RESUMO
The Golgi complex is a highly dynamic and tightly regulated cellular organelle with essential roles in the processing as well as the sorting of proteins and lipids. Its structure undergoes rapid disassembly and reassembly during normal physiological processes, including cell division, migration, polarization, differentiation, and cell death. Golgi dispersal or fragmentation also occurs in pathological conditions, such as neurodegenerative diseases, infectious diseases, congenital disorders of glycosylation diseases, and cancer. In this review, current knowledge about both structural organization and morphological alterations in the Golgi in physiological and pathological conditions is summarized together with the methodologies that help to reveal its structure.
Assuntos
Complexo de Golgi , Doenças Neurodegenerativas , Humanos , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Divisão Celular , Transporte Proteico , Doenças Neurodegenerativas/metabolismoRESUMO
Distal hereditary motor neuropathies (HMNs) and axonal Charcot-Marie-Tooth neuropathy (CMT2) are clinically and genetically heterogeneous diseases characterized primarily by motor neuron degeneration and distal weakness. The genetic cause for about half of the individuals affected by HMN/CMT2 remains unknown. Here, we report the identification of pathogenic variants in GBF1 (Golgi brefeldin A-resistant guanine nucleotide exchange factor 1) in four unrelated families with individuals affected by sporadic or dominant HMN/CMT2. Genomic sequencing analyses in seven affected individuals uncovered four distinct heterozygous GBF1 variants, two of which occurred de novo. Other known HMN/CMT2-implicated genes were excluded. Affected individuals show HMN/CMT2 with slowly progressive distal muscle weakness and musculoskeletal deformities. Electrophysiological studies confirmed axonal damage with chronic neurogenic changes. Three individuals had additional distal sensory loss. GBF1 encodes a guanine-nucleotide exchange factor that facilitates the activation of members of the ARF (ADP-ribosylation factor) family of small GTPases. GBF1 is mainly involved in the formation of coatomer protein complex (COPI) vesicles, maintenance and function of the Golgi apparatus, and mitochondria migration and positioning. We demonstrate that GBF1 is present in mouse spinal cord and muscle tissues and is particularly abundant in neuropathologically relevant sites, such as the motor neuron and the growth cone. Consistent with the described role of GBF1 in Golgi function and maintenance, we observed marked increase in Golgi fragmentation in primary fibroblasts derived from all affected individuals in this study. Our results not only reinforce the existing link between Golgi fragmentation and neurodegeneration but also demonstrate that pathogenic variants in GBF1 are associated with HMN/CMT2.
Assuntos
Axônios/metabolismo , Doença de Charcot-Marie-Tooth/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Debilidade Muscular/genética , Atrofia Muscular Espinal/genética , Anormalidades Musculoesqueléticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Axônios/patologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/patologia , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Heterozigoto , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Debilidade Muscular/diagnóstico , Debilidade Muscular/metabolismo , Debilidade Muscular/patologia , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Anormalidades Musculoesqueléticas/diagnóstico , Anormalidades Musculoesqueléticas/metabolismo , Anormalidades Musculoesqueléticas/patologia , Mutação , Linhagem , Cultura Primária de Células , Medula Espinal/anormalidades , Medula Espinal/metabolismoRESUMO
Organelles can easily be disrupted by intracellular and extracellular factors. Studies on ER and mitochondria indicate that a wide range of responses are elicited upon organelle disruption. One response thought to be of particular importance is autophagy. Cells can target entire organelles into autophagosomes for removal. This wholesale nature makes autophagy a robust means for eliminating compromised organelles. Recently, it was demonstrated that the Golgi apparatus is a substrate of autophagy. On the other hand, various reports have shown that components traffic away from the Golgi for elimination in an autophagosome-independent manner when the Golgi apparatus is stressed. Future studies will reveal how these different pieces of machinery coordinate to drive Golgi degradation. Quantitative measurements will be needed to determine how much autophagy contributes to the maintenance of the Golgi apparatus.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Autofagossomos/metabolismo , Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Controle de QualidadeRESUMO
BACKGROUND INFORMATION: Aberrant production of amyloid beta (Aß) causes disruption of intracellular calcium homeostasis, a crucial factor in the pathogenesis of Alzheimer's disease. Calcium is required for the fusion and trafficking of vesicles. Previously, we demonstrated that Sec31A, a main component for coat protein complex II (COPII) vesicles at ER exit sites (ERES), is modulated by O-GlcNAcylation. O-GlcNAcylation, a unique and dynamic protein glycosylation process, modulates the formation of COPII vesicles. RESULTS: In this study, we observed that disrupted calcium levels affected the formation of COPII vesicles in ERES through calcium-triggered O-GlcNAcylation of Sec31A. Additionally, we found that Aß impaired ERES through Aß-disturbed calcium homeostasis and O-GlcNAcylation of Sec31A in neuronal cells. Furthermore, we identified that Aß disrupted the ribbon-like structure of Golgi. Golgi fragmentation by Aß was rescued by up-regulation of O-GlcNAcylaion levels using Thiamet G (ThiG), an O-GlcNAcase inhibitor. Additionally, we observed that the Golgi reassembly stacking proteins having a function in Golgi stacking showed attenuation at COPII vesicles following Aß treatment. CONCLUSIONS: This study demonstrated that Aß impaired Sec31A targeting to ERES through altered Sec31A O-GlcNAcylation triggered by disruption of intracellular calcium homeostasis. SIGNIFICANCE: The findings of this study suggested that protection of ERES or Sec31 O-GlcNAcylation may offer a promising novel avenue for development of AD therapeutics.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Células HeLa , Humanos , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2T487I patient spinal cord tissues. Both the ER-Golgi intermediate (ERGIC) compartment and the Golgi become disorganised and fragmented. This activates ER stress and inhibits ER-associated degradation. Hence, this study highlights perturbations in secretory protein trafficking and ER homeostasis as pathogenic mechanisms associated with ALS/FTD-associated forms of UBQLN2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Estresse do Retículo Endoplasmático , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Transporte ProteicoRESUMO
Parkinson's disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α-σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies.
Assuntos
Doença de Parkinson/patologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
In vertebrates, individual Golgi stacks are joined into a compact ribbon structure; however, the relevance of a ribbon structure has been elusive. Here, we exploit the finding that the membrane tether of the trans-Golgi network, GCC88 (encoded by GCC1), regulates the balance between Golgi mini-stacks and the Golgi ribbon. Loss of Golgi ribbons in stable cells overexpressing GCC88 resulted in compromised mechanistic target of rapamycin (mTOR) signaling and a dramatic increase in LC3-II-positive autophagosomes, whereas RNAi-mediated depletion of GCC88 restored the Golgi ribbon and reduced autophagy. mTOR was absent from dispersed Golgi mini-stacks whereas recruitment of mTOR to lysosomes was unaffected. We show that the Golgi ribbon is a site for localization and activation of mTOR, a process dependent on the ribbon structure. We demonstrate a strict temporal sequence of fragmentation of Golgi ribbon, loss of Golgi mTOR and subsequent increased autophagy. Golgi ribbon fragmentation has been reported in various neurodegenerative diseases and we demonstrate the potential relevance of our findings in neuronal cells using a model of neurodegeneration. Overall, this study highlights a role for the Golgi ribbon in pathways central to cellular homeostasis.This article has an associated First Person interview with the first author of the paper.
Assuntos
Autofagia , Complexo de Golgi/metabolismo , Mamíferos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neuroblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Proteínas tau/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.
Assuntos
Autofagia , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Hepacivirus/fisiologia , Membranas Intracelulares/metabolismo , Replicação Viral/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP/genética , Complexo de Golgi/genética , Complexo de Golgi/virologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Membranas Intracelulares/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação/genéticaRESUMO
In contrast to the booming production and application of nanomaterials, research on the toxicological impacts and possible hazards of nanoparticles to tissues and organs is still in its infancy. Golgi apparatus is one of the most important organelles in cells and plays a key role in intracellular protein processing. The structural integrity of Golgi is vital for its normal function, and Golgi disturbance could result in a wide range of diseases and disorders. In this study, for the first time we found gold nanoparticles (Au NPs) induced size-dependent cytoplasmic calcium increase and Golgi fragmentation, which hampers normal Golgi functions, leads to abnormal protein processing, and causes cellular adhesion decrease, while cell viability was not significantly compromised. Additionally, early renal pathological changes were induced in vivo. This work is significant to nanoparticle research because it illustrates the important role of size on Au NP-induced changes in Golgi morphology and their consequences in vitro and in vivo, which has important implications for the biological applications of nanomaterials.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Ouro , Complexo de Golgi , Rim , Nanopartículas Metálicas/química , Animais , Adesão Celular/efeitos dos fármacos , Avaliação de Medicamentos , Feminino , Ouro/química , Ouro/farmacocinética , Ouro/farmacologia , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its pathophysiological cascade is still enigmatic. More than 90% of ALS patients suffer from sporadic ALS, which makes it specifically demanding to generate appropriate model systems. One interesting aspect considering the seeding, spreading and further disease development of ALS is the cerebrospinal fluid (CSF). We therefore asked whether CSF from sporadic ALS patients is capable of causing disease typical changes in human patient-derived spinal MN cultures and thus could represent a novel model system for sporadic ALS. By using induced pluripotent stem cell (iPSC)-derived MNs from healthy controls and monogenetic forms of ALS we could demonstrate a harmful effect of ALS-CSF on healthy donor-derived human MNs. Golgi fragmentation-a typical finding in lower organism models and human postmortem tissue-was induced solely by addition of ALS-CSF, but not control-CSF. No other neurodegenerative hallmarks-including pathological protein aggregation-were found, underpinning Golgi fragmentation as early event in the neurodegenerative cascade. Of note, these changes occurred predominantly in MNs, the cell type primarily affected in ALS. We thus present a novel way to model early features of sporadic ALS.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Complexo de Golgi/patologia , Neurônios Motores/patologia , Agregação Patológica de Proteínas , Medula Espinal/fisiopatologia , Adulto , Esclerose Lateral Amiotrófica/metabolismo , Líquido Cefalorraquidiano , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Medula Espinal/metabolismoRESUMO
The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.
Assuntos
Sobrevivência Celular , Complexo de Golgi/metabolismo , Estresse Fisiológico , Fatores de Ribosilação do ADP/efeitos dos fármacos , Fatores de Ribosilação do ADP/metabolismo , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Naftóis/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologiaRESUMO
Alpha-synuclein (aSyn) is the major protein component of Lewy bodies and Lewy neurites, the typical pathological hallmarks in Parkinson's disease (PD) and Dementia with Lewy bodies. aSyn is capable of inducing transcriptional deregulation, but the precise effect of specific aSyn mutants associated with familial forms of PD, remains unclear. Here, we used transgenic mice overexpressing human wild-type (WT) or A30P aSyn to compare the transcriptional profiles of the two animal models. We found that A30P aSyn promotes strong transcriptional deregulation and increases DNA binding. Interestingly, COL4A2, a major component of basement membranes, was found to be upregulated in both A30P aSyn transgenic mice and in dopaminergic neurons expressing A30P aSyn, suggesting a crucial role for collagen related genes in aSyn-induced toxicity. Finally, we observed that A30P aSyn alters Golgi morphology and increases the susceptibility to endoplasmic reticulum (ER) stress in dopaminergic cells. In total, our findings provide novel insight into the putative role of aSyn on transcription and on the molecular mechanisms involved, thereby opening novel avenues for future therapeutic interventions in PD and other synucleinopathies.
Assuntos
Colágeno Tipo IV/biossíntese , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Fragmentos de Peptídeos/biossíntese , alfa-Sinucleína/biossíntese , Animais , Células Cultivadas , Colágeno Tipo IV/genética , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , alfa-Sinucleína/genéticaRESUMO
Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to control serotonin 5-HT1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/ultraestrutura , Transporte Proteico , Ratos , Proteínas de Transporte Vesicular/genéticaRESUMO
Golgi fragmentation is a highly regulated process that allows division of the Golgi complex between the two daughter cells. The mitotic reorganization of the Golgi is accompanied by a temporary block in Golgi functioning, as protein transport in and out of the Golgi stops. Our group has previously demonstrated the involvement of the alternatively spliced variants ERK1c and MEK1b (ERK1 is also known as MAPK3, and MEK1 as MAP2K1) in mitotic Golgi fragmentation. We had also found that ERK1c translocates to the Golgi at the G2 to M phase transition, but the molecular mechanism underlying this recruitment remains unknown. In this study, we narrowed the translocation timing to prophase and prometaphase, and elucidated its molecular mechanism. We found that CDK1 phosphorylates Ser343 of ERK1c, thereby allowing the binding of phosphorylated ERK1c to a complex that consists of PI4KIIIß (also known as PI4KB) and the 14-3-3γ dimer (encoded by YWHAB). The stability of the complex is regulated by protein kinase D (PKD)-mediated phosphorylation of PI4KIIIß. The complex assembly induces the Golgi shuttling of ERK1c, where it is activated by MEK1b, and induces Golgi fragmentation. Our work shows that protein shuttling to the Golgi is not completely abolished at the G2 to M phase transition, thus integrating several independent Golgi-regulating processes into one coherent pathway.
Assuntos
Proteínas 14-3-3/metabolismo , Complexo de Golgi/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mitose/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células HeLa , Humanos , Proteína Quinase 3 Ativada por Mitógeno/genética , Multimerização ProteicaRESUMO
BACKGROUND: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. METHODS: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RESULTS: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. CONCLUSION: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.
Assuntos
Neoplasias da Mama/metabolismo , Complexo de Golgi/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Proteínas rho de Ligação ao GTPRESUMO
The acquisition of proper dendrite morphology is a crucial aspect of neuronal development towards the formation of a functional network. The role of the extracellular matrix and its cellular receptors in this process has remained enigmatic. We report that the CD44 adhesion molecule, the main hyaluronan receptor, is localized in dendrites and plays a crucial inhibitory role in dendritic tree arborization in vitro and in vivo. This novel function is exerted by the activation of Src tyrosine kinase, leading to the alteration of Golgi morphology. The mechanism operates during normal brain development, but its inhibition might have a protective influence on dendritic trees under toxic conditions, during which the silencing of CD44 expression prevents dendritic shortening induced by glutamate exposure. Overall, our results indicate a novel role for CD44 as an essential regulator of dendritic arbor complexity in both health and disease.
Assuntos
Córtex Cerebral/enzimologia , Dendritos/enzimologia , Ácido Glutâmico/toxicidade , Complexo de Golgi/enzimologia , Hipocampo/enzimologia , Receptores de Hialuronatos/metabolismo , Neurogênese , Quinases da Família src/metabolismo , Animais , Animais Recém-Nascidos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/imunologia , Dendritos/efeitos dos fármacos , Dendritos/imunologia , Ativação Enzimática , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Complexo de Golgi/imunologia , Células HEK293 , Células HeLa , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Hipocampo/imunologia , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Masculino , Morfogênese , Mutação , Interferência de RNA , Ratos , Ratos Wistar , Transdução de Sinais , Transfecção , Quinases da Família src/genéticaRESUMO
The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97-p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135 function requires membrane association and p97 interaction, both of which are inhibited in mitosis by VCIP135 phosphorylation. We found that wild-type VCIP135, but not its phosphomimetic mutants, rescues Golgi structure in VCIP135-depleted cells. Our results demonstrate that VCIP135 phosphorylation regulates its Golgi membrane association and p97 interaction, and thus contributes to the tight control of the Golgi disassembly and reassembly process during the cell cycle.
Assuntos
Ciclo Celular/genética , Endopeptidases/genética , Complexo de Golgi/metabolismo , Fusão de Membrana/genética , beta Carioferinas/genética , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Teste de Complementação Genética , Células HeLa , Humanos , Mutação , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , beta Carioferinas/metabolismoRESUMO
BACKGROUND: It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. METHODS: HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM EtOH for 72 hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM). RESULTS: First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive. CONCLUSIONS: Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.
Assuntos
Etanol/farmacologia , Glicosilação/efeitos dos fármacos , Complexo de Golgi/enzimologia , Fígado/enzimologia , Animais , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Hepatócitos/metabolismo , Humanos , Masculino , Manosidases/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , RatosRESUMO
ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Oxirredutases do Álcool/metabolismo , Brefeldina A/metabolismo , Proteínas de Ligação a DNA/metabolismo , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Oxirredutases do Álcool/química , Animais , Sítios de Ligação , Ligação Competitiva , Western Blotting , Brefeldina A/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , NAD/química , NAD/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , RatosRESUMO
This study aimed to investigate the effects of H2O2-induced oxidative stress on cell viability and survival, as well as changes in the distribution of Golgi apparatus and in the level of Golgi reassembly and stacking protein 65 (GRASP65). Cell viability of cultured N2a cells treated with H2O2 was measured by the MTT assay. Apoptosis was measured by flow cytometry analyses. Cells labeled by indirect immunofluorescence were observed under confocal microscope to detect any Golgi morphological alterations; electron microscopy of Golgi apparatus was also done. Expression of GRASP65 and phospho-GRASP65 was examined by immunoblotting. H2O2 treatment reduced the cell viability and raised the cell mortality of N2a cells in a time-dependent manner. Notable changes were only observed in the distribution and morphology of Golgi apparatus at 6 h after H2O2 treatment. The expression of GRASP65 showed no significant changes at different time points; the phosphorylated GRASP65 level was significantly increased after H2O2 treatment, peaked at 3 h, and finally dropped at 6 h. Taken together, GRASP65 phosphorylation may have a critical role in inducing cell death at the early stage after H2O2 treatment, while its role in H2O2-induced Golgi morphological changes may be complex.