RESUMEN
Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Munc18/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmisión Sináptica , Animales , Estimulación Eléctrica , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores , Femenino , Células HEK293 , Hipocampo/fisiología , Humanos , Técnicas In Vitro , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Fosforilación , Embarazo , Ratas Wistar , Estrés Psicológico/metabolismoRESUMEN
Synaptotagmin-1 (Syt1) is the principal Ca(2+) sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQ-induced docking was strictly PI(4,5)P2-dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca(2+)-triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain--and, by inference, Syt1-mediated membrane crosslinking--is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion. SIGNIFICANCE STATEMENT: This study provides new insights in how the two opposite sides of the C2B domain of Synaptotagmin-1 participate in secretory vesicle fusion, and in more upstream steps, especially vesicle docking. We show that the "bottom" surface of the C2B domain is required for triggering fusion, but not for docking. Synaptotagmin-1 promotes docking by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. Mutations in the C2B bottom surface (R398/399) selectively disrupt the latter. These mutations help to discriminate protein regions involved in different aspects of Synaptotagmin-1 function in docking and fusion.
Asunto(s)
Células Cromafines/metabolismo , Mutación/genética , Vesículas Sinápticas/genética , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Células Cromafines/ultraestructura , Embrión de Mamíferos , Femenino , Masculino , Fusión de Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Proteínas SNARE/metabolismo , Vías Secretoras/genética , Transmisión Sináptica/genética , Vesículas Sinápticas/ultraestructuraRESUMEN
Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i.e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective, consistent with an upstream role for PICK1. Disrupting lipid binding of the BAR domain (2K-E mutation) or of the PDZ domain (CC-GG mutation) was sufficient to reproduce the secretion phenotype of the null mutant. The same mutations are known to eliminate PICK1 function in receptor trafficking, indicating that the multiple functions of PICK1 involve a conserved mechanism. Summarized, our findings demonstrate that PICK1 functions in vesicle biogenesis and is necessary to maintain normal vesicle numbers and size.
Asunto(s)
Glándulas Suprarrenales/citología , Proteínas Portadoras/metabolismo , Células Cromafines/citología , Exocitosis/fisiología , Proteínas Nucleares/metabolismo , Vesículas Secretoras/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Proteínas Portadoras/genética , Catecolaminas/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Células Cromafines/ultraestructura , Exocitosis/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteínas Nucleares/genética , Transporte de Proteínas/fisiología , Vesículas Secretoras/genética , Vesículas Secretoras/ultraestructura , Capacitancia Vascular/genéticaRESUMEN
BACKGROUND: Adrenal chromaffin cells are a widely used model system to study regulated exocytosis and other membrane-associated processes. Alterations in the amount and localization of the proteins involved in these processes can be visualized with fluorescent probes that report the effect of different stimuli or genetic modifications. However, the quantitative analysis of such images remains difficult, especially when focused on specific locations, such as the plasma membrane. NEW METHOD: We developed an image analysis algorithm, named plasma membrane analysis in chromaffin cells (PlasMACC). PlasMACC enables automatic detection of the plasma membrane region and quantitative analysis of multi-fluorescent signals from spherical cells. PlasMACC runs in the image analysis software ImageJ environment, it is user-friendly and freely available. RESULTS: PlasMACC delivers detailed information about intensity, thickness and density of fluorescent signals at the plasma membrane of both living and fixed cells. Individual signals can be compared between cells and different signals within one cell can be correlated. PlasMACC can process conventional laser-scanning confocal images as well as data obtained by super-resolution methods such as structured illumination microscopy. COMPARISON WITH EXISTING METHOD(S): By comparing PlasMACC to methods currently used in the field, we show more consistent quantitative data due to the fully automated algorithm. PlasMACC also provides an expanded set of novel analysis parameters. CONCLUSION: PlasMACC enables a detailed quantification of fluorescent signals at the plasma membrane of spherical cells in an unbiased and reliable fashion.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Células Cromafines/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Actinas/genética , Glándulas Suprarrenales/metabolismo , Algoritmos , Animales , Antígeno CD146/genética , Antígeno CD146/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Citosol/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Ratones Noqueados , Ratones Transgénicos , Potasio/metabolismo , Validación de Programas de ComputaciónRESUMEN
The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short-term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca(2+)-channel trafficking, but is dispensable for transmitter release.
Asunto(s)
Proteínas Qb-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Canales de Calcio/metabolismo , Estructuras del Núcleo Celular/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Ratones , Ratones Mutantes , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608. Biological significance In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring a potential role in the physiology of the brain. These extracellular vesicles provide a rich source of candidate biomarkers, representing a brain "fluid biopsy". Most interestingly, the involvement of extracellular vesicles in transferring toxic proteins such as α-synuclein and ß-amyloid has been postulated as one of the mechanisms involved in the spreading of neurodegeneration to different brain areas. In line with this, we show that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and the prion protein. Delineating the protein composition of extracellular vesicles in CSF is a first and crucial step to comprehend their origin and their function in the central nervous system and to establish their biomarker potential.
Asunto(s)
Proteínas del Líquido Cefalorraquídeo/análisis , Bases de Datos de Proteínas , Proteoma , Precursor de Proteína beta-Amiloide/sangre , Biomarcadores/sangre , Encéfalo/metabolismo , Proteínas del Líquido Cefalorraquídeo/química , Cromatografía Liquida , Biología Computacional , Humanos , Microscopía Electrónica , Enfermedades Neurodegenerativas/sangre , Enfermedades Neurodegenerativas/metabolismo , Priones , Proteómica , Espectrometría de Masas en TándemRESUMEN
We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (vitrification of sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Microscopía de Contraste de Fase/métodos , Microtomía/métodos , Vitrificación , Animales , Humanos , Hidrazinas , Lisosomas/ultraestructura , Ratones , Microscopía Fluorescente/métodos , Monocitos/citología , Neuronas/citología , Sinapsis/ultraestructuraRESUMEN
Synaptotagmin-1 and -7 constitute the main calcium sensors mediating SNARE-dependent exocytosis in mouse chromaffin cells, but the role of a closely related calcium-binding protein, Doc2b, remains enigmatic. We investigated its role in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca(2+)]i following stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized release, and limits out-of-phase secretion.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Vesículas Secretoras/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Células Cultivadas , Células Cromafines/ultraestructura , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/ultraestructura , Sinaptotagmina I/metabolismoRESUMEN
SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.
Asunto(s)
Transporte de Proteínas , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/metabolismo , Vesículas Transportadoras/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células Cromafines/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/genética , Sinaptotagmina I/química , Sinaptotagmina I/genéticaRESUMEN
The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin-proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Potenciales Postsinápticos Excitadores/fisiología , Proteínas de Unión al GTP/genética , Guanilato-Quinasas/genética , Hipocampo/citología , Proteínas de la Membrana/genética , Microscopía Electrónica , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/ultraestructura , Fenotipo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Ubiquitina/metabolismoRESUMEN
The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found that neurons lacking the BEACH (beige-Chediak/Higashi) domain protein Neurobeachin (Nbea) had strongly reduced synaptic responses caused by a reduction in surface levels of glutamate and GABA(A) receptors. In the absence of Nbea, immature AMPA receptors accumulated early in the biosynthetic pathway, and mature N-methyl-d-aspartate, kainate, and GABA(A) receptors did not reach the synapse, whereas maturation and surface expression of other membrane proteins, synapse formation, and presynaptic function were unaffected. These data show that Nbea regulates synaptic transmission under basal conditions by targeting neurotransmitter receptors to synapses.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Ácido Kaínico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Proteínas Portadoras/genética , Proteínas de la Membrana , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/fisiología , Receptores de GABA-A/genética , Receptores de Ácido Kaínico/genética , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/genéticaRESUMEN
Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Vesículas Secretoras/metabolismo , Animales , Células Cultivadas , Femenino , Hipocampo/fisiología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Terminales Presinápticos/fisiologíaRESUMEN
Munc18-1 was originally described as an essential docking factor in chromaffin cells. Recent findings showed that Munc18-1 has an additional role in the regulation of the cortical F-actin network, which is thought to function as a physical barrier preventing secretory vesicles from access to their release sites under resting conditions. In our review, we discuss whether this function is evolutionarily conserved in all Sec1/Munc18-like (SM) proteins. In addition, we introduce a new quantification method that improves the analysis of cortical filamentous actin (F-actin) in comparison with existing methods. Since the docking process is highly evolutionarily conserved in the SM protein superfamily, we use our novel quantification method to investigate whether the F-actin-regulating function is similarly conserved among SM proteins. Our preliminary data suggest that the regulation of cortical F-actin is a shared function of SM proteins, and we propose a way to gain more insight in the molecular mechanism underlying the Munc18-1-mediated cortical F-actin regulation.
Asunto(s)
Actinas/fisiología , Células Cromafines/fisiología , Proteínas Munc18/fisiología , Vesículas Secretoras/fisiología , Animales , Células Cultivadas , Células Cromafines/citología , Técnicas Citológicas/métodos , Ratones , Ratones Noqueados , Proteínas Munc18/genética , Vesículas Secretoras/genéticaRESUMEN
Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.
Asunto(s)
Proteínas Munc18/fisiología , Proteínas SNARE/fisiología , Transmisión Sináptica/fisiología , Animales , Ratones , Ratones Noqueados , Neuronas/fisiología , Mutación Puntual , Unión Proteica , Vesículas SinápticasRESUMEN
Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.
Asunto(s)
Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Hipocampo/fisiopatología , Hipocampo/ultraestructura , Fenotipo , Proteómica , Sinapsis/metabolismo , Actinas/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/fisiopatología , Región CA1 Hipocampal/ultraestructura , Diferenciación Celular , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/fisiología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/fisiopatología , Técnicas de Inactivación de Genes , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Neuritas/metabolismo , Plasticidad Neuronal/fisiología , Seudópodos/metabolismo , Sinapsis/patología , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patología , Espectrometría de Masas en TándemRESUMEN
Exocytosis requires formation of SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.
Asunto(s)
Exocitosis/fisiología , Fusión de Membrana/fisiología , Proteína 25 Asociada a Sinaptosomas/fisiología , Animales , Membrana Celular/fisiología , Células Cromafines/fisiología , Vesículas Citoplasmáticas/fisiología , Proteínas Fluorescentes Verdes/genética , Ratones , Mutación , Proteínas SNARE/fisiología , Proteína 25 Asociada a Sinaptosomas/genéticaRESUMEN
The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD(-/-) ) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.
Asunto(s)
Células Cromafines , Vesículas Transportadoras/fisiología , Proteínas de Unión al GTP rab3/metabolismo , Animales , Western Blotting , Investigaciones con Embriones , Ratones , Ratones Noqueados , Biogénesis de Organelos , Isoformas de ProteínasRESUMEN
Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses.
Asunto(s)
Células Cromafines/metabolismo , Vesículas Secretoras/metabolismo , Sinapsis/metabolismo , Animales , Hormonas/metabolismo , Ratones , Neurotransmisores/metabolismo , Unión ProteicaRESUMEN
Docking, the stable association of secretory vesicles with the plasma membrane, is considered to be the necessary first step before vesicles gain fusion-competence, but it is unclear how vesicles dock. In adrenal medullary chromaffin cells, access of secretory vesicles to docking sites is controlled by dense F-actin (filamentous actin) beneath the plasma membrane. Recently, we found that, in the absence of Munc18-1, the number of docked vesicles and the thickness of cortical F-actin are affected. In the present paper, I discuss the possible mechanism by which Munc18-1 modulates cortical F-actin and how it orchestrates the docking machinery via an interaction with syntaxin-1. Finally, a comparison of Munc18's role in embryonic mouse and adult bovine chromaffin cell model systems will be made to clarify observed differences in cortical F-actin as well as docking phenotypes.
Asunto(s)
Membrana Celular/metabolismo , Vesículas Secretoras/metabolismo , Actinas/metabolismo , Animales , Células Cromafines/citología , Células Cromafines/metabolismo , Citoesqueleto/metabolismo , Fusión de Membrana/fisiología , Proteínas Munc18/metabolismo , Vesículas Secretoras/ultraestructura , Sinapsis/metabolismo , Sintaxina 1/metabolismoRESUMEN
Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.