RESUMEN
Small Rab GTPases, the largest group of small monomeric GTPases, regulate vesicle trafficking in cells, which are integral to many cellular processes. Their role in neurological diseases, such as cancer and inflammation have been extensively studied, but their implication in kidney disease has not been researched in depth. Rab3a and its effector Rabphillin-3A (Rph3A) expression have been demonstrated to be present in the podocytes of normal kidneys of mice rats and humans, around vesicles contained in the foot processes, and they are overexpressed in diseases with proteinuria. In addition, the Rab3A knockout mice model induced profound cytoskeletal changes in podocytes of high glucose fed animals. Likewise, RphA interference in the Drosophila model produced structural and functional damage in nephrocytes with reduction in filtration capacities and nephrocyte number. Changes in the structure of cardiac fiber in the same RphA-interference model, open the question if Rab3A dysfunction would produce simultaneous damage in the heart and kidney cells, an attractive field that will require attention in the future.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Riñón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Células Epiteliales/metabolismo , Humanos , Riñón/patología , Glomérulos Renales/metabolismo , Proteínas del Tejido Nervioso/fisiología , Podocitos/metabolismo , Proteínas de Transporte Vesicular/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab3A/fisiología , Rabfilina-3ARESUMEN
Rab3A is a small G-protein of the Rab family that is involved in the late steps of exocytosis. Here, we studied the role of Rab3A and its relationship with Munc13-1 and Munc18-1 during vesicle priming. Phorbol 12-myristate 13-acetate (PMA) is known to enhance the percentage of fusion-competent vesicles and this is mediated by protein kinase C (PKC)-independent Munc13-1 activation and PKC-dependent dissociation of Munc18-1 from syntaxin 1a. Our results show that the effects of PMA varied in cells overexpressing Rab3A or mutants of Rab3A and in cells with Rab3A knockdown. When Munc13-1 was overexpressed in Rab3A knockdown cells, secretion was completely inhibited. In cells overexpressing a Rab-interacting molecule (RIM)-binding deficient Munc13-1 mutant, 128-Munc13-1, the effects of Rab3A on PMA-induced secretion was abolished. The effect of PMA, which disappeared in cells overexpressing GTP-Rab3A (Q81L), could be reversed by co-expressing Munc18-1 but not its mutant R39C, which is unable to bind to syntaxin 1a. In cells overexpressing Munc18-1, manipulation of Rab3A activity had no effect on secretion. Finally, Munc18-1 enhanced the dissociation of Rab3A, and such enhancement correlated with exocytosis. In summary, our results support the hypothesis that the Rab3A cycle is coupled with the activation of Munc13-1 via RIM, which accounts for the regulation of secretion by Rab3A. Munc18-1 acts downstream of Munc13-1/RIM/Rab3A and interacts with syntaxin 1a allowing vesicle priming. Furthermore, Munc18-1 promotes Rab3A dissociation from vesicles, which then results in fusion.
Asunto(s)
Exocitosis/fisiología , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Secretoras/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Microscopía Confocal , Proteínas Munc18/genética , Proteínas del Tejido Nervioso/genética , Células PC12 , Fotoblanqueo , Unión Proteica , Transporte de Proteínas , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Secretoras/ultraestructura , Transfección , Proteína de Unión al GTP rab3A/genética , Proteína de Unión al GTP rab3A/metabolismoRESUMEN
Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca(2+)-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.
Asunto(s)
Calcio/fisiología , Exocitosis/fisiología , Genes Sobrepuestos , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/fisiología , Proteínas de Unión al GTP rab/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Células Cultivadas , Exocitosis/genética , Guanosina Difosfato/genética , Guanosina Difosfato/fisiología , Guanosina Trifosfato/genética , Guanosina Trifosfato/fisiología , Hipocampo/metabolismo , Proteoma/genética , Proteoma/fisiología , Ratas , Fracciones Subcelulares/metabolismo , Vesículas Sinápticas/genética , Proteínas de Unión al GTP rab/genética , Proteína de Unión al GTP rab3A/genéticaRESUMEN
Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Recently mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GTPase-activating protein were found to cause Warburg Micro syndrome and Martsolf syndrome, respectively, both of which exhibit mental retardation. We have found that loss of p130 in mice results in inhibition of Ca2+-dependent glutamate release from cerebrocortical synaptosomes and alters short-term plasticity in the hippocampal CA1 region, probably through the accumulation of the GTP-bound form of Rab3A. Here, we describe the procedures for the measurement of the GTP-bound pool of Rab3A with pull-down assay using mouse brains and the biochemical method for the measurement of glutamate release from mouse synaptosomes.
Asunto(s)
Proteína de Unión al GTP rab3A/fisiología , Anomalías Múltiples/genética , Animales , Calcio/metabolismo , Cricetinae , Ácido Glutámico/metabolismo , Humanos , Ratones , Sinaptosomas/metabolismo , Síndrome , Proteínas de Unión al GTP rab3/deficiencia , Proteína de Unión al GTP rab3A/análisisRESUMEN
Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1.
Asunto(s)
Encéfalo/fisiología , Células Cromafines/fisiología , Guanidina/metabolismo , Proteínas Munc18/metabolismo , Vesículas Secretoras/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Clonación Molecular , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Noqueados , Ratones Mutantes , Vesículas Secretoras/ultraestructura , Virus de los Bosques Semliki/genética , Proteína de Unión al GTP rab3A/genéticaRESUMEN
The acrosome reaction is a regulated Ca2+-dependent secretion event required for sperm-egg interaction. Previous studies indicate that the process requires Rab3-dependent tethering of membranes, SNARE complex assembly, and Ca2+-mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane-permeant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine-containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine-rich peptide, were able to translocate into sperm. Moreover, membrane-permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplasmic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocytosis. In conclusion, arginine-containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.
Asunto(s)
Acrosoma/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Exocitosis/fisiología , Espermatozoides/metabolismo , Proteína de Unión al GTP rab3A/fisiología , Acrosoma/enzimología , Animales , Células CHO , Cricetinae , Cricetulus , Activación Enzimática/fisiología , Femenino , Guanosina Trifosfato/fisiología , Humanos , Masculino , Prenilación de Proteína , Espermatozoides/enzimologíaRESUMEN
Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.
Asunto(s)
Melanotrofos/metabolismo , Proteínas de Unión al GTP rab3/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Capacidad Eléctrica , Exocitosis/fisiología , Inmunohistoquímica , Hibridación in Situ , Inyecciones , Melanotrofos/efectos de los fármacos , Melanotrofos/fisiología , Microscopía Confocal , Oligonucleótidos Antisentido/farmacología , Técnicas de Placa-Clamp , Hipófisis/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/metabolismo , Distribución Tisular , Proteínas de Unión al GTP rab3/metabolismo , Proteína de Unión al GTP rab3A/administración & dosificación , Proteína de Unión al GTP rab3A/metabolismo , Proteína de Unión al GTP rab3A/farmacologíaRESUMEN
Recent studies have suggested that two small GTPases, Rab3A and Rab27A, play a key role in the late steps of dense-core vesicle exocytosis in endocrine cells; however, neither the precise mechanisms by which these two GTPases regulate dense-core vesicle exocytosis nor the functional relationship between them is clear. In this study, we expressed a number of different Rab proteins, from Rab1 to Rab41 in PC12 cells and systematically screened them for those that are specifically localized on dense-core vesicles. We found that four Rabs (Rab3A, Rab27A, Rab33A, Rab37) are predominantly targeted to dense-core vesicles in PC12 cells, and that three of them (Rab3A, Rab27A, Rab33A) are endogenously expressed on dense-core vesicles. We further investigated the effect of silencing each Rab with specific small interfering RNA on vesicle dynamics by total internal reflection fluorescence microscopy in a single PC12 cell. Silencing either Rab3A or Rab27A in PC12 cells significantly decreased the number of dense-core vesicles docked to the plasma membrane without altering the kinetics of individual exocytotic events, whereas silencing of Rab33A had no effect at all. Simultaneous silencing of Rab3A and Rab27A caused a significantly greater decrease in number of vesicles docked to the plasma membrane. Our findings indicate that Rab3A and Rab27A cooperatively regulate docking step(s) of dense-core vesicles to the plasma membrane.
Asunto(s)
Exocitosis/fisiología , Proteínas de Unión al GTP rab/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Exocitosis/efectos de los fármacos , Células PC12 , ARN Interferente Pequeño/farmacología , Ratas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/fisiología , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rab/biosíntesis , Proteínas rab27 de Unión a GTP , Proteína de Unión al GTP rab3A/antagonistas & inhibidores , Proteína de Unión al GTP rab3A/biosíntesisRESUMEN
Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+ sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+ required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.
Asunto(s)
Hipocampo/citología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab3/fisiología , Proteína de Unión al GTP rab3A/fisiología , Animales , Cadmio/farmacología , Calcio/farmacología , Señalización del Calcio , Células Cultivadas , Potenciales Evocados/fisiología , Ratones , Ratones Noqueados , Factores de Tiempo , Proteínas de Unión al GTP rab3/deficiencia , Proteínas de Unión al GTP rab3/genética , Proteína de Unión al GTP rab3A/deficiencia , Proteína de Unión al GTP rab3A/genéticaRESUMEN
The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle--the acrosome--is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/alpha-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/alpha-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)-specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.
Asunto(s)
Acrosoma/fisiología , Calcio/farmacología , Exocitosis/fisiología , Proteínas SNARE/metabolismo , Espermatozoides/metabolismo , Proteína de Unión al GTP rab3A/fisiología , Reacción Acrosómica/fisiología , Toxinas Botulínicas/farmacología , Toxinas Botulínicas Tipo A , Calcimicina/farmacología , Calcio/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Etilenodiaminas/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Permeabilidad/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Espermatozoides/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagminas/fisiología , Toxina Tetánica/farmacología , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Neuronal communication is tightly regulated by presynaptic signaling, thereby temporarily and locally secreting one or more transmitters in order to exert propagation or modulation of network activity. In the last 2 decades our insight into the molecular regulation of presynaptic transmitter vesicle traffic and fusion has exponentionally grown due to the identification of specific functional interactions between presynaptic proteins involved in these processes. In addition, a plethora of extracellular and intracellular messengers regulate neurotransmitter release, occasionally leading to short- or long-term adaptations of the synapse to altered environmental signals. Important in this respect is the ability of various nerve terminals to diverge their output by differentiation in secretion of co-localized transmitters. This divergence in presynaptic signaling may converge in the postsynaptic target neuron or spread to neighbouring cells. In this review differential presynaptic signaling mechanisms will be related to their potential divergent roles in transmitter release.
Asunto(s)
Neurotransmisores/metabolismo , Terminales Presinápticos/fisiología , Transmisión Sináptica/fisiología , Animales , Señalización del Calcio/fisiología , Humanos , Fosfatos de Inositol/fisiología , Microscopía Electrónica , Modelos Biológicos , Nucleótidos Cíclicos/fisiología , Fosforilación , Terminales Presinápticos/ultraestructura , Vesículas Secretoras/fisiología , Vesículas Sinápticas/fisiología , Proteína de Unión al GTP rab3A/fisiologíaRESUMEN
The active zone protein RIM1alpha is required both for maintaining normal probability of neurotransmitter release and for long-term presynaptic potentiation at brain synapses. We now demonstrate that RIM1alpha(-/-) mice exhibit normal coordination and anxiety-related behaviors but display severely impaired learning and memory. Mice with a synaptotagmin 1 mutation, which selectively lowers release probability, and mice with Rab3A deletion, which selectively abolishes presynaptic long-term potentiation, do not exhibit this abnormality. Our data suggest that a decrease in release probability or a loss of presynaptic LTP alone is not sufficient to cause major behavioral alterations, but the combination of presynaptic abnormalities in RIM1alpha(-/-) mice severely alters learning and memory.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas de Unión al GTP/fisiología , Memoria/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Conducta Animal , Western Blotting/métodos , Encéfalo/metabolismo , Condicionamiento Clásico/fisiología , Miedo/fisiología , Hibridación in Situ/métodos , Aprendizaje/fisiología , Aprendizaje por Laberinto/fisiología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Actividad Motora/genética , Desempeño Psicomotor/fisiología , Tiempo de Reacción/genética , Percepción Espacial/fisiología , Conducta Espacial/fisiología , Sinaptotagmina I , Sinaptotagminas , Factores de Tiempo , Proteína de Unión al GTP rab3A/fisiologíaRESUMEN
Members of the Rab family of monomeric GTPases have been implicated in vesicle trafficking, and Rab3A, located on synaptic vesicles in neurones and secretory vesicles in neuroendocrine cells, is likely to be involved in vesicle fusion leading to neurotransmitter release. A hydrolysis-deficient mutant of Rab3A, Rab3AQ81L, has been shown to potently inhibit hormone release. Here we show that the inhibition of hormone release by Rab3AQ81L is activity-dependent. Bovine adrenal chromaffin cells were induced to express Rab3AQ81L and green fluorescent protein by adenoviral gene transfer of a bicistronic construct. Fluorescent cells were stimulated with single depolarizations and trains of depolarizing pulses in whole cell perforated patch clamp recordings, and exocytosis was detected with cell capacitance measurements and carbon fibre amperometry. When single depolarizations were used to evoke exocytosis, cells expressing Rab3AQ81L showed a 50% reduction in response amplitude. When trains of brief depolarizations (10 or 40 ms) were used to evoke exocytosis, responses rapidly declined to zero in cells expressing Rab3AQ81L. Wild-type Rab3A had effects similar to Rab3AQ81L, causing significant inhibition of exocytosis only during repetitive stimulation. Expression of Rab5A did not alter exocytosis evoked by single depolarizations or repetitive stimulation. Applying a long duration depolarization in the middle of a stimulus train revealed that exocytotic efficacy (capacitance increase per amount of calcium influx) was not decreased in Rab3AQ81L-expressing cells. Instead, the activity-dependent increase in exocytotic efficacy observed in control cells did not occur in Rab3AQ81L-expressing cells. Our results suggest that Rab3A in the GTP bound conformation prevents activity-dependent facilitation.
Asunto(s)
Células Cromafines/fisiología , Exocitosis/fisiología , Proteína de Unión al GTP rab3A/fisiología , Adenoviridae/genética , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Catecolaminas/metabolismo , Bovinos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Estimulación Eléctrica , Potenciales de la Membrana/fisiología , Microelectrodos , Modelos Neurológicos , Técnicas de Placa-Clamp , Plásmidos/genética , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Proteína de Unión al GTP rab3A/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/fisiologíaRESUMEN
Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over-expression of enhanced green fluorescent protein-tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge-coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.
Asunto(s)
Exocitosis , Microscopía Fluorescente/métodos , Vesículas Secretoras/química , Animales , Células Cultivadas , Proteínas Fluorescentes Verdes , Luz , Proteínas Luminiscentes , Fusión de Membrana , Microscopía Fluorescente/instrumentación , Células PC12 , Ratas , Vesículas Secretoras/metabolismo , Proteína de Unión al GTP rab3A/fisiologíaRESUMEN
Gene profiling in the central nervous system presents unique challenges due to the unprecedented heterogeneity of cells, systems and functions in time and space. We have employed a multidisciplinary approach using whole cell patch clamp recording and transcriptional analysis to define the genomic basis of trophin-induced hippocampal synaptic plasticity. Transcriptional analysis of single cells by linear amplification of antisense RNA has added a new dimension of sensitivity and selectivity to the study of the complex and heterogeneous population of neurons. We describe different gene expression profiling techniques that offer novel approaches to monitoring thousands of genes in parallel, fostering identification of circuits involved in learning and memory.
Asunto(s)
Encéfalo/fisiología , Hipocampo/fisiología , Factores de Crecimiento Nervioso/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Transcripción Genética , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Amplificación de Genes , Perfilación de la Expresión Génica/métodos , Genoma , Ratones , Ratones Noqueados/genética , ARN sin Sentido/genética , Ratas , Ratas Sprague-Dawley , Proteína de Unión al GTP rab3A/genética , Proteína de Unión al GTP rab3A/fisiologíaAsunto(s)
Exocitosis , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Proteína de Unión al GTP rab3A/fisiología , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Catecolaminas/metabolismo , Electrofisiología , Cinética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación , Células PC12 , Ratas , Serotonina/farmacocinética , Proteínas de Transporte de Serotonina en la Membrana Plasmática , TransfecciónRESUMEN
Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.
Asunto(s)
Ritmo Circadiano/genética , Mutación Puntual , Privación de Sueño/fisiopatología , Proteína de Unión al GTP rab3A/genética , Alelos , Animales , Western Blotting , Etilnitrosourea , Femenino , Homeostasis , Locomoción/genética , Locomoción/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Trastornos del Sueño-Vigilia/genética , Proteína de Unión al GTP rab3A/fisiologíaRESUMEN
Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J cells.
Asunto(s)
Páncreas/citología , Páncreas/metabolismo , Proteínas de Unión al GTP rab3/biosíntesis , Proteína de Unión al GTP rab3A/biosíntesis , Amilasas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Colecistoquinina/farmacología , Citosol/metabolismo , Dexametasona/farmacología , Exocitosis , Immunoblotting , Inmunohistoquímica , Microscopía Confocal , Plásmidos/metabolismo , Isoformas de Proteínas , Ratas , Transfección , Proteínas de Unión al GTP rab3/fisiología , Proteína de Unión al GTP rab3A/fisiologíaRESUMEN
The expression of rab3A and rab3D isoforms in the enteroendocrine, cholecystokinin-secreting, cell lines STC-1 and GLUTag is here demonstrated. In contrast, rab3B is undetectable in these two cell lines, and rab3C is only slightly expressed in GLUTag cells. Using a transient co-transfection system with human growth hormone as reporter protein, we show that overexpression of the GTPase-deficient mutant rab3AQ81L, but not rab3DQ81L, significantly decreases human growth hormone secretory responses to various agonists in STC-1 cells. These results indicate that endocrine cell lines of intestinal origin express rab3A and rab3D proteins, but the GTP-bound form of rab3A only acts as a negative modulator in the control of cholecystokinin secretion from STC-1 cells.