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1.
J Biol Chem ; 286(46): 40276-86, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21926173

RESUMO

The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Multimerização Proteica/fisiologia , Membrana Celular/química , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Células HeLa , Humanos , Estrutura Quaternária de Proteína
2.
Mol Cell Neurosci ; 46(2): 409-18, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21111824

RESUMO

Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system.


Assuntos
Exossomos/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Exossomos/ultraestrutura , Glutamina/metabolismo , Microscopia Eletrônica de Transmissão , Neurônios/ultraestrutura , Ratos , Sinapses/ultraestrutura
3.
J Cell Sci ; 123(Pt 17): 2943-54, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20699355

RESUMO

The highly conserved ESCRT-III complex is responsible for deformation and cleavage of membranes during endosomal trafficking and other cellular activities. In humans, dominant mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD). The decade-long process leading to this cortical degeneration is not well understood. One possibility is that, akin to other neurodegenerative diseases, the pathogenic protein affects the integrity of dendritic spines and synapses before any neuronal death. Using confocal microscopy and 3D reconstruction, we examined whether expressing the FTD-linked mutants CHMP2B(intron5) and CHMP2B(Delta10) in cultured hippocampal neurons modified the number or structure of spines. Both mutants induced a significant decrease in the proportion of large spines with mushroom morphology, without overt degeneration. Furthermore, CHMP2B(Delta10) induced a drop in frequency and amplitude of spontaneous excitatory postsynaptic currents, suggesting that the more potent synapses were lost. These effects seemed unrelated to changes in autophagy. Depletion of endogenous CHMP2B by RNAi resulted in morphological changes similar to those induced by mutant CHMP2B, consistent with dominant-negative activity of pathogenic mutants. Thus, CHMP2B is required for spine growth. Taken together, these results demonstrate that a mutant ESCRT-III subunit linked to a human neurodegenerative disease can disrupt the normal pattern of spine development.


Assuntos
Espinhas Dendríticas/patologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Demência Frontotemporal/genética , Proteínas do Tecido Nervoso/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Dendritos/metabolismo , Dendritos/patologia , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Mutação , Ratos
4.
J Neurosci Res ; 87(1): 12-25, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18711726

RESUMO

In cultured hippocampal neurons and in adult brain, the splicing regulatory protein Sam68 is partially relocated to the somatodendritic domain and associates with dendritic polysomes. Transfer to the dendrites is activity-dependent. We have investigated the repertoire of neuronal mRNAs to which Sam68 binds in vivo. By using coimmunoprecipitation and microarray screening techniques, Sam68 was found to associate with a number of plasticity-related mRNA species, including Eef1a1, an activity-responsive mRNA coding for translation elongation factor eEF1A. In cortical neuronal cultures, translation of the Eef1a1 mRNA was strongly induced by neuronal depolarisation and correlated with enhanced association of Sam68 with polysomal mRNAs. The possible function of Sam68 in Eef1a1 mRNA utilization was studied by expressing a dominant-negative, cytoplasmic Sam68 mutant (GFP-Sam68DeltaC) in cultured hippocampal neurons. The level of eEF1A was lower in neurons expressing GFP-Sam68DeltaC than in control neurons, supporting the proposal that endogenous Sam68 may contribute to the translational efficiency of the Eef1a1 mRNA. These findings are discussed in the light of the complex, potentially crucial regulation of eEF1A biosynthesis during long-term synaptic change.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Fator 1 de Elongação de Peptídeos/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Hipocampo/citologia , Humanos , Imunoprecipitação/métodos , Ligação Proteica/fisiologia , Biossíntese de Proteínas , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Transfecção/métodos
5.
Neurosci Lett ; 379(3): 152-7, 2005 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-15843054

RESUMO

Long-term synaptic change in the cortex and the hippocampus is believed to require the highly localized delivery and translation of mRNAs in the dendritic shafts and spines. The molecular interactions that underlie local signalling between synapses and mRNAs are still largely undefined. After purification from total brain extracts, the NMDA receptor is known to be associated with numerous proteins, including the multifunctional RNA-binding factor TLS (also called FUS). In non-neural tissue, TLS is a vital nuclear protein with roles in DNA repair, homologous recombination, transcriptional regulation and pre-mRNA processing. We have examined the distribution of TLS in hippocampal neurones, both in the adult brain and in mature primary cultures, using subcellular fractionation and immunofluorescence techniques. TLS immunoreactivity is largely excluded from the neuronal nucleus and is found in the cytosol and in somatodendritic particles. In some of these particles, TLS colocalizes with Sam68, a nuclear RNA-binding protein that we previously showed is incorporated into dendritic RNA granules. Some of the TLS clusters also colocalize with NMDA receptor clusters. Finally, TLS clusters are occasionally seen within spine heads. The apparent removal of TLS from the nucleus might result in specific patterns of mRNA transcription or splicing in hippocampal neurones. TLS may also contribute to steering, anchoring or regulating mRNAs at synaptic sites.


Assuntos
Núcleo Celular/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/citologia , Neurônios/citologia , Proteína FUS de Ligação a RNA/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting/métodos , Fracionamento Celular/métodos , Células Cultivadas , Proteínas de Ligação a DNA , Embrião de Mamíferos , Imunofluorescência/métodos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo
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