RESUMO
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.
Assuntos
Membrana Celular/metabolismo , Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Sinaptotagmina I/metabolismo , Sintaxina 1/metabolismo , Animais , Técnicas de Inativação de Genes , Camundongos , Proteínas Munc18/metabolismo , Sintaxina 1/genéticaRESUMO
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.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Munc18/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica , Animais , Estimulação Elétrica , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores , Feminino , Células HEK293 , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação , Gravidez , Ratos Wistar , Estresse Psicológico/metabolismoRESUMO
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.
Assuntos
Proteínas Qb-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Animais , Canais de Cálcio/metabolismo , Estruturas do Núcleo Celular/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Camundongos , Camundongos Mutantes , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
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.
Assuntos
Células Cromafins/metabolismo , Mutação/genética , Vesículas Sinápticas/genética , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Células Cromafins/ultraestrutura , Embrião de Mamíferos , Feminino , Masculino , Fusão de Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Proteínas SNARE/metabolismo , Via Secretória/genética , Transmissão Sináptica/genética , Vesículas Sinápticas/ultraestruturaRESUMO
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.
Assuntos
Proteínas Munc18/fisiologia , Proteínas SNARE/fisiologia , Transmissão Sináptica/fisiologia , Animais , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Mutação Puntual , Ligação Proteica , Vesículas SinápticasRESUMO
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.
Assuntos
Glândulas Suprarrenais/citologia , Proteínas de Transporte/metabolismo , Células Cromafins/citologia , Exocitose/fisiologia , Proteínas Nucleares/metabolismo , Vesículas Secretórias/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Proteínas de Transporte/genética , Catecolaminas/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Células Cromafins/ultraestrutura , Exocitose/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/genética , Transporte Proteico/fisiologia , Vesículas Secretórias/genética , Vesículas Secretórias/ultraestrutura , Capacitância Vascular/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Células Cromafins/ultraestrutura , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Vesículas Secretórias/ultraestrutura , Sinaptotagmina I/metabolismoRESUMO
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.
Assuntos
Transporte Proteico , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Vesículas Transportadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Células Cromafins/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/genética , Sinaptotagmina I/química , Sinaptotagmina I/genéticaRESUMO
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.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Microscopia de Contraste de Fase/métodos , Microtomia/métodos , Vitrificação , Animais , Humanos , Hidrazinas , Lisossomos/ultraestrutura , Camundongos , Microscopia de Fluorescência/métodos , Monócitos/citologia , Neurônios/citologia , Sinapses/ultraestruturaRESUMO
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.
Assuntos
Células Cromafins , Vesículas Transportadoras/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Western Blotting , Pesquisas com Embriões , Camundongos , Camundongos Knockout , Biogênese de Organelas , Isoformas de ProteínasRESUMO
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.
Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Fenótipo , Proteômica , Sinapses/metabolismo , Actinas/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Região CA1 Hipocampal/ultraestrutura , Diferenciação Celular , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Técnicas de Inativação de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Neuritos/metabolismo , Plasticidade Neuronal/fisiologia , Pseudópodes/metabolismo , Sinapses/patologia , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patologia , Espectrometria de Massas em TandemRESUMO
Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.
Assuntos
Diglicerídeos/fisiologia , Plasticidade Neuronal/fisiologia , Proteína Quinase C/fisiologia , Receptores Pré-Sinápticos/fisiologia , Transdução de Sinais/fisiologia , Animais , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Células Cromafins/metabolismo , Diglicerídeos/metabolismo , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Cinética , Lentivirus/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutação/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Ésteres de Forbol/farmacologia , Fosforilação , Gravidez , Proteína Quinase C/antagonistas & inibidores , Receptores Pré-Sinápticos/ultraestruturaRESUMO
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.
Assuntos
Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Sinapses/metabolismo , Animais , Hormônios/metabolismo , Camundongos , Neurotransmissores/metabolismo , Ligação ProteicaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Vesículas Secretórias/metabolismo , Actinas/metabolismo , Animais , Células Cromafins/citologia , Células Cromafins/metabolismo , Citoesqueleto/metabolismo , Fusão de Membrana/fisiologia , Proteínas Munc18/metabolismo , Vesículas Secretórias/ultraestrutura , Sinapses/metabolismo , Sintaxina 1/metabolismoRESUMO
Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.
Assuntos
Exocitose/fisiologia , Fusão de Membrana/fisiologia , Proteínas Munc18/metabolismo , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Células Cromafins/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Munc18/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Vesículas Sinápticas/genéticaRESUMO
Neuroendocrine cells like chromaffin cells and PC-12 cells are established models for transport, docking and secretion of secretory vesicles. In micrographs, these vesicles are recognized by their electron dense core. The analysis of secretory vesicle distribution is usually performed manually, which is labour-intensive and subject to human bias and error. We have developed an algorithm to analyze secretory vesicle distribution and docking in electron micrographs. Our algorithm automatically detects the vesicles and calculates their distance to the plasma membrane on basis of the pixel coordinates, ensuring that all vesicles are counted and the shortest distance is measured. We validated the algorithm on a several preparations of endocrine cells. The algorithm was highly accurate in recognizing secretory vesicles and calculating their distribution including vesicle-docking analysis. Furthermore, the algorithm enabled the extraction of parameters that cannot be measured manually like vesicle clustering. Taking together, the algorithm facilitates and expands the unbiased and efficient analysis of secretory vesicle distribution and docking.
Assuntos
Células Cromafins/ultraestrutura , Processamento Eletrônico de Dados/métodos , Vesículas Secretórias/fisiologia , Vesículas Secretórias/ultraestrutura , Algoritmos , Animais , Células Cultivadas , Células Cromafins/metabolismo , Embrião de Mamíferos , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Reprodutibilidade dos TestesRESUMO
Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light-electron microscopy.
Assuntos
Células Cultivadas/citologia , Animais , Polaridade Celular , Células Cultivadas/metabolismo , Células Cultivadas/ultraestrutura , Imunofluorescência , Secções Congeladas , Gelatina , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Células PC12 , Proteínas R-SNARE , Ratos , Inclusão do TecidoRESUMO
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.
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Bases de Dados de Proteínas , Proteoma , Precursor de Proteína beta-Amiloide/sangue , Biomarcadores/sangue , Encéfalo/metabolismo , Proteínas do Líquido Cefalorraquidiano/química , Cromatografia Líquida , Biologia Computacional , Humanos , Microscopia Eletrônica , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/metabolismo , Príons , Proteômica , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Reconhecimento Automatizado de Padrão/métodos , Actinas/genética , Glândulas Suprarrenais/metabolismo , Algoritmos , Animais , Antígeno CD146/genética , Antígeno CD146/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Citosol/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Camundongos Knockout , Camundongos Transgênicos , Potássio/metabolismo , Validação de Programas de ComputadorRESUMO
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.