Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
Cell ; 186(24): 5308-5327.e25, 2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-37922900

RESUMO

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.


Assuntos
Oócitos , Proteínas , Gravidez , Animais , Feminino , Oócitos/metabolismo , Proteínas/metabolismo , Embrião de Mamíferos/metabolismo , Citoesqueleto , Ribossomos , Desenvolvimento Embrionário , Mamíferos
2.
J Physiol ; 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-39367860

RESUMO

The synaptic vesicle cluster (SVC) is an essential component of chemical synapses, which provides neurotransmitter-loaded vesicles during synaptic activity, at the same time as also controlling the local concentrations of numerous exo- and endocytosis cofactors. In addition, the SVC hosts molecules that participate in other aspects of synaptic function, from cytoskeletal components to adhesion proteins, and affects the location and function of organelles such as mitochondria and the endoplasmic reticulum. We argue here that these features extend the functional involvement of the SVC in synapse formation, signalling and plasticity, as well as synapse stabilization and metabolism. We also propose that changes in the size of the SVC coalesce with changes in the postsynaptic compartment, supporting the interplay between pre- and postsynaptic dynamics. Thereby, the SVC could be seen as an 'all-in-one' regulator of synaptic structure and function, which should be investigated in more detail, to reveal molecular mechanisms that control synaptic function and heterogeneity.

3.
Int J Mol Sci ; 21(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105896

RESUMO

Munc13 isoforms are constituents of the presynaptic compartment of chemical synapses, where they govern important steps in preparing synaptic vesicles for exocytosis. The role of Munc13-1, -2 and -3 is well documented in brain neurons, but less is known about their function and distribution among the neurons of the retina and their conventional and ribbon-type chemical synapses. Here, we examined the retinae of Munc13-1-, -2-, and -3-EXFP knock-in (KI) mice with a combination of immunocytochemistry, physiology, and electron microscopy. We show that knock-in of Munc13-EXFP fusion proteins did not affect overall retinal anatomy or synapse structure, but slightly affected synaptic transmission. By labeling Munc13-EXFP KI retinae with specific antibodies against Munc13-1, -2 and -3, we found that unlike in the brain, most retinal synapses seem to operate with a single Munc13 isoform. A surprising exception to this rule was type 6 ON bipolar cells, which expressed two Munc13 isoforms in their synaptic terminals, ubMunc13-2 and Munc13-3. The results of this study provide an important basis for future studies on the contribution of Munc13 isoforms in visual signal processing in the mammalian retina.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/fisiologia , Sinapses/fisiologia , Animais , Eletrorretinografia , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Retina/citologia , Retina/ultraestrutura , Transmissão Sináptica/fisiologia
4.
J Cell Sci ; 128(4): 638-44, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25609709

RESUMO

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.


Assuntos
Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Membrana/genética , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Tomografia com Microscopia Eletrônica , Exocitose/fisiologia , Feminino , Células Ciliadas Auditivas Internas/citologia , Audição/genética , Audição/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp
5.
J Neurosci ; 34(44): 14687-96, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25355221

RESUMO

Munc13-3 is a presynaptic protein implicated in vesicle priming that is strongly expressed in cerebellar granule cells (GCs). Mice deficient of Munc13-3 (Munc13-3(-/-)) show an increased paired-pulse ratio (PPR), which led to the hypothesis that Munc13-3 increases the release probability (pr) of vesicles. In the present study, we analyzed unitary synaptic connections between GCs and basket cells in acute cerebellar slices from wild-type and Munc13-3(-/-) mice. Unitary EPSCs recorded from Munc13-3(-/-) GCs showed normal kinetics and synaptic latency but a significantly increased PPR and fraction of synaptic failures. A quantal analysis revealed that neither the charge of single quanta nor the binominal parameter N were affected by loss of Munc13-3 but that pr was almost halved in Munc13-3(-/-). Neither presynaptic Ca(2+) influx was affected by deletion of Munc13-3 nor replenishment of the readily releasable vesicle pool. However, a high concentration of EGTA led to a reduction in EPSCs that was significantly stronger in Munc13-3(-/-). We conclude that Munc13-3 is responsible for an additional step of molecular and/or positional "superpriming" that substantially increases the efficacy of Ca(2+)-triggered release.


Assuntos
Cerebelo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos , Transmissão Sináptica/fisiologia
6.
Cerebellum ; 14(3): 264-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25617111

RESUMO

Munc13-3 is a member of the Munc13 family of synaptic vesicle priming proteins and mainly expressed in cerebellar neurons. Munc13-3 null mutant (Munc13-3 (-/-)) mice show decreased synaptic release probability at parallel fiber to Purkinje cell, granule cell to Golgi cell, and granule cell to basket cell synapses and exhibit a motor learning deficit at highest rotarod speeds. Since we detected Munc13-3 immunoreactivity in the dentate gyrus, as reported here for the first time, and current studies indicated a crucial role for the cerebellum in hippocampus-dependent spatial memory, we systematically investigated Munc13-3 (-/-) mice versus wild-type littermates of both genders with respect to hippocampus-related cognition and a range of basic behaviors, including tests for anxiety, sensory functions, motor performance and balance, sensorimotor gating, social interaction and competence, and repetitive and compulsive behaviors. Neither basic behavior nor hippocampus-dependent cognitive performance, evaluated by Morris water maze, hole board working and reference memory, IntelliCage-based place learning including multiple reversals, and fear conditioning, showed any difference between genotypes. However, consistent with a disturbed cerebellar reflex circuitry, a reliable reduction in the acoustic startle response in both male and female Munc13-3 (-/-) mice was found. To conclude, complete deletion of Munc13-3 leads to a robust decrease in the acoustic startle response. This readout of a fast cerebellar reflex circuitry obviously requires synaptic vesicle priming by Munc13-3 for full functionality, in contrast to other behavioral or cognitive features, where a nearly perfect compensation of Munc13-3 deficiency by related synaptic proteins has to be assumed.


Assuntos
Comportamento Animal/fisiologia , Cerebelo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Reflexo de Sobressalto/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Ansiedade/psicologia , Cerebelo/fisiologia , Cognição/fisiologia , Feminino , Hipocampo/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Desempenho Psicomotor/fisiologia , Comportamento Social , Memória Espacial/fisiologia , Transmissão Sináptica/fisiologia
7.
bioRxiv ; 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37662300

RESUMO

Neurotransmitter is released from dedicated sites of synaptic vesicle fusion within a synapse. Following fusion, the vacated sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance. Here, we find that replacement vesicles are clustered around this region by Intersectin-1. Specifically, Intersectin-1 forms dynamic molecular condensates with Endophilin A1 near release sites and sequesters vesicles around this region. In the absence of Intersectin-1, vesicles within 20 nm of the plasma membrane are reduced, and consequently, vacated sites cannot be replenished rapidly, leading to depression of synaptic transmission. Similarly, mutations in Intersectin-1 that disrupt Endophilin A1 binding result in similar phenotypes. However, in the absence of Endophilin, this replacement pool of vesicles is available but cannot be accessed, suggesting that Endophilin A1 is needed to mobilize these vesicles. Thus, our work describes a distinct physical region within a synapse where replacement vesicles are harbored for release site replenishment.

8.
Sci Adv ; 9(25): eadf6222, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37343100

RESUMO

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot resolve how the actions of individual complexes integrate. We used cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation, and environment. Our detailed morphological characterization suggests that sequential synaptic vesicle states precede neurotransmitter release, where Munc13-comprising bridges localize vesicles <10 nanometers and soluble N-ethylmaleimide-sensitive factor attachment protein 25-comprising bridges <5 nanometers from the plasma membrane, the latter constituting a molecularly primed state. Munc13 activation supports the transition to the primed state via vesicle bridges to plasma membrane (tethers), while protein kinase C promotes the same transition by reducing vesicle interlinking. These findings exemplify a cellular function performed by an extended assembly comprising multiple molecularly diverse complexes.


Assuntos
Transmissão Sináptica , Vesículas Sinápticas , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Fusão de Membrana , Membrana Celular/metabolismo , Neurotransmissores/metabolismo
9.
Neuron ; 110(2): 248-265.e9, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-34767769

RESUMO

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.


Assuntos
Dopamina , Transmissão Sináptica , Corpo Estriado , Dopamina/metabolismo , Exocitose , Sinapses/metabolismo
10.
Philos Trans R Soc Lond B Biol Sci ; 376(1821): 20190759, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33550951

RESUMO

Neurosecretory vesicles are highly specialized trafficking organelles that store neurotransmitters that are released at presynaptic nerve endings and are, therefore, important for animal cell-cell signalling. Despite considerable anatomical and functional diversity of neurons in animals, the protein composition of neurosecretory vesicles in bilaterians appears to be similar. This similarity points towards a common evolutionary origin. Moreover, many putative homologues of key neurosecretory vesicle proteins predate the origin of the first neurons, and some even the origin of the first animals. However, little is known about the molecular toolkit of these vesicles in non-bilaterian animals and their closest unicellular relatives, making inferences about the evolutionary origin of neurosecretory vesicles extremely difficult. By comparing 28 proteins of the core neurosecretory vesicle proteome in 13 different species, we demonstrate that most of the proteins are present in unicellular organisms. Surprisingly, we find that the vesicular membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein synaptobrevin is localized to the vesicle-rich apical and basal pole in the choanoflagellate Salpingoeca rosetta. Our 3D vesicle reconstructions reveal that the choanoflagellates S. rosetta and Monosiga brevicollis exhibit a polarized and diverse vesicular landscape reminiscent of the polarized organization of chemical synapses that secrete the content of neurosecretory vesicles into the synaptic cleft. This study sheds light on the ancestral molecular machinery of neurosecretory vesicles and provides a framework to understand the origin and evolution of secretory cells, synapses and neurons. This article is part of the theme issue 'Basal cognition: multicellularity, neurons and the cognitive lens'.


Assuntos
Evolução Biológica , Coanoflagelados/fisiologia , Proteínas R-SNARE/metabolismo , Vesículas Sinápticas/fisiologia
11.
Elife ; 102021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33749593

RESUMO

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Sinaptotagminas/metabolismo , Animais , Células Cromafins/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Exocitose , Fusão de Membrana , Camundongos , Camundongos Endogâmicos C57BL
12.
Nat Commun ; 12(1): 7129, 2021 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-34880248

RESUMO

The brain extracellular matrix (ECM) consists of extremely long-lived proteins that assemble around neurons and synapses, to stabilize them. The ECM is thought to change only rarely, in relation to neuronal plasticity, through ECM proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. Using multiple ECM labeling and imaging assays, from super-resolution optical imaging to nanoscale secondary ion mass spectrometry, both in culture and in brain slices, we find that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within ~3 days, in an activity-dependent fashion. Interfering with the recycling process perturbs severely neuronal function, strongly reducing synaptic vesicle exo- and endocytosis. We conclude that the neuronal ECM can be remodeled frequently through mechanisms that involve endocytosis and recycling of ECM proteins.


Assuntos
Endocitose , Matriz Extracelular/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Tenascina/metabolismo , Animais , Encéfalo/metabolismo , Epitopos , Proteínas da Matriz Extracelular/metabolismo , Complexo de Golgi , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia
13.
Neuron ; 108(5): 843-860.e8, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991831

RESUMO

Electron microscopy can resolve synapse ultrastructure with nanometer precision, but the capture of time-resolved, activity-dependent synaptic membrane-trafficking events has remained challenging, particularly in functionally distinct synapses in a tissue context. We present a method that combines optogenetic stimulation-coupled cryofixation ("flash-and-freeze") and electron microscopy to visualize membrane trafficking events and synapse-state-specific changes in presynaptic vesicle organization with high spatiotemporal resolution in synapses of cultured mouse brain tissue. With our experimental workflow, electrophysiological and "flash-and-freeze" electron microscopy experiments can be performed under identical conditions in artificial cerebrospinal fluid alone, without the addition of external cryoprotectants, which are otherwise needed to allow adequate tissue preservation upon freezing. Using this approach, we reveal depletion of docked vesicles and resolve compensatory membrane recycling events at individual presynaptic active zones at hippocampal mossy fiber synapses upon sustained stimulation.


Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Membranas Sinápticas/fisiologia , Membranas Sinápticas/ultraestrutura , Animais , Técnicas de Introdução de Genes/métodos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica/métodos , Microtomia/métodos , Técnicas de Cultura de Órgãos , Transporte Proteico/fisiologia
14.
Cell Rep ; 30(11): 3632-3643.e8, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32187536

RESUMO

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.


Assuntos
Hipocampo/fisiologia , Hipocampo/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Animais , AMP Cíclico/metabolismo , Potenciais Pós-Sinápticos Excitadores , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musgosas Hipocampais/fisiologia , Fibras Musgosas Hipocampais/ultraestrutura , Neurotransmissores/metabolismo , Técnicas de Cultura de Órgãos , Vesículas Secretórias/fisiologia , Vesículas Secretórias/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Fixação de Tecidos
15.
J Cell Biol ; 218(3): 1011-1026, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30782781

RESUMO

Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Vesículas Sinápticas/genética
16.
Methods Mol Biol ; 1538: 215-231, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27943193

RESUMO

Transmission electron microscopy serves as a valuable tool for synaptic structure-function analyses aimed at identifying morphological features or modifications associated with specific developmental stages or dysfunctional synaptic states. By utilizing cryo-preparation techniques to minimize the introduction of structural artifacts during sample preparation, and electron tomography to reconstruct the 3D ultrastructural architecture of a synapse, the spatial organization and morphological properties of synaptic organelles and subcompartments can be quantified with unparalleled precision. In this chapter, we present an experimental approach combining organotypic slice culture, high-pressure freezing, automated freeze-substitution, and electron tomography to investigate spatial relationships between synaptic vesicles and active zone release sites in synapses from lethal mouse mutants.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Hipocampo/citologia , Hipocampo/ultraestrutura , Imageamento Tridimensional/métodos , Sinapses/ultraestrutura , Animais , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Eletrônica de Transmissão/métodos
17.
Elife ; 62017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28598330

RESUMO

SUMO1-conjugation of proteins at neuronal synapses is considered to be a major post-translational regulatory process in nerve cell and synapse function, but the published evidence for SUMO1-conjugation at synapses is contradictory. We employed multiple genetic mouse models for stringently controlled biochemical and immunostaining analyses of synaptic SUMO1-conjugation. By using a knock-in reporter mouse line expressing tagged SUMO1, we could not detect SUMO1-conjugation of seven previously proposed synaptic SUMO1-targets in the brain. Further, immunostaining of cultured neurons from wild-type and SUMO1 knock-out mice showed that anti-SUMO1 immunolabelling at synapses is non-specific. Our findings indicate that SUMO1-conjugation of synaptic proteins does not occur or is extremely rare and hence not detectable using current methodology. Based on our data, we discuss a set of experimental strategies and minimal consensus criteria for the validation of SUMOylation that can be applied to any SUMOylation substrate and SUMO isoform.


Assuntos
Proteína SUMO-1/metabolismo , Sumoilação , Sinapses/metabolismo , Animais , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Camundongos , Proteína SUMO-1/genética , Coloração e Rotulagem
18.
Neuron ; 94(2): 304-311.e4, 2017 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-28426965

RESUMO

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.


Assuntos
Cálcio/metabolismo , Dendritos/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Sinapses/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Sinapses/fisiologia
19.
J Cell Biol ; 216(4): 1143-1161, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28264913

RESUMO

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Linhagem Celular , Hipocampo/metabolismo , Camundongos , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Transmissão Sináptica/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo
20.
Cell Rep ; 15(10): 2239-2250, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239031

RESUMO

Complexin (Cplx) proteins modulate the core SNARE complex to regulate exocytosis. To understand the contributions of Cplx to signaling in a well-characterized neural circuit, we investigated how Cplx3, a retina-specific paralog, shapes transmission at rod bipolar (RB)→AII amacrine cell synapses in the mouse retina. Knockout of Cplx3 strongly attenuated fast, phasic Ca(2+)-dependent transmission, dependent on local [Ca(2+)] nanodomains, but enhanced slower Ca(2+)-dependent transmission, dependent on global intraterminal [Ca(2+)] ([Ca(2+)]I). Surprisingly, coordinated multivesicular release persisted at Cplx3(-/-) synapses, although its onset was slowed. Light-dependent signaling at Cplx3(-/-) RB→AII synapses was sluggish, owing largely to increased asynchronous release at light offset. Consequently, propagation of RB output to retinal ganglion cells was suppressed dramatically. Our study links Cplx3 expression with synapse and circuit function in a specific retinal pathway and reveals a role for asynchronous release in circuit gain control.


Assuntos
Exocitose , Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retina/citologia , Retina/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/farmacologia , Exocitose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Corpos Multivesiculares/efeitos dos fármacos , Corpos Multivesiculares/metabolismo , Proteínas do Tecido Nervoso/deficiência , Retina/efeitos dos fármacos , Células Bipolares da Retina/efeitos dos fármacos , Células Bipolares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA