RESUMO
OBJECTIVES: Spinal cord stimulation (SCS) has been shown to be an effective and safe option to treat patients with intractable pain in the general population. Our study examined the experience of US veterans with SCS. METHODS: We reviewed electronic health records and conducted phone interviews with 65 veterans who had SCS from 2008 to 2020 at the Southeastern Louisiana Veterans Health Care System (SLVHCS). Our primary outcome measure was veteran would recommend SCS to peers. Secondary outcomes were improvements in activities of daily living and ability to decrease opioid pain medications. RESULTS: A majority (77%) of veterans recommended SCS to their peers. Statistical difference was seen in 16 of 18 categories of activities of daily living based on the Pain Outcomes Questionnaire. No permanent neurologic deficits or deaths were associated with SCS use. There were no neurological sequelae. Three patients (5%) developed skin dehiscence postimplant and were treated with explant surgery but all were eager to get a new SCS implanted. CONCLUSION: Veterans at SLVHCS were satisfied with their experience using SCS and few experienced adverse effects.
RESUMO
Complex Regional Pain Syndrome (CRPS) is a musculoskeletal pain condition that often develops after limb injury and/or immobilization. Although the exact mechanisms underlying CRPS are unknown, the syndrome is associated with central and autonomic nervous system dysregulation and peripheral hyperalgesia symptoms. These symptoms also manifest in alcoholic neuropathy, suggesting that the two conditions may be pathophysiologically accretive. Interestingly, people assigned female at birth (AFAB) appear to be more sensitive to both CRPS and alcoholic neuropathy. To better understand the biobehavioral mechanisms underlying these conditions, we investigated a model of combined CRPS and alcoholic neuropathy in female rats. Animals were pair-fed either a Lieber-DeCarli alcohol liquid diet or a control diet for ten weeks. CRPS was modeled via unilateral hind limb cast immobilization for seven days, allowing for the other limb to serve as a within-subject control for hyperalgesia measures. To investigate the role of circulating ovarian hormones on pain-related behaviors, half of the animals underwent ovariectomy (OVX). Using the von Frey procedure to record mechanical paw withdrawal thresholds, we found that cast immobilization and chronic alcohol drinking separately and additively produced mechanical hyperalgesia observed 3 days after cast removal. We then examined neuroadaptations in AMPA GluR1 and NMDA NR1 glutamate channel subunits, extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB) in bilateral motor and cingulate cortex across all groups. Consistent with increased pain-related behavior, chronic alcohol drinking increased GluR1, NR1, ERK, and CREB phosphorylation in the cingulate cortex. OVX did not alter any of the observed effects. Our results suggest accretive relationships between CRPS and alcoholic neuropathy symptoms and point to novel therapeutic targets for these conditions.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Giro do Cíngulo/efeitos dos fármacos , Hiperalgesia/fisiopatologia , Sistema de Sinalização das MAP Quinases , Consumo de Bebidas Alcoólicas/fisiopatologia , Animais , Estimulantes do Sistema Nervoso Central/toxicidade , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Etanol/farmacologia , Etanol/toxicidade , Feminino , Giro do Cíngulo/metabolismo , Elevação dos Membros Posteriores/efeitos adversos , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Nociceptividade , Ratos , Ratos Endogâmicos F344 , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Primary cilia are cellular antennae that receive and transduce extracellular cues. These microtubule-rich structures are comprised of at least three distinct ciliary compartments: basal bodies, transition zone, and axoneme. Septins have been implicated in cilia function at the transition zone, but accumulating evidence suggests that they localize predominantly within the axoneme. Here, we describe three fixation conditions that preserve the substructure of primary cilia and demonstrate known ciliary proteins that localize to these distinct ciliary substructures. Finally, we show immunostaining and live microscopy methods to detect septins within the axoneme.
Assuntos
Técnicas de Cultura de Células/métodos , Cílios/ultraestrutura , Microscopia de Fluorescência/métodos , Septinas/isolamento & purificação , Axonema/química , Axonema/ultraestrutura , Corpos Basais/química , Corpos Basais/ultraestrutura , Linhagem Celular , Cílios/química , Humanos , Microtúbulos/química , Microtúbulos/ultraestrutura , Septinas/químicaRESUMO
Mass measurements of 68Ge, 68As, and 68Se have been obtained with the Canadian Penning Trap mass spectrometer. The results determine the mass excess of 68Se as -54 232(19) keV, the first measurement with a precision and reliability sufficient to address the light-curve and energy output of x-ray bursts as well as the abundances of the elements synthesized. Under typical conditions used for modeling x-ray bursts, 68Se is found to cause a significant delay in the rp process nucleosynthesis.
RESUMO
In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of 25 kDa homologue, synaptosome-associated protein of 23 kDa, is also expressed in the rat choroid plexus, which was confirmed by reverse-transcriptase polymerase chain reaction. Our findings suggest that synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein are involved in secretion of polypeptides from the choroid plexus epithelium. The presence of synaptosome-associated protein of 25 kDa and its homologue as well as multiple isoforms of vesicle-associated membrane protein in choroidal epithelium may play a role in the apical versus basolateral targeting of secretory vesicles.
Assuntos
Plexo Corióideo/fisiologia , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Animais , Western Blotting , Plexo Corióideo/química , Epitélio/química , Epitélio/fisiologia , Exocitose/fisiologia , Imuno-Histoquímica , Masculino , Fusão de Membrana/fisiologia , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Testes de Precipitina , Proteínas R-SNARE , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 25 Associada a SinaptossomaRESUMO
Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.
Assuntos
Colecistocinina/farmacologia , Exocitose , Proteínas do Tecido Nervoso , Pâncreas/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Animais , Membrana Celular/metabolismo , Colecistocinina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Munc18 , Pancreatite/etiologia , Proteína Quinase C/fisiologia , Proteínas/análise , Ratos , Proteínas SNARE , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcgamma receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Oncogênicas/metabolismo , Fagocitose/fisiologia , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Moléculas de Adesão Celular/genética , Linhagem Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Proteínas de Membrana/genética , Camundongos , Proteínas dos Microfilamentos/genética , Monócitos/citologia , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Proteína da Síndrome de Wiskott-Aldrich , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Engulfment of particles by phagocytes involves remodeling of the plasma membrane. We review recent work that suggests that focal exocytosis of endomembranes plays an important role in pseudopod extension during phagocytosis.
Assuntos
Membrana Celular/fisiologia , Fagocitose/fisiologia , Fagossomos/fisiologia , Actinas , Animais , Membrana Celular/microbiologia , Exocitose/fisiologia , Humanos , Lisossomos/fisiologia , Proteínas Opsonizantes , Receptores de IgG/fisiologia , Salmonella typhimurium/fisiologiaRESUMO
Proteins that bind to SNAREs may regulate their function. One such protein, VAP-33, was first discovered in Aplysia californica and has two mammalian homologues, VAP-A and VAP-B. VAP-A has been implicated in vesicle targeting to the plasma membrane based on its location in polarized cells and its ability to bind VAMP in vitro. Here, we demonstrate that VAP-A is a widely expressed resident of the ER/Golgi intermediate compartment in COS-7 cells. Moreover, we demonstrate that VAMP-binding and VAP-dimerization require both the N- and C-terminal domains of VAP-A and also that VAP-A binds to a wide range of SNAREs and fusion-related proteins including syntaxin 1A, rbet1, rsec22, alphaSNAP, and NSF. Together, these results suggest that VAP-A is not a regulator of a specific VAMP, but rather may play a more general role in SNARE-mediated vesicle traffic between the ER and Golgi in nonpolarized cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/metabolismo , Células COS , Proteínas de Transporte/química , Linhagem Celular , Dimerização , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas Sensíveis a N-Etilmaleimida , Proteínas do Tecido Nervoso/metabolismo , Proteínas Qc-SNARE , Proteínas R-SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Sintaxina 1 , Distribuição TecidualRESUMO
Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta-cells inhibited Ca(2+)-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca(2+) (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca(2+)-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca(2+) influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca(2+)-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca(2+) or cAMP potentiation.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Cálcio/metabolismo , AMP Cíclico/metabolismo , Exocitose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Toxina Tetânica/farmacologia , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Células Clonais , Exocitose/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina , Insulinoma , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Pancreáticas , Técnicas de Patch-Clamp , Proteínas R-SNARE , Proteínas Recombinantes/metabolismo , Proteína 25 Associada a Sinaptossoma , Transfecção , Células Tumorais CultivadasRESUMO
INTRODUCTION: Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined. AIMS: To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted. METHODOLOGY AND RESULTS: High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin-phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE-stimulated amylase release from adenovirus-cytomegalovirus (AdCMV)-SNAP-23 or AdCMV-SNAP-25-infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE-evoked enzyme secretion from AdCMV-SNAP-23deltaC8- and AdCMV-SNAP-25(1-197)-infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV-SNAP-25AC8- or AdCMV-SNAP-25(1-197)-infected acini were co-infected with wild-type SNAP-23 or SNAP-25. CONCLUSION: From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Colecistocinina/fisiologia , Exocitose/fisiologia , Pâncreas/fisiologia , Sincalida/análogos & derivados , Adenoviridae/genética , Amilases/metabolismo , Animais , Exocitose/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Ratos , Ratos Sprague-Dawley , Deleção de Sequência , Sincalida/farmacologia , Proteína 25 Associada a SinaptossomaRESUMO
NEM-sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF-1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identified as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.
Assuntos
Proteínas de Transporte/genética , Paralisia/genética , Proteínas de Transporte Vesicular , Animais , Western Blotting , Proteínas de Transporte/análise , Drosophila , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Imuno-Histoquímica , Larva , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação/genética , Proteínas Sensíveis a N-Etilmaleimida , Junção Neuromuscular/efeitos dos fármacos , Proteínas SNARE , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
The wing of Drosophila melanogaster has long been used as a model system to characterize intermolecular interactions important in development. Implicit in our understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. We have therefore chosen the Drosophila wing to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and we constructed a mutant form of NSF whose expression we directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, we find that the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, we used this phenotype to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. We conclude that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways will act as a sensitive reporter of partial membrane-trafficking disruption.
Assuntos
Drosophila/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular , Asas de Animais/crescimento & desenvolvimento , Animais , Proteínas de Transporte/metabolismo , Drosophila/anatomia & histologia , Drosophila/genética , Proteínas de Drosophila , Mutação , Proteínas Sensíveis a N-Etilmaleimida , Fenótipo , Transporte Proteico , Proteínas Qa-SNARE , Proteínas R-SNARE , Receptores Notch , Proteínas SNARE , Distribuição Tecidual , Asas de Animais/anatomia & histologiaRESUMO
Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.
Assuntos
Potenciação de Longa Duração/fisiologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Glicina/metabolismo , Glicina/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Camundongos , Neurônios/citologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresAssuntos
Proteínas de Ciclo Celular , Exocitose , GTP Fosfo-Hidrolases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/isolamento & purificação , Perfilação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Ligação Proteica , Proteínas Qa-SNARE , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE , Septinas , Vesículas Sinápticas/enzimologia , TransfecçãoRESUMO
Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.
Assuntos
Proteínas de Transporte/fisiologia , Fagocitose , Salmonella typhimurium/patogenicidade , Proteínas de Transporte Vesicular , Animais , Células COS , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/ultraestrutura , Cricetinae , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Mutação , Proteínas Sensíveis a N-Etilmaleimida , Toxina Tetânica/farmacologia , Transfecção , Vacúolos/microbiologia , Proteína 3 Associada à Membrana da VesículaRESUMO
A hallmark of calcium-triggered synaptic transmission is the cooperative relationship between calcium and the amount of transmitter released. This relationship is thought to be important for improving the efficiency of synaptic vesicle exocytosis. Although it is generally held that cooperativity arises from the interaction of multiple calcium ions with a single calcium-sensing molecule, the precise molecular basis of this phenomenon is not known. The SNARE proteins are known to be critical for synaptic vesicle exocytosis. We therefore tested for a contribution of SNARE proteins to cooperativity by genetically reducing the levels of syntaxin IA and neuronal-synaptobrevin in Drosophila. Surprisingly, we found that reducing these SNARE proteins also reduced Ca(2+) cooperativity. Thus, SNARE proteins are important for determining the cooperative relationship between calcium and synaptic transmission.
Assuntos
Cálcio/fisiologia , Proteínas de Membrana/fisiologia , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular , Animais , Drosophila , Imunofluorescência , Proteínas SNARERESUMO
Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.
Assuntos
Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Actinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4 , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/genética , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas R-SNARE , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxina Tetânica/metabolismo , Proteína 3 Associada à Membrana da VesículaRESUMO
Phagocytosis involves the receptor-mediated extension of plasmalemmal protrusions, called pseudopods, which fuse at their tip to engulf a particle. Actin polymerizes under the nascent phagosome and may propel the protrusion of pseudopods. Alternatively, membrane extension could result from the localized insertion of intracellular membranes into the plasmalemma next to the particle. Here we show focal accumulation of VAMP3-containing vesicles, likely derived from recycling endosomes, in the vicinity of the nascent phagosome. Using green fluorescent protein (GFP) as both a fluorescent indicator and an exofacial epitope tag, we show that polarized fusion of VAMP3 vesicles precedes phagosome sealing. It is therefore likely that targeted delivery of endomembranes contributes to the elongation of pseudopods. In addition to mediating pseudopod formation, receptor-triggered focal secretion of endosomes may contribute to polarized membrane extension in processes such as lamellipodial elongation or chemotaxis.
Assuntos
Exocitose , Proteínas de Membrana/metabolismo , Fagossomos/metabolismo , Animais , Antígenos CD/metabolismo , Células CHO , Cricetinae , Endossomos/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Proteínas de Membrana Lisossomal , Fusão de Membrana , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Microscopia de Fluorescência , Fagocitose , Pseudópodes/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína 3 Associada à Membrana da VesículaRESUMO
Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMP-associated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulin-dependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events.