and tracking. Users adapt the workflows to the problem at hand by interactively providing sparse training annotations for a nonlinear classifier. ilastik can process data in up to five dimensions (3D, time and number of channels). Its computational back end runs operations on-demand wherever possible, allowing for interactive prediction on data larger than RAM. Once the classifiers are trained, ilastik workflows can be applied to new data from the command line without further user interaction. We describe all ilastik workflows in detail, including three case studies and a discussion on the expected performance.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Translocador Nuclear Receptor Aril Hidrocarboneto/fisiologia , Proliferação de Células , Colágeno/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos
9.
Nat Commun ; 10(1): 4399, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562315

RESUMO

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.


Assuntos
Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Células Cultivadas , Embrião de Mamíferos/citologia , Retículo Endoplasmático/ultraestrutura , Fibroblastos/citologia , Células HeLa , Humanos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Ligação Proteica , Ratos , Proteínas rho de Ligação ao GTP/genética
10.
Biomed Pharmacother ; 117: 109053, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176169

RESUMO

Chronic obstructive pulmonary disease (COPD) often causes diaphragm dysfunction, which is the main contributor to neuro-muscular respiratory failure and associated with the prognosis of COPD. However, the morphological changes in diaphragm during the development of COPD are complicated and underlying mechanisms have not been absolutely elucidated. Considering smoking is one of the most leading and important risk factors for development of COPD, we set out to investigate the effects of smoking on muscle fibre remodeling and underlying mechanisms in diaphragms. Rats were randomly exposed to cigarette smoke (CS) for one of three durations of 4, 8, and 12 weeks. CS exposure resulted in increased percentage of type I muscle fibres, enhanced apoptosis index, endoplasmic reticulum (ER) dilation, elevated expression of glucose-regulated protein 78, C/EBP homologous protein, caspase-12, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), cytochrome c oxidase 4, Sdhb, p53 and a reduction in fibre diameters in rat diaphragms. The results indicated that CS exposure induced a shift to muscle fibres with aerobic metabolism in predominance in rat diaphragms, which may be dependent on the regulation of PGC-1α and p53. Additionally, ER stress (ERS) associated apoptosis may contribute to the pathogenesis of diaphragmatic muscle atrophy induced by CS exposure.


Assuntos
Diafragma/patologia , Fibras Musculares Esqueléticas/patologia , Fumar/efeitos adversos , Animais , Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Enfisema/complicações , Enfisema/patologia , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Feminino , Mitocôndrias/metabolismo , Atrofia Muscular/patologia , Miosinas/metabolismo , Oxirredução , Pneumonia/complicações , Pneumonia/patologia , Ratos Sprague-Dawley , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo
11.
mBio ; 10(3)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186324

RESUMO

Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.


Assuntos
Retículo Endoplasmático/ultraestrutura , Enterovirus/fisiologia , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica de Varredura , Replicação Viral , Animais , Retículo Endoplasmático/virologia , Infecções por Enterovirus , Complexo de Golgi/virologia , Processamento de Imagem Assistida por Computador , Gotículas Lipídicas/ultraestrutura , Células Vero
12.
Nat Commun ; 10(1): 2370, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147549

RESUMO

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Forma das Organelas/genética , Autofagia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico/genética
13.
Methods Mol Biol ; 1992: 231-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148042

RESUMO

Optical tweezers allow for noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi)quantitative force measurements within a living cell can be performed. In this chapter, we provide practical tips for setting up such experiments paying special attention to the technical considerations for integrating optical tweezers into a confocal microscope. Next, we describe experimental approaches we have taken to trap intracellular structures in plant cells.


Assuntos
Microscopia Confocal/instrumentação , Pinças Ópticas , Células Vegetais/ultraestrutura , Plantas/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Desenho de Equipamento , Microscopia Confocal/métodos
14.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083507

RESUMO

Some single-stranded positive-sense RNA [ssRNA(+)] viruses, including Flavivirus, generate specific organelle-like structures in the host endoplasmic reticulum (ER). These structures are called virus replication organelles and consist of two distinct subdomains, the vesicle packets (VPs) and the convoluted membranes (CMs). The VPs are clusters of small vesicle compartments and are considered to be the site of viral genome replication. The CMs are electron-dense amorphous structures observed in proximity to the VPs, but the exact roles of CMs are mostly unknown. Several recent studies have revealed that flaviviruses recruit several host factors that are usually used for the biogenesis of other conventional organelles and usurp their function to generate virus replication organelles. In the current review, we summarize recent studies focusing on the role of host factors in the formation of virus replication organelles and discuss how these intricate membrane structures are organized.


Assuntos
Retículo Endoplasmático/metabolismo , Flavivirus/fisiologia , Biogênese de Organelas , RNA Viral/metabolismo , Replicação Viral/fisiologia , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Retículo Endoplasmático/ultraestrutura
15.
Cell Biol Int ; 43(8): 846-851, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31115951

RESUMO

Lelio Orci has made seminal contributions to our understanding of pancreatic islet structure and function. He introduced quantitative criteria to structural analysis in the study of endocrine pancreas in a series of works performed in collaboration with Albert Renold, Roger Unger, and Donald Steiner. Orci has moved islet cell morphology from the primitive era of histochemistry and electron microscopy into the modern era of cell biology, applying the most advanced techniques and covering every aspect of normal and pathological structure-function relationships. In collaboration with James Rothman in New York and Randy Schekman in Berkley, Orci discovered that the transport steps from the endoplasmic reticulum to the Golgi complex, and within the Golgi, are mediated by two sets of vesicles coated with protein envelopes different from clathrin.


Assuntos
Retículo Endoplasmático , Complexo de Golgi , Ilhotas Pancreáticas , Distinções e Prêmios , Transporte Biológico , Clatrina/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , História do Século XX , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Itália , Suíça
16.
Microsc Res Tech ; 82(8): 1339-1344, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31070847

RESUMO

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium-maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD-model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium-maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium-maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD-model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS.


Assuntos
Colo/citologia , Células Epiteliais/ultraestrutura , Doenças Inflamatórias Intestinais/patologia , Animais , Colo/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Células Epiteliais/patologia , Células Caliciformes/ultraestrutura , Complexo de Golgi/patologia , Complexo de Golgi/ultraestrutura , Doenças Inflamatórias Intestinais/induzido quimicamente , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mucinas , Ratos , Ácido Trinitrobenzenossulfônico/administração & dosagem
17.
Toxicol Lett ; 311: 37-48, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029751

RESUMO

Polybrominated diphenyl ether-153 (BDE-153) has been demonstrated to induce neuronal apoptosis in rat cerebral cortex and primary neurons, however, the roles of mitochondria and endoplasmic reticulum (ER) remain unclear in the BDE-153-induced neuronal apoptosis. To this purpose, we observed the mitochondria and ER ultrastructure changes in the neuronal apoptosis in rats following BDE-153 treatment, detected the mitochondrial membrane potential (MMP), Ca2+-Mg2+-ATP enzyme activity, and the changes of mitochondria and ER apoptosis related molecules in rat cerebral cortex and in primary neurons following BDE-153 treatment. Results showed that compared to the control group, neuronal apoptosis was significantly increased in a dose-dependent manner in rat cerebral cortex and in primary neurons following BDE-153 treatment. In comparison with control, BDE-153 treatment induced remarkable ultrastructural changes in ER rather than in mitochondria, and the severity of ER damage was worse with the increasing BDE-153 dose. Meanwhile, ER apoptosis related molecules including caspase-12 (at mRNA level), cleaved caspase-12 (at protein level), and Tmem132a (at mRNA and protein levels) were significantly increased in the cerebral cortex in rats following BDE-153 treatment, while procaspase-12 protein was significantly decreased, comparing with control. In contrast, mitochondria apoptosis related molecules (MMP, Ca2+-Mg2+-ATP enzyme activity, cyt-C protein, caspase-3, 8, 9 mRNA, caspase-8, 9 enzyme activities) did not significantly changed in the cerebral cortex of rats or in primary neurons following BDE-153 treatment, except for the elevated caspase-3 mRNA and enzyme activity. Therefore, we conclude that BDE-153 induced neuronal apoptosis was dependent on p53, and mediated more by ER than mitochondria in the cerebral cortex of rats and in primary neurons. The findings suggest that ER is a potential sensitive target of BDE-153 neurotoxicity, providing a scientific evidence for the mechanism and intervention study on PBDE's neurotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Degeneração Neural , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Bifenil Polibromatos/toxicidade , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
J Steroid Biochem Mol Biol ; 190: 234-241, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991093

RESUMO

Oxysterols, oxidized derivatives of cholesterol found in LDL and atherosclerotic plaques, trigger several biological responses involved in the initiation and progression of atherosclerosis. Endothelial dysfunction, which occurs when vascular homeostasis is altered, plays a key role in the pathogenesis of several metabolic diseases. The contribution of endoplasmic reticulum (ER) stress to endothelial disfunction is a relatively recent area of investigation. There is a well-established link between LDL oxidation and ER stress but the role played by specific products of lipid oxidation into this interaction is still to be defined. The present study shows that secosterol-B (SEC-B), 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ßcarboxaldehyde, a cholesterol autoxidation product recently identified in the atherosclerotic plaque, is able to induce ER stress in HUVEC cells, as revealed by significant expansion and change of structure. At low doses, i.e. 1 and 5 µM, cells try to cope with this stress by activating autophagy and the ubiquitin proteasome system in the attempt to restore ER function. However, at higher doses, i.e. 20 µM, cell apoptosis occurs in a pathway that involves early phosphorylation of eIF2α and NF-kB activation, suggesting that the adaptive program fails and the cell activates the apoptotic program. These findings provide additional insight about the role of oxysterols in endothelial dysfunction and its potential involvement in atherosclerotic pathophysiology.


Assuntos
Colesterol/análogos & derivados , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Apoptose , Autofagia , Colesterol/metabolismo , Colesterol/farmacologia , Retículo Endoplasmático/ultraestrutura , Células Endoteliais/ultraestrutura , Fator de Iniciação 2 em Eucariotos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , NF-kappa B/metabolismo
19.
Ultrastruct Pathol ; 43(2-3): 99-109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30966840

RESUMO

Hydrosalpinx is a disease commonly observed in women and characterized by the obstruction which is in the shape of a fluid-filled sac at the distal part of tuba uterina closed to the ovary. In this study, we aimed to obtain endometrial tissue samples from the hydrosalpinx patients, before and after the surgical treatment and compare these endometrial tissue samples by using light and electron microscope. Endometrial tissue samples were obtained from the 24 women with bilateral hydrosalpinx range 19-46 years before and after the surgical treatment, and normal endometrial tissues were collected from five women without hydrosalpinx and evaluated as a control group. In endometrial samples of hydrosalpinx patients; it was observed that large and unregulated interstitial spaces representing the organellar destruction, membranous whorl structures associated with organelle destruction, thinning in the surface epithelium, decreasing in numbers of microvillus and pinopodes in microvilli cells, increasing in heterochromatin and picnotic changes in the nucleus, expansion, and vacuolization in the endoplasmic reticulum cisternae in the apical cytoplasm and intraepithelial macrophages and lymphocytes were rised in number. Although mild structural changes were observed in endometrial tissues obtained after surgical treatment of hydrosalpinx, surface epithelium, glandular and stromal cell structures were more similar to control endometrial specimens. In conclusion; serious structural changes have occurred in endometrial tissues of hydrosalpinx patients. These structural abnormalities have removed after surgical treatment so it is considered that surgical treatment is effective in patients with hydrosalpinx.


Assuntos
Endométrio/anormalidades , Endométrio/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Microscopia Eletrônica , Adulto , Elétrons , Endométrio/cirurgia , Epitélio/patologia , Feminino , Humanos , Pessoa de Meia-Idade
20.
Mol Biol Cell ; 30(12): 1377-1389, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995177

RESUMO

The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitose , Sequência de Aminoácidos , Cromatina/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/química , Metáfase , Domínios Proteicos
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