RESUMO
The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Montagem de Clatrina , Mutação/genética , Proteínas Adaptadoras de Transporte Vesicular , Animais , Antígenos CD/metabolismo , Sítios de Ligação , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular , Clatrina/metabolismo , Citometria de Fluxo , Proteínas de Membrana Lisossomal , Substâncias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Fenótipo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Cones de Crescimento/metabolismo , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Axônios/metabolismo , Células COS , Adesão Celular , Células Cultivadas , Chlorocebus aethiops , Exocitose , Fusão de Membrana , Metaloendopeptidases/metabolismo , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Toxina Tetânica/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Proteínas de Transporte Vesicular , Animais , Transporte Biológico , Encéfalo/embriologia , Encéfalo/metabolismo , Caderinas/metabolismo , Caderinas/fisiologia , Compartimento Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Grânulos Citoplasmáticos/fisiologia , Embrião de Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Microscopia de Vídeo , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Neuritos/fisiologia , Neurônios/fisiologia , Células PC12 , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas R-SNARE , RNA Interferente Pequeno/farmacologia , Ratos , Proteínas SNARE , Transdução de Sinais , Frações SubcelularesRESUMO
Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.
Assuntos
Membrana Celular/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Células Musculares/metabolismo , Hipertonia Muscular/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Etilmaleimida/farmacologia , Transportador de Glucose Tipo 4 , Proteínas de Transporte de Monossacarídeos/genética , Células Musculares/citologia , Hipertonia Muscular/patologia , Proteínas Musculares/genética , Mioblastos/citologia , Mioblastos/metabolismo , Neurotoxinas/farmacologia , Proteínas R-SNARE , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas SNARERESUMO
Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/metabolismo , Complexo 3 de Proteínas Adaptadoras , Animais , Brefeldina A/farmacologia , Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Eletrofisiologia , Camundongos , Camundongos Transgênicos , Fibras Musgosas Hipocampais/efeitos dos fármacos , Pressão Osmótica , Sensibilidade e Especificidade , Sinapses/efeitos dos fármacos , Transmissão Sináptica , Técnicas de Cultura de Tecidos , Fatores de Transcrição/genéticaRESUMO
Intracellular membrane trafficking in eukaryotes involves the budding of vesicles from a donor compartment, their translocation, and subsequent fusion with a target membrane. This last step has been shown to involve SNARE proteins, classified into two categories, vesicular (v)-SNAREs and target (t)-SNAREs. It is the pairing of v- and t-SNAREs that is responsible for bringing the lipid bilayers together for membrane fusion. Key to the discovery of SNAREs is the sensitivity of their neuronal synaptic prototypes, which mediate the release of neurotransmitters, to clostridial neurotoxins. In this review, we focus on tetanus neurotoxin-sensitive and tetanus neurotoxin-insensitive v-SNAREs, in particular synaptobrevin and cellubrevin, both tetanus neurotoxin-sensitive and Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, also called VAMP7). The brevins are characterized by an RD sequence in the middle of their SNARE motif whereas TI-VAMP has an RG sequence. These two categories of exocytic v-SNAREs define two important routes to and from the plasma membrane: one sensitive, the other insensitive to tetanus neurotoxin. We also discuss the central role of the endosomal system that could be considered, as already suggested for Rab proteins, as a mosaic of v-SNAREs, thus raising the question of whether or not these two routes can merge, and if so, how and where.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/farmacologia , Toxina Tetânica/farmacologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Endossomos/metabolismo , Exocitose , Humanos , Bicamadas Lipídicas/química , Fusão de Membrana , Proteínas de Membrana/química , Modelos Biológicos , Proteínas R-SNARE , Proteínas SNARE , Transdução de Sinais , Vesículas Sinápticas/química , Proteína 3 Associada à Membrana da Vesícula , Proteínas de Transporte Vesicular/efeitos dos fármacosRESUMO
To assess if membrane diffusion could affect the kinetics of receptor recruitment at adhesive contacts, we transfected neurons with green fluorescent protein-tagged immunoglobin cell adhesion molecules of varying length (25-180 kD), and measured the lateral mobility of single quantum dots bound to those receptors at the cell surface. The diffusion coefficient varied within a physiological range (0.1-0.5 microm(2)/s), and was inversely proportional to the size of the receptor. We then triggered adhesive contact formation by placing anti-green fluorescent protein-coated microspheres on growth cones using optical tweezers, and measured surface receptor recruitment around microspheres by time-lapse fluorescence imaging. The accumulation rate was rather insensitive to the type of receptor, suggesting that the long-range membrane diffusion of immunoglobin cell adhesion molecules is not a limiting step in the initiation of neuronal contacts.
Assuntos
Biofísica/métodos , Membrana Celular , Neurônios/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Difusão , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Imunoglobulinas/química , Cinética , Microscopia de Fluorescência , Peso Molecular , Ligação Proteica , Ratos , Fatores de Tempo , TransfecçãoRESUMO
Membrane fusion depends on the formation of a complex of four SNARE motifs, three that bear a central glutamine and are localized in the target membrane (t-SNARE) and one that bears an arginine and is localized in the donor vesicle (v-SNARE). We have characterized the arginine 56 to proline mutant (R56P) of synaptobrevin-2 (Sb). SbR56P was blocked at the plasma membrane in association with the endogenous plasma membrane t-SNARE due to an inhibition of SNARE complex dissociation, suggesting that the plasma membrane is its first target. Cell surface blockade of SbR56P could be rescued by coexpression of synaptophysin, a partner of Sb. Sb was blocked at the plasma membrane but SNARE complexes were unaffected in cells expressing defective dynamin, indicating that the phenotype of SbR56P was not due to an internalization defect. When expressed in neurons, SbR56P localized both to axonal and dendritic plasma membranes, showing that both domains are initial targets of Sb. The R56P mutation affects a highly conserved position in v-SNAREs, and might thus provide a general tool for identifying their first target membranes.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Mutação/genética , Proteínas de Transporte Vesicular/metabolismo , Animais , Arginina/genética , Axônios/metabolismo , Células Cultivadas , Dendritos/metabolismo , Células HeLa , Hipocampo/citologia , Temperatura Alta/efeitos adversos , Humanos , Íons/química , Cinética , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Prolina/genética , Ligação Proteica , Proteínas R-SNARE , Coelhos , Ratos , Proteínas SNARE , Termodinâmica , Proteínas de Transporte Vesicular/químicaRESUMO
SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] proteins are essential for membrane fusion but their regulation is not yet fully understood. We have previously shown that the amino-terminal Longin domain of the v-SNARE TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein)/VAMP7 plays an inhibitory role in neurite outgrowth. The goal of this study was to investigate the regulation of TI-VAMP as a model of v-SNARE regulation. We show here that the Longin domain (LD) plays a dual role. First, it negatively regulates the ability of TI-VAMP and of a Longin/Synaptobrevin chimera to participate in SNARE complexes. Second, it interacts with the adaptor complex AP-3 and this interaction targets TI-VAMP to late endosomes. Accordingly, in mocha cells lacking AP-3 delta, TI-VAMP is retained in an early endosomal compartment. Furthermore, TI-VAMPc, an isoform of TI-VAMP lacking part of the LD, does not interact with AP-3, and therefore is not targeted to late endosomes; however, this shorter LD still inhibits SNARE-complex formation. These findings support a mechanism controlling both localization and function of TI-VAMP through the LD and clathrin adaptors. Moreover, they point to the amino-terminal domains of SNARE proteins as multifunctional modules responsible for the fine tuning of SNARE function.