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1.
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
2.
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
3.
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
4.
Microscopy (Oxf) ; 68(3): 243-253, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30860257

RESUMO

This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 µm or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus.


Assuntos
Arilsulfatases/metabolismo , Complexo de Golgi/ultraestrutura , NAD/química , Osteoclastos/ultraestrutura , Pirofosfatases/metabolismo , Animais , Retículo Endoplasmático Rugoso/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Membrana Nuclear/metabolismo , Osmio/química , Ratos , Ratos Wistar
5.
Nat Commun ; 10(1): 735, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760704

RESUMO

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.


Assuntos
Autofagia , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Receptores de Peptídeos/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Transporte Proteico , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
6.
PLoS One ; 14(2): e0211408, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30759123

RESUMO

Cell motility is critical to biological processes from wound healing to cancer metastasis to embryonic development. The involvement of organelles in cell motility is well established, but the role of organelle positional reorganization in cell motility remains poorly understood. Here we present an automated image analysis technique for tracking the shape and motion of Golgi bodies and cell nuclei. We quantify the relationship between nuclear orientation and the orientation of the Golgi body relative to the nucleus before, during, and after exposure of mouse fibroblasts to a controlled change in cell substrate topography, from flat to wrinkles, designed to trigger polarized motility. We find that the cells alter their mean nuclei orientation, in terms of the nuclear major axis, to increasingly align with the wrinkle direction once the wrinkles form on the substrate surface. This change in alignment occurs within 8 hours of completion of the topographical transition. In contrast, the position of the Golgi body relative to the nucleus remains aligned with the pre-programmed wrinkle direction, regardless of whether it has been fully established. These findings indicate that intracellular positioning of the Golgi body precedes nuclear reorientation during mouse fibroblast directed migration on patterned substrates. We further show that both processes are Rho-associated kinase (ROCK) mediated as they are abolished by pharmacologic ROCK inhibition whereas mouse fibroblast motility is unaffected. The automated image analysis technique introduced could be broadly employed in the study of polarization and other cellular processes in diverse cell types and micro-environments. In addition, having found that the nuclei Golgi vector may be a more sensitive indicator of substrate features than the nuclei orientation, we anticipate the nuclei Golgi vector to be a useful metric for researchers studying the dynamics of cell polarity in response to different micro-environments.


Assuntos
Movimento Celular , Núcleo Celular/ultraestrutura , Complexo de Golgi/ultraestrutura , Imagem com Lapso de Tempo/métodos , Animais , Polaridade Celular , Células Cultivadas , Fibroblastos , Camundongos
7.
J Cell Sci ; 132(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30709970

RESUMO

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi.


Assuntos
Autoantígenos/genética , Centrossomo/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Septinas/genética , Células 3T3-L1 , Animais , Autoantígenos/metabolismo , Transporte Biológico , Compartimento Celular , Linhagem Celular , Centrossomo/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Complexo de Golgi/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Células Jurkat/metabolismo , Células Jurkat/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Septinas/antagonistas & inibidores , Septinas/metabolismo , Transdução de Sinais
8.
J Cell Biol ; 218(3): 1055-1065, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659100

RESUMO

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lipídeos de Membrana/metabolismo , Receptores de Esteroides/metabolismo , Motivos de Aminoácidos , Transporte Biológico Ativo/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Lipídeos de Membrana/genética , Microscopia Eletrônica , Receptores de Esteroides/genética
9.
Cells ; 8(1)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646582

RESUMO

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.


Assuntos
Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Anticorpos de Domínio Único/imunologia , Animais , Células COS , Cromatina/química , Cromatina/ultraestrutura , DNA/química , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Proteínas Luminescentes/imunologia , Mitocôndrias/química , Mitocôndrias/ultraestrutura
10.
Fungal Genet Biol ; 123: 78-86, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30550852

RESUMO

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.


Assuntos
Aspergillus nidulans/ultraestrutura , Complexo I de Proteína do Envoltório/química , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Aspergillus nidulans/química , Aspergillus nidulans/genética , Transporte Biológico/genética , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/química , Complexo de Golgi/química , Microscopia de Fluorescência , Mutação , Fenótipo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética
11.
Mol Biol Cell ; 30(4): 478-490, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30566031

RESUMO

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.


Assuntos
Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Fusão de Membrana/efeitos dos fármacos , Nocodazol/farmacologia , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
12.
Biochem Biophys Res Commun ; 509(1): 222-226, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30587338

RESUMO

Condylar cartilage is a joint cartilage essential for smooth jaw movement. The importance of ciliary proteins in condylar cartilage development has been reported. However, little is known about how ciliary proteins control the homeostasis of condylar cartilage. Here we show that intraflagellar transport 20 (IFT20), a ciliary protein, is required for the maintenance of cartilaginous matrix in condylar cartilage. Utilizing NG2-CreER mice expressed in condylar cartilage, we deleted Ift20 by tamoxifen treatment at juvenile-to-adult stages. In wild-type condylar cartilage, IFT20 was robustly produced in the cis-Golgi, but deletion of Ift20 by tamoxifen induction of NG2-CreER (Ift20:NG2-CreER) resulted in reduced cell proliferation and decreased Golgi size in condylar cartilage. Importantly, while the primary cilia were present in cartilage cells in the condylar layers of wild-type mice, no primary cilia were present in the Ift20:NG2-CreER condylar layers. Consistent with this finding, ciliary-mediated Hedgehog signaling was severely attenuated in Ift20 mutant chondrocytes, and thus the production levels of type X collagen were significantly reduced in Ift20:NG2-CreER mice. These results suggest that IFT20 is required for Golgi size and Hedgehog signaling to maintain cartilaginous matrix in condylar cartilage. Our study highlights the unique function of IFT20 in the homeostasis of condylar cartilage.


Assuntos
Proteínas de Transporte/metabolismo , Cartilagem Articular/metabolismo , Proteínas Hedgehog/metabolismo , Animais , Proteínas de Transporte/genética , Cartilagem Articular/ultraestrutura , Linhagem Celular , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Deleção de Genes , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Camundongos Knockout , Transdução de Sinais
13.
JCI Insight ; 3(23)2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30518689

RESUMO

Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.


Assuntos
Acondroplasia/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Acondroplasia/patologia , Transporte Biológico Ativo/genética , Proliferação de Células , Sobrevivência Celular , Colesterol/análise , Proteínas do Citoesqueleto , Retículo Endoplasmático/ultraestrutura , Feminino , Feto , Fibroblastos/patologia , Doenças Genéticas Inatas/genética , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Humanos , Mutação , Linhagem , Fenótipo , Análise de Sequência de Proteína , Esteróis/análise
14.
Cell Death Dis ; 9(12): 1170, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518913

RESUMO

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a key role in biosynthesis of vitamin K2 and coenzyme Q10 using geranylgeranyl diphosphate (GGPP). However, the mechanism by which UBIAD1 participates in tumorigenesis remains unknown. This study show that UBIAD1 interacts with H-Ras, retains H-Ras in the Golgi apparatus, prevents H-Ras trafficking from the Golgi apparatus to the plasma membrane, blocks the aberrant activation of Ras/MAPK signaling, and inhibits the proliferation of bladder cancer cells. In addition, GGPP was required to maintain the function of UBIAD1 in regulating the Ras/ERK signaling pathway. A Drosophila model was employed to confirm the function of UBIAD1/HEIX in vivo. The activation of Ras/ERK signaling at the plasma membrane induced melanotic masses in Drosophila larvae. Our study suggests that UBIAD1 serves as a tumor suppressor in cancer and tentatively reveals the underlying mechanism of melanotic mass formation in Drosophila.


Assuntos
Dimetilaliltranstransferase/genética , Proteínas de Drosophila/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Genes Reporter , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Larva/citologia , Larva/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Fosfatos de Poli-Isoprenil/farmacologia , Ligação Proteica , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Transfecção
15.
Elife ; 72018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30499774

RESUMO

It is unclear how the two principal functions of the Golgi complex, processing and transport, are spatially organized. Studying such spatial organization by optical imaging is challenging, partially due to the dense packing of stochastically oriented Golgi stacks. Using super-resolution microscopy and markers such as Giantin, we developed a method to identify en face and side views of individual nocodazole-induced Golgi mini-stacks. Our imaging uncovered that Golgi enzymes preferentially localize to the cisternal interior, appearing as a central disk or inner-ring, whereas components of the trafficking machinery reside at the periphery of the stack, including the cisternal rim. Interestingly, conventional secretory cargos appeared at the cisternal interior during their intra-Golgi trafficking and transiently localized to the cisternal rim before exiting the Golgi. In contrast, bulky cargos were found only at the rim. Our study therefore directly demonstrates the spatial separation of processing and transport functions within the Golgi complex.


Assuntos
Transporte Biológico , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/química , Transporte Proteico/efeitos dos fármacos , Complexo de Golgi/química , Células HeLa , Humanos , Nocodazol/farmacologia
16.
Nat Microbiol ; 3(12): 1377-1384, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30397340

RESUMO

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.


Assuntos
Acetiltransferases/genética , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Enzimas Desubiquitinantes/química , Complexo de Golgi/metabolismo , Processamento de Proteína Pós-Traducional , Células A549 , Acetilação , Acetiltransferases/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Enzimas Desubiquitinantes/genética , Regulação Bacteriana da Expressão Gênica , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Células Vero
17.
J Alzheimers Dis ; 65(4): 1185-1207, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30124450

RESUMO

Abnormal fibrillary aggregation of tau protein is a pathological condition observed in Alzheimer's disease and other tauopathies; however, the presence and pathological significance of early non-fibrillary aggregates of tau remain under investigation. In cell and animal models expressing normal or modified tau, toxic effects altering the structure and function of several membranous organelles have also been reported in the absence of fibrillary structures; however, how these abnormalities are produced is an issue yet to be addressed. In order to obtain more insights into the mechanisms by which tau may disturb intracellular membranous elements, we transiently overexpressed human full-length tau and several truncated tau variants in cultured neuroblastoma cells. After 48 h of transfection, either full-length or truncated tau forms produced significant fragmentation of the Golgi apparatus (GA) with no changes in cell viability. Noteworthy is that in the majority of cells exhibiting dispersion of the GA, a ring-shaped array of cortical or perinuclear microtubule (Mt) bundles was also generated under the expression of either variant of tau. In contrast, Taxol treatment of non-transfected cells increased the amount of Mt bundles but not sufficiently to produce fragmentation of the GA. Tau-induced ring-shaped Mt bundles appeared to be well-organized and stable structures because they were resistant to Nocodazole post-treatment and displayed a high level of tubulin acetylation. These results further indicate that a mechanical force generated by tau-induced Mt-bundling may be responsible for Golgi fragmentation and that the repeated domain region of tau may be the main promoter of this effect.


Assuntos
Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microtúbulos/metabolismo , Neuroblastoma/ultraestrutura , Proteínas tau/metabolismo , Brefeldina A/farmacologia , Metabolismo dos Carboidratos/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação/genética , Neuroblastoma/patologia , Nocodazol/farmacologia , Compostos Orgânicos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transfecção , Proteínas tau/genética
18.
J Cell Physiol ; 233(12): 9145-9158, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29968908

RESUMO

Ultrastructural changes on the apical surface of the luminal epithelium of the uterus are known as pinopodes. Their morphology in species and in special species is associated with different results about size, duration, and percentage of surface area covered by pinopodes. The content of pinopodes is different in rodents and humans. In mice and rats pinopodes have many vacuoles and no organelle that extends to the actin stalk above the microvilli. Human pinopodes do not have a large vacuole and contain the golgi complex, a rough endoplasmic reticulum, secretory vesicles, and mitochondria that extend from the entire cell surface. It has been suggested that pinopodes are good markers of endometrial receptivity and implantation window. There are several molecular markers related to the presence of pinopodes, including integrins, leukemia inhibiting factor (LIF), l-selectin, HOXA10, glutaredoxin, glycodelinA, heparin-binding epidermal growth factor, mucins, and microRNAs (miRNAs). Multiple lines of evidence have indicated that miRNAs could affect the expression of LIF and pinopodes in the endometrium and these molecules play key roles in implantation window processes. Here, we have summarized the morphology and function of pinopodes. Moreover, we have highlighted several molecules in relation to pinopodes that could be used as biomarkers.


Assuntos
Biomarcadores/metabolismo , Epitélio/ultraestrutura , MicroRNAs/genética , Útero/ultraestrutura , Animais , Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Rugoso/ultraestrutura , Epitélio/metabolismo , Feminino , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Proteínas de Homeodomínio/genética , Humanos , Selectina L/genética , Fator Inibidor de Leucemia/genética , Camundongos , Ratos , Útero/metabolismo
19.
Autophagy ; 14(10): 1761-1778, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29969945

RESUMO

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl- channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.


Assuntos
Autofagia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Proteínas rab de Ligação ao GTP/metabolismo
20.
Nat Commun ; 9(1): 2816, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026494

RESUMO

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here, we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via sequential elongation and expansion processes. This starts with a synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial-cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.


Assuntos
Cartilagem/metabolismo , Esôfago/metabolismo , Morfogênese/genética , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Traqueia/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular , Polaridade Celular , Embrião de Mamíferos , Esôfago/citologia , Esôfago/crescimento & desenvolvimento , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso/citologia , Miócitos de Músculo Liso/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Traqueia/citologia , Traqueia/crescimento & desenvolvimento , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
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