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1.
Nat Commun ; 12(1): 2034, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795673

RESUMO

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mapas de Interação de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
2.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
3.
Nat Commun ; 12(1): 614, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504762

RESUMO

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.


Assuntos
Isocitrato Desidrogenase/genética , Mutação/genética , Organelas/metabolismo , Fosfolipídeos/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Glioblastoma/patologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Modelos Biológicos , Oligodendroglioma/patologia , Estearoil-CoA Dessaturase/metabolismo
4.
J Neurochem ; 157(3): 450-466, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33259669

RESUMO

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.


Assuntos
Transporte Biológico/genética , Proteínas Munc18/deficiência , Proteínas Munc18/genética , Animais , Morte Celular , Membrana Celular/metabolismo , Células Cultivadas , Toxina da Cólera/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/patologia , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Neurônios/ultraestrutura , Proteínas SNARE/deficiência , Proteínas SNARE/genética
5.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33301566

RESUMO

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.


Assuntos
Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteólise , Brefeldina A/farmacologia , Linhagem Celular , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Ácidos Indolacéticos/farmacologia , Interfase/efeitos dos fármacos , Nocodazol/farmacologia , Proteólise/efeitos dos fármacos
6.
Methods Mol Biol ; 2217: 301-311, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215388

RESUMO

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5ß1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/genética , Integrina alfa5beta1/genética , Junções Íntimas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico , Polaridade Celular , Cultura em Câmaras de Difusão , Células Endoteliais/ultraestrutura , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Integrina alfa5beta1/metabolismo , Microscopia de Fluorescência/métodos , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Junções Íntimas/ultraestrutura
7.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374830

RESUMO

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors SNAREs was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome compositionofTKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Membrana Celular/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico
8.
Sci Rep ; 10(1): 16604, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024151

RESUMO

Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A', that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A' isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A' act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.


Assuntos
Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Isoenzimas/metabolismo , Microscopia Eletrônica
9.
Exp Cell Res ; 396(2): 112324, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065114

RESUMO

In previous work, we showed that highly proliferative cells and cancer cells, but not cells with normal growth rate, have tubules rich in alpha-1,2 fucosylated epitopes that extend radially from the nucleus to the cell periphery and form an unusual uptake system. The importance of alpha-1,2 fucosylation in forming tubules was demonstrated by proving that down-regulating the two corresponding fucosyltransferases (FUT1 and FUT2) causes tubule fragmentation. Here, we present evidence that in the prostate cancer cell line DU145, the tubules arise in actively growing cells from vesicles in the medial and trans elements of a partially fragmented Golgi complex, while in not actively growing cells the tubules become completely independent from the Golgi complex. Formation and elongation of the tubules proved to depend on the actin cytoskeleton, since the alpha-1,2 fucosylated protein(s) segregate with the cytoskeleton proteins, and not in the membrane fraction, as do the Golgi markers and other fucosylated proteins, while depolymerization of the actin filaments causes tubule fragmentation and shifting of the alpha-1,2 fucosylated proteins into the membrane fraction.


Assuntos
Actinas/metabolismo , Fucose/metabolismo , Complexo de Golgi/metabolismo , Neoplasias da Próstata/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Citocalasinas/farmacologia , Epitopos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Lectinas/metabolismo , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/ultraestrutura
10.
Int J Biol Macromol ; 164: 3632-3644, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871120

RESUMO

Golgi Reassembly and Stacking Proteins (GRASPs) were firstly described as crucial elements in determining the structure of the Golgi complex. However, data have been accumulating over the years showing GRASPs can participate in various cell processes beyond the Golgi maintenance, including cell adhesion and migration, autophagy and unconventional secretion of proteins. A comprehensive understanding of the GRASP functions requires deep mechanistic knowledge of its structure and dynamics, especially because of the unique structural plasticity observed for many members of this family coupled with their high promiscuity in mediating protein-protein interactions. Here, we critically review data regarding the structural biophysics of GRASPs in the quest for understanding the structural determinants of different functionalities. We dissect GRASP structure starting with the full-length protein down to its separate domains (PDZ1, PDZ2 and SPR) and outline some structural features common to all members of the GRASP family (such as the presence of many intrinsically disordered regions). Although the impact of those exquisite properties in vivo will still require further studies, it is possible, from our review, to pinpoint factors that must be considered in future interpretation of data regarding GRASP functions, thus bringing somewhat new perspectives to the field.


Assuntos
Biofísica , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/ultraestrutura , Conformação Proteica , Cristalografia por Raios X , Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura
11.
Nat Chem Biol ; 16(12): 1361-1367, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32958953

RESUMO

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.


Assuntos
Autofagossomos/metabolismo , Azidas/química , Colina/análogos & derivados , Retículo Endoplasmático/metabolismo , Fosfatidilcolinas/metabolismo , Coloração e Rotulagem/métodos , Autofagossomos/ultraestrutura , Transporte Biológico , Carbocianinas/metabolismo , Química Click/métodos , Retículo Endoplasmático/ultraestrutura , Corantes Fluorescentes/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Imagem Molecular/métodos , Fosfatidilcolinas/química , Rodamina 123/metabolismo
12.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 319(4): G432-G442, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32755306

RESUMO

Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes.NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.


Assuntos
Apresentação do Antígeno/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fígado/virologia , Acetaldeído , Estresse do Retículo Endoplasmático/genética , Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Antígeno HLA-A2/análise , Células Hep G2 , Vírus da Hepatite B/genética , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Fígado/imunologia , RNA Mensageiro/análise , Transfecção , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética
14.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751072

RESUMO

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental, or stress signals. Sorting of transporters from their site of synthesis, the endoplasmic reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the multivesicular bodies (MVB)/lysosomes/vacuole system. In specific cases, internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review, we present evidence that shows that transporter traffic to the PM takes place through Golgi bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale of why transporters and possibly other housekeeping membrane proteins 'avoid' routes of polar trafficking.


Assuntos
Aspergillus nidulans/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Aspergillus nidulans/genética , Membrana Celular/ultraestrutura , Endocitose/genética , Retículo Endoplasmático/ultraestrutura , Endossomos/metabolismo , Endossomos/ultraestrutura , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Complexo de Golgi/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Via Secretória/genética , Vacúolos/metabolismo , Vacúolos/ultraestrutura
15.
FEBS Lett ; 594(19): 3086-3094, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32668013

RESUMO

The Golgi is surrounded by a ribosome-excluding matrix. Recently, we reported that the cis-Golgi-localized golgin GM130 can phase-separate to form dynamic, liquid-like condensates in vitro and in vivo. Here, we show that the overexpression of each of the remaining cis (golgin160, GMAP210)- and trans (golgin97, golgin245, GCC88, GCC185)-golgins results in novel protein condensates. Focused ion beam scanning electron microscopy (FIB-SEM) images of GM130 condensates reveal a complex internal organization with branching aqueous channels. Pairs of golgins overexpressed in the same cell form distinct juxtaposed condensates. These findings support the hypothesis that, in addition to their established roles as vesicle tethers, phase separation may be a common feature of the golgin family that contributes to Golgi organization.


Assuntos
Autoantígenos/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Autoantígenos/química , Autoantígenos/ultraestrutura , Sobrevivência Celular , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Imagem com Lapso de Tempo , Rede trans-Golgi/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(30): 17820-17831, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661174

RESUMO

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (Pam Myh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam 0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by Pam Myh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by Pam Myh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.


Assuntos
Amidina-Liases/metabolismo , Grânulos Citoplasmáticos/metabolismo , Átrios do Coração/metabolismo , Oxigenases de Função Mista/metabolismo , Vesículas Secretórias/metabolismo , Amidina-Liases/genética , Animais , Fator Natriurético Atrial/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Knockout , Oxigenases de Função Mista/genética , Monócitos/metabolismo , Células Musculares/metabolismo , Vesículas Secretórias/ultraestrutura
17.
Sci Rep ; 10(1): 11314, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647231

RESUMO

Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.


Assuntos
Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Proteínas Luminescentes/química , Mitocôndrias/ultraestrutura , Tetróxido de Ósmio/química , Animais , Células COS , Chlorocebus aethiops , Fluorescência , Corantes Fluorescentes , Células HEK293 , Células HeLa , Humanos , Coloração e Rotulagem
18.
Biochem Biophys Res Commun ; 528(3): 447-452, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32499114

RESUMO

The conserved oligomeric Golgi (COG) complex, which consists of eight subunits named COG1-COG8, is highly conserved with homologous subunits present in most eukaryotic species. In yeast and mammalian, the COG complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Although homologs of COG subunits have been identified in Arabidopsis, the functions of the complex and its subunits remain to be fully elucidated. In this study, we have utilized genetic and cytologic approaches to characterize the role of the COG6 subunit. We showed that a mutation in COG6 caused male transmission defect due to aberrant pollen tube growth. At the subcellular level, Golgi bodies exhibited altered morphology in cog6 pollen and cell wall components were incorrectly deposited in pollen tubes. COG6 fused to green fluorescent protein (GFP), which complemented the aberrant growth of cog6 pollen tubes, was localized to the Golgi apparatus. We propose that COG6, as a subunit of the COG complex, modulates Golgi morphology and vesicle trafficking homeostasis during pollen tube growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Genes de Plantas , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica de Transmissão , Mutação , Plantas Geneticamente Modificadas , Tubo Polínico/genética
19.
J Cell Biol ; 219(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32406500

RESUMO

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/metabolismo , Via Secretória/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Imagem Óptica , Transporte Proteico , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
20.
Sci Adv ; 6(14): eaay9572, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32270040

RESUMO

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Ribossomos/metabolismo , Animais , Transporte Biológico , Microscopia Crioeletrônica , Vesículas Citoplasmáticas/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Imagem Molecular , Especificidade de Órgãos , Ratos , Ribossomos/ultraestrutura , Estresse Fisiológico
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