RESUMEN
Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that-like SynCAM 1-MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABAA receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.
Asunto(s)
Proteínas de la Membrana , Densidad Postsináptica , Proteínas de la Membrana/metabolismo , Densidad Postsináptica/metabolismo , Receptores AMPA/metabolismo , Receptores de GABA-A , Sinapsis/metabolismoRESUMEN
Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia-mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/- mice exhibited increased expression of IL-1beta and IL-18 compared to wild-type microglia. Becn1+/-APPPS1 mice also contained enhanced IL-1beta levels. The investigation of the IL-1beta/IL-18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in Becn1+/- microglia. Super-resolation microscopy revealed a very close association of NLRP3 aggregates and LC3-positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.
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Autofagia , Beclina-1/fisiología , Inflamación/inmunología , Microglía/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Placa Amiloide/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/fisiología , Animales , Autofagosomas , Citocinas/metabolismo , Femenino , Inflamasomas , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Presenilina-1/fisiologíaRESUMEN
Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach "DNA-PAINT" robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by "Exchange-PAINT", which requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure, we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3-6 nm) and multicolor registration of dual- and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.
Asunto(s)
Nanoestructuras , Imagen Individual de Molécula , ADN/química , Microscopía Fluorescente/métodos , Oligonucleótidos/química , Imagen Individual de Molécula/métodosRESUMEN
Pathological aggregation of amyloid-ß (Aß) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aß plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aß1-40 without changes in Aß plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.
Asunto(s)
Enfermedad de Alzheimer , Interleucina-12/deficiencia , Subunidad p19 de la Interleucina-23/deficiencia , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Interleucina-12/genética , Subunidad p40 de la Interleucina-12/deficiencia , Subunidad p40 de la Interleucina-12/genética , Subunidad p19 de la Interleucina-23/genética , Masculino , Ratones , Ratones Transgénicos , Placa AmiloideRESUMEN
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.
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Autoantígenos/genética , Centrosoma/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Septinas/genética , Células 3T3-L1 , Animales , Autoantígenos/metabolismo , Transporte Biológico , Compartimento Celular , Línea Celular , Centrosoma/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Aparato de Golgi/ultraestructura , Células HEK293 , Células HeLa , Humanos , Células Jurkat/metabolismo , Células Jurkat/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Septinas/antagonistas & inhibidores , Septinas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.
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Equilibrio Ácido-Base/efectos de los fármacos , Receptores de Vasopresinas/efectos de los fármacos , Vasopresinas/farmacología , Equilibrio Ácido-Base/genética , Animales , Células Cultivadas/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Inmunohistoquímica , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratas Brattleboro , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Vasopresinas/genética , Sensibilidad y Especificidad , Urinálisis/métodosRESUMEN
Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.
Asunto(s)
Endocitosis , Fosfatos de Fosfatidilinositol/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Nexinas de Clasificación/metabolismo , Factores de TiempoRESUMEN
Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Regulación de la Expresión Génica , Neurotransmisores/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Encéfalo/metabolismo , Tronco Encefálico/metabolismo , Endocitosis , Eliminación de Gen , Ratones , Ratones Noqueados , Microscopía Confocal , Péptidos/química , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Sinapsis/metabolismo , Transmisión Sináptica , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.
Asunto(s)
Retículo Endoplásmico/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Membrana Celular/metabolismo , Dimerización , Células HEK293 , Humanos , Ratas , Receptores de Hormona Liberadora de Corticotropina/químicaRESUMEN
The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39â dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15â nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40â nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.
Asunto(s)
Encéfalo/ultraestructura , Color , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Vesículas Sinápticas/ultraestructura , Animales , Encéfalo/citología , Ratones , Estructura Molecular , Células 3T3 NIHRESUMEN
Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.
Asunto(s)
Células Endoteliales , Nanopartículas Magnéticas de Óxido de Hierro , Humanos , Células Endoteliales/metabolismo , Endotelio , Perfilación de la Expresión Génica , Colágeno/metabolismo , Estrés Mecánico , Células CultivadasRESUMEN
AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15â kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.
Asunto(s)
Infecciones por Coxsackievirus , Citocinas , Metabolismo Energético , Glucólisis , Mitocondrias Cardíacas , Miocitos Cardíacos , Ubiquitinas , Animales , Humanos , Masculino , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Enterovirus Humano B/patogenicidad , Enterovirus Humano B/metabolismo , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/patología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Ubiquitinas/metabolismo , Ubiquitinas/genéticaRESUMEN
BACKGROUND INFORMATION: Single molecule-based super-resolution methods have become important tools to study nanoscale structures in cell biology. However, the complexity of multi-colour applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy (dSTORM) offers a simple way to perform single molecule super-resolution imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-colour dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-colour imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent (OFF) states. RESULTS: We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM (SD-dSTORM). In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biological nanostructures. In addition, SD-dSTORM allows the use of commercially available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localisation. CONCLUSIONS: SD-dSTORM presents a significant advance towards user-friendly single molecule localisation-based super-resolution microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-colour dSTORM.
Asunto(s)
Carbocianinas/química , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Microtúbulos/ultraestructura , Algoritmos , Animales , Línea Celular , Chlorocebus aethiops , Color , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador/instrumentación , Rayos Láser , Luz , Microscopía Fluorescente/instrumentaciónRESUMEN
Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3-induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second 'sensitizing' stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII-dependent manner. This mechanism was also observed by in vivo C-fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Nociceptores/fisiología , Umbral del Dolor/fisiología , Agonistas Adrenérgicos beta/farmacología , Análisis de Varianza , Animales , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Inositol 1,4,5-Trifosfato/farmacología , Isoproterenol/farmacología , Masculino , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/fisiología , Neuronas/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Proteína Quinasa C-epsilon/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Rianodina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Canales Catiónicos TRPV/metabolismo , Tionucleótidos/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity.
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Disulfuros , Enterovirus Humano B , Dimerización , Disulfuros/metabolismo , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Células HeLa , Humanos , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.
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Infecciones por Coxsackievirus , Infecciones por Enterovirus , Enterovirus , Animales , Cisteína , Enterovirus Humano B/fisiología , Células HeLa , Humanos , Ratones , Virulencia , Replicación ViralRESUMEN
Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.
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Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Exocitosis/fisiología , Aparato de Golgi/metabolismo , Transporte de Proteínas , Animales , Anticuerpos/metabolismo , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Polaridad Celular , Perros , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Fusión de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Microinyecciones , Microscopía Confocal , Microscopía Fluorescente , Microtúbulos/metabolismo , Nocodazol/farmacología , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.
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Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/fisiología , Animales , Proteínas Portadoras/fisiología , Fibroblastos/fisiología , Forminas , Lisofosfolípidos/farmacología , Ratones , Proteínas Asociadas a Microtúbulos/fisiología , Células 3T3 NIH , Unión Proteica , Transfección , Proteínas de Unión al GTP rhoRESUMEN
OBJECTIVE: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic ß-cells. METHODS: A ß-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. RESULTS: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from ß-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from ß-cell-specific Arfrp1 knockout mice. CONCLUSION: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Femenino , Proteínas de la Matriz de Golgi/genética , Células HeLa , Humanos , Masculino , Ratones , Ratones Noqueados , Transporte de Proteínas , Proteínas SNARE/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.