RESUMEN
Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
RESUMEN
Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (â¼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
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Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Conexinas/química , Conexinas/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Conexinas/genética , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Indoles/farmacocinética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Fosforilación , Serina/genética , Serina/metabolismoRESUMEN
AIMS: Dynamin-2 is a large GTPase, a member of the dynamin superfamily that regulates membrane remodelling and cytoskeleton dynamics. Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM), a congenital neuromuscular disorder characterised by progressive weakness and atrophy of the skeletal muscles. Cognitive defects have been reported in some DNM2-linked CNM patients suggesting that these mutations can also affect the central nervous system (CNS). Here we studied how a dynamin-2 CNM-causing mutation influences the CNS function. METHODS: Heterozygous mice harbouring the p.R465W mutation in the dynamin-2 gene (HTZ), the most common causing autosomal dominant CNM, were used as disease model. We evaluated dendritic arborisation and spine density in hippocampal cultured neurons, analysed excitatory synaptic transmission by electrophysiological field recordings in hippocampal slices, and evaluated cognitive function by performing behavioural tests. RESULTS: HTZ hippocampal neurons exhibited reduced dendritic arborisation and lower spine density than WT neurons, which was reversed by transfecting an interference RNA against the dynamin-2 mutant allele. Additionally, HTZ mice showed defective hippocampal excitatory synaptic transmission and reduced recognition memory compared to the WT condition. CONCLUSION: Our findings suggest that the dynamin-2 p.R465W mutation perturbs the synaptic and cognitive function in a CNM mouse model and support the idea that this GTPase plays a key role in regulating neuronal morphology and excitatory synaptic transmission in the hippocampus.
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Dinamina II , Miopatías Estructurales Congénitas , Animales , Ratones , Modelos Animales de Enfermedad , Dinamina II/genética , Dinamina II/metabolismo , Músculo Esquelético/metabolismo , Mutación , Miopatías Estructurales Congénitas/genética , Neuronas/metabolismo , Transmisión SinápticaRESUMEN
Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.
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Dinamina II , Miopatías Estructurales Congénitas , Dinamina II/genética , Dinamina II/metabolismo , Exocitosis , Mutación con Ganancia de Función , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Ionomicina , Músculo Esquelético/metabolismo , Mutación , Mioblastos/metabolismo , Miopatías Estructurales Congénitas/metabolismoRESUMEN
Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.
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Células Cromafines/metabolismo , Conexinas/metabolismo , Exocitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptor Cross-Talk/fisiología , Receptores Purinérgicos P2X7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Quelantes del Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Bovinos , Línea Celular Tumoral , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Humanos , Ratones , Receptor Cross-Talk/efectos de los fármacosRESUMEN
Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela's inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.
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Acetilcisteína/administración & dosificación , Antioxidantes/administración & dosificación , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Cinturas/dietoterapia , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Índice de Masa Corporal , Modelos Animales de Enfermedad , Humanos , Peroxidación de Lípido/efectos de los fármacos , Ratones , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Resultado del TratamientoRESUMEN
ß-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.
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Actinas/metabolismo , Canales de Calcio Tipo L/metabolismo , Células Cromafines/metabolismo , Regulación hacia Abajo/fisiología , Dominios Homologos src/fisiología , Animales , Bovinos , Células Cultivadas , Subunidades de Proteína/metabolismo , Transporte de Proteínas/fisiología , ConejosRESUMEN
Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca2+-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.
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Actinas/metabolismo , Disferlina/química , Distrofia Muscular de Cinturas/metabolismo , Mioblastos/citología , Citoesqueleto de Actina/metabolismo , Actinas/genética , Adolescente , Adulto , Línea Celular , Disferlina/genética , Disferlina/metabolismo , Femenino , Humanos , Masculino , Distrofia Muscular de Cinturas/genética , Mioblastos/metabolismo , Dominios ProteicosRESUMEN
The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells.
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Médula Suprarrenal/metabolismo , Gránulos Cromafines/metabolismo , Exocitosis , Animales , Canales de Calcio/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
INTRODUCTION: MRI characterization of dysferlinopathy has been mostly limited to the lower limbs. We aimed to broaden the MRI description of dysferlinopathy and to correlate it with objective measures of motor dysfunction. METHODS: Sequential whole-body axial MRI was performed in 27 patients with genetically confirmed dysferlinopathy classified according to disease duration. Spearman correlations of fatty infiltration scores versus Motor Function Measure (MFM) were calculated. RESULTS: Significant fatty infiltration was symmetrically present in early stages mainly in the posterior compartments of legs and thighs, thigh adductors, pelvic girdle, and some paravertebral muscles and the subscapularis. Later, fatty infiltration involved leg and thigh anterior compartments, arms and forearms, paravertebral, and trunk muscles. MRI infiltration score correlated positively with disease duration and negatively with MFM scale. CONCLUSIONS: We expand MRI characterization of dysferlinopathy and provide evidence for use of MRI scoring combined with motor functional scales to assess the natural course of disease. Muscle Nerve, 2016 Muscle Nerve 54: 203-210, 2016.
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Imagen por Resonancia Magnética , Distrofia Muscular de Cinturas/diagnóstico por imagen , Distrofia Muscular de Cinturas/fisiopatología , Imagen de Cuerpo Entero , Adolescente , Niño , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/etiología , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Músculo Esquelético/diagnóstico por imagen , Estudios Retrospectivos , Estadísticas no Paramétricas , Adulto JovenRESUMEN
Dynamin-2 is a pleiotropic GTPase whose best-known function is related to membrane scission during vesicle budding from the plasma or Golgi membranes. In the nervous system, dynamin-2 participates in synaptic vesicle recycling, post-synaptic receptor internalization, neurosecretion, and neuronal process extension. Some of these functions are shared with the other two dynamin isoforms. However, the involvement of dynamin-2 in neurological illnesses points to a critical function of this isoform in the nervous system. In this regard, mutations in the dynamin-2 gene results in two congenital neuromuscular disorders. One of them, Charcot-Marie-Tooth disease, affects myelination and peripheral nerve conduction, whereas the other, Centronuclear Myopathy, is characterized by a progressive and generalized atrophy of skeletal muscles, yet it is also associated with abnormalities in the nervous system. Furthermore, single nucleotide polymorphisms located in the dynamin-2 gene have been associated with sporadic Alzheimer's disease. In the present review, we discuss the pathogenic mechanisms implicated in these neurological disorders.
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Dinamina II/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/anomalías , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Dinamina II/genética , Endocitosis , Humanos , Músculo Esquelético/patología , Mutación , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.
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Adenosina Trifosfato , Catecolaminas , Células Cromafines , Exocitosis , Receptores Purinérgicos P2X7 , Animales , Receptores Purinérgicos P2X7/metabolismo , Células Cromafines/metabolismo , Células Cromafines/efectos de los fármacos , Bovinos , Adenosina Trifosfato/metabolismo , Ratones , Catecolaminas/metabolismo , Exocitosis/efectos de los fármacos , Células PC12 , Ratas , Calcio/metabolismo , Comunicación Autocrina , Ratones Endogámicos C57BL , Células Cultivadas , MasculinoRESUMEN
In this work entomological material and nomenclatural types of the Ibero-Balearic species of Tentyria Latreille, 1802 genus are revised. A reordination in groups of species and a new classification of valid species are proposed. Thirteen groups of species including 31 species and six subspecies have been established. Seven new species of Iberian Tentyria are described and figured, including two from Portugal: Tentyria stupefacta sp. nov. and Tentyria faroensis sp. nov.; and five species from Spain: Tentyria espanoli sp. nov., Tentyria kochi sp. nov., Tentyria striatorugosa sp. nov., Tentyria castrotovari sp. nov. and Tentyria pseudogaditana sp. nov. Three new subspecies: Tentyria sinuatocollis ssp. escalerai nov., Tentyria velox ssp. serrana nov., Tentyria sublaevis ssp. cognata nov., are also described. The true identity of 14 species historically confused is argued: Tentyria curculionoides (Herbst, 1799); Tentyria peiroleri Solier, 1835; Tentyria incerta Solier, 1835; Tentyria levis Solier, 1835; Tentyria sinuatocollis Rosenhauer, 1856; Tentyria prolixa Rosenhauer, 1856; Tentyria rugosostriata Kraatz, 1865; Tentyria corrugata Rosenhauer, 1856; Tentyria velox Chevrolat, 1865; Tentyria sublaevis Kraatz, 1865; Tentyria heydeni Haag-Rutenberg, 1870; Tentyria lateritia Reitter, 1900; Tentyria castiliana Koch, 1944; and Tentyria aragonica Koch, 1944. The taxonomic status and the name Tentyria subrugosa Solier, 1835 have been modified because it is a primary homonym of Tentyria subrugosa Besser, 1832. Tentyria elongata Waltl, 1835 is a primary homonym of Tentyria elongata Gebler, 1829 (= Anatolica angustata (Steven, 1828)). The taxonomic status of Tentyria grossa ssp. basalis Schaufuss, 1869 is also re-established. Lectotypes of 14 species are designated; seven synonymies are established (one of them from the Italian fauna). The distribution of species is indicated and, agreeing with the new taxonomical classification, a key of species is also provided.
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Escarabajos , Animales , Distribución AnimalRESUMEN
Dynamin superfamily proteins (DSPs) comprise a large group of GTP-ases that orchestrate membrane fusion and fission, and cytoskeleton remodeling in different cell-types. At the central nervous system, they regulate synaptic vesicle recycling and signaling-receptor turnover, allowing the maintenance of synaptic transmission. In the presynapses, these GTP-ases control the recycling of synaptic vesicles influencing the size of the ready-releasable pool and the release of neurotransmitters from nerve terminals, whereas in the postsynapses, they are involved in AMPA-receptor trafficking to and from postsynaptic densities, supporting excitatory synaptic plasticity, and consequently learning and memory formation. In agreement with these relevant roles, an important number of neurological disorders are associated with mutations and/or dysfunction of these GTP-ases. Along the present review we discuss the importance of DSPs at synapses and their implication in different neuropathological contexts.
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Neuronas , Transmisión Sináptica , Dinaminas/metabolismo , Humanos , Plasticidad Neuronal , Neuronas/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
Muscular dystrophies (MDs) are a heterogeneous group of congenital neuromuscular disorders whose clinical signs include myalgia, skeletal muscle weakness, hypotonia, and atrophy that leads to progressive muscle disability and loss of ambulation. MDs can also affect cardiac and respiratory muscles, impairing life-expectancy. MDs in clude Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy. These and other MDs are caused by mutations in genes that encode proteins responsible for the structure and function of skeletal muscles, such as components of the dystrophin-glycoprotein-complex that connect the sarcomeric-actin with the extracellular matrix, allowing contractile force transmission and providing stability during muscle contraction. Consequently, in dystrophic conditions in which such proteins are affected, muscle integrity is disrupted, leading to local inflammatory responses, oxidative stress, Ca2+-dyshomeostasis and muscle degeneration. In this scenario, dysregulation of connexin hemichannels seem to be an early disruptor of the homeostasis that further plays a relevant role in these processes. The interaction between all these elements constitutes a positive feedback loop that contributes to the worsening of the diseases. Thus, we discuss here the interplay between inflammation, oxidative stress and connexin hemichannels in the progression of MDs and their potential as therapeutic targets.
RESUMEN
Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.
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Células Cromafines/metabolismo , Dinaminas/metabolismo , Exocitosis/fisiología , Sinaptofisina/metabolismo , Animales , Anticuerpos/farmacología , Bovinos , Células Cultivadas , Células Cromafines/ultraestructura , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/metabolismo , Dinaminas/genética , Dinaminas/inmunología , Electroquímica/métodos , Exocitosis/efectos de los fármacos , Inmunoprecipitación/métodos , Microinyecciones , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sinaptofisina/química , Sinaptofisina/genética , Sinaptofisina/inmunología , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
This study is part of the Follow up Restoration Program of animal communities that colonize the Guadiamar River Basin. In 1998, the area was affected by a release of toxic sludge after the retention walls of the Aznalcóllar Mines (southern Iberian Peninsula) broke. The main objective of this study was to assess the current state of the population of Tenebrionidae, one of the most representative groups of edaphic Coleoptera inhabiting the Guadiamar River Basin. This paper analyses the progress made by the darkling beetle community six years after the disaster occurred and the Restoration Program was implemented. The study is based on faunistic data from systematic sampling carried out for six years to monitor plots distributed across the damaged area. To make an overall assessment of the tenebrionid fauna in relation to adjacent areas qualitative and quantitative ecological indices were applied, and temporal follow up and biogeographical comparisons were also made. The results indicate that, on the whole, tenebrionid fauna was somewhat affected by the Aznalcóllar Mine spill, and that a greater loss of fauna was detected closer to the accident site. The analysis of the temporal population dynamic suggests that the most affected zones are undergoing a process of re-colonization. However, this process varies widely by species and has not yet reached the expected levels of a non-affected river basin in the southern Iberian Peninsula.
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Biodiversidad , Escarabajos , Restauración y Remediación Ambiental , Animales , Geografía , Sustancias Peligrosas , Residuos Industriales , Minería , Ríos , EspañaRESUMEN
AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.
Asunto(s)
Señalización del Calcio/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Animales , Calcio/metabolismo , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Femenino , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp/métodosRESUMEN
High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.
Asunto(s)
Dinamina II/genética , Miopatías Estructurales Congénitas/patología , Dominios Proteicos/genética , Multimerización de Proteína/genética , Arginina/genética , Cristalografía por Rayos X , Dinamina II/metabolismo , Dinamina II/ultraestructura , Humanos , Simulación de Dinámica Molecular , Mutación Missense , Miopatías Estructurales Congénitas/genética , Conformación Proteica en Hélice alfa/genética , Triptófano/genéticaRESUMEN
In humans, Down Syndrome (DS) is a condition caused by partial or full trisomy of chromosome 21. Genes present in the DS critical region can result in excess gene dosage, which at least partially can account for DS phenotype. Although regulator of calcineurin 1 (RCAN1) belongs to this region and its ectopic overexpression in neurons impairs transmitter release, synaptic plasticity, learning and memory, the relative contribution of RCAN1 in a context of DS has yet to be clarified. In the present work, we utilized an in vitro model of DS, the CTb neuronal cell line derived from the brain cortex of a trisomy 16 (Ts16) fetal mouse, which reportedly exhibits acetylcholine release impairments compared to CNh cells (a neuronal cell line established from a normal littermate). We analyzed single exocytotic events by using total internal reflection fluorescence microscopy (TIRFM) and the vesicular acetylcholine transporter fused to the pH-sensitive green fluorescent protein (VAChT-pHluorin) as a reporter. Our analyses showed that, compared with control CNh cells, the trisomic CTb cells overexpress RCAN1, and they display a reduced number of Ca2+-induced exocytotic events. Remarkably, RCAN1 knockdown increases the extent of exocytosis at levels comparable to those of CNh cells. These results support a critical contribution of RCAN1 to the exocytosis process in the trisomic condition.