RESUMEN
Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Western Blotting , Calcio/farmacología , Cruzamientos Genéticos , Relación Dosis-Respuesta a Droga , Drosophila , Proteínas de Drosophila , Electrofisiología , Genes Recesivos , Inmunohistoquímica , Proteínas de la Membrana/fisiología , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Neuromuscular/embriología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Neurotransmisores/metabolismo , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas SNARE , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteína 25 Asociada a Sinaptosomas , Temperatura , Factores de Tiempo , Transformación GenéticaRESUMEN
Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.
Asunto(s)
Drosophila melanogaster/fisiología , Exocitosis/fisiología , Neuropéptidos/biosíntesis , Animales , Factor Natriurético Atrial/metabolismo , Axones/fisiología , Western Blotting , Estimulación Eléctrica , Exocitosis/genética , Genotipo , Inmunohistoquímica , Larva , Microscopía Fluorescente , Neuropéptidos/genética , Neuropéptidos/metabolismo , Plásmidos/genética , Terminales Presinápticos/fisiología , Precursores de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
Although teleost fish have higher levels of brain aromatase activity than any other vertebrate group, its function remains speculative, and no study has identified its cellular basis. A previous study determined aromatase activity in a vocal fish, the plainfin midshipman (Porichthys notatus), and found highest levels in the telencephalon and lower levels in the sonic hindbrain, which was dimorphic between and within (males) sexes. We have now localized aromatase-containing cells in the midshipman brain both by immunocytochemistry using teleost-specific aromatase antibodies and by in situ hybridization using midshipman-specific aromatase probes. Aromatase-immuno-reactivity and mRNA hybridization signal are consistent with relative levels of aromatase activity in different brain regions: concentrated in the dimorphic sonic motor nucleus, in a band just beneath the periaqueductal gray in the midbrain, in ventricular regions in the hypothalamus, and highest levels in the telencephalon especially in preoptic and ventricular areas. Surprisingly, double-label immunofluorescence does not show aromatase-immunoreactive colocalization in neurons, but instead in radial glia throughout the brain. This is the first study to identify aromatase expression mostly, if not entirely, in glial cells under normal rather than brain injury-dependent conditions. The abundance of aromatase in teleosts may represent an adaptation linked to continual neurogenesis that is known to occur throughout an individual's lifetime among fishes. The localization of aromatase within the intersexually and intrasexually dimorphic vocal-motor circuit further implies a function in the expression of alternative male reproductive phenotypes and, more generally, the development of natural, individual variation of specific brain nuclei.
Asunto(s)
Aromatasa/biosíntesis , Encéfalo/enzimología , Neuroglía/enzimología , ARN Mensajero/biosíntesis , Animales , Especificidad de Anticuerpos , Aromatasa/genética , Batrachoidiformes , Encéfalo/citología , Encéfalo/fisiología , Técnica del Anticuerpo Fluorescente , Hipotálamo/citología , Hipotálamo/enzimología , Inmunohistoquímica , Hibridación in Situ , Mesencéfalo/citología , Mesencéfalo/enzimología , Neuroglía/citología , Sustancia Gris Periacueductal/citología , Sustancia Gris Periacueductal/enzimología , Caracteres Sexuales , Telencéfalo/citología , Telencéfalo/enzimología , Vocalización Animal/fisiologíaRESUMEN
A novel Drosophila mutant named Tripped-and-fell (Taf) was isolated in a F1 screen for dominant temperature sensitive paralytics. Recombination mapping using multiply marked chromosomes and P elements have pinpointed the locus of Taf to polytene band 93 on the right arm of the third chromosome (3R). When exposed to restrictive temperatures, both Taf heterozygotes and homozygotes paralyzed; however, homozygotes paralyzed at lower temperatures and took longer to recover than heterozygotes. There are also positive correlations between recovery time from paralysis and both duration and temperature of exposure. Electroretinograms (ERGs) revealed that both homozygotes and heterozygotes have a grossly normal light response at 22 degrees C, but at 37 degrees C, the ERGs from both homozygotes and heterozygotes are unable to maintain a normal sustained depolarization and have a reduced off-transient potential. The severity of the ERG repolarization phenotype is greater in homozygotes than in heterozygotes.
Asunto(s)
Actividad Motora/genética , Mutación , Parálisis/genética , Visión Ocular/genética , Animales , Mapeo Cromosómico , Drosophila melanogaster/genética , Electrorretinografía , Femenino , Genes Dominantes , Masculino , Fenotipo , Transmisión Sináptica/genética , TemperaturaRESUMEN
Technological advances in electrophysiology and molecular biology in the last two decades have led to great progress in ion channel research. The invention of the patch-clamp recording technique has enabled the characterization of the biophysical and pharmacological properties of single channels. Rapid progress in the development of molecular biology techniques and their application to ion channel research led to the cloning, in the 1980s, of genes encoding all major classes of voltage- and ligand-gated ionic channels. It has become clear that operationally defined channel types represent extended families of ionic channels. Several experimental approaches have been developed to test whether there is a correlation between the detection of particular ion channel subunit mRNAs and the electrophysiological response to a pharmacological or electrical stimulus in a cell. In one method, whole-cell patch-clamp recording is performed on a cell in culture or tissue-slice preparation. The biophysical and pharmacological properties of the ionic channels of interest are characterized and the cytoplasmic contents of the recorded cell are then harvested into the patch pipette. In a variant of this method, the physiological properties of a cell are characterized with a two-electrode voltage clamp and, following the recording, the entire cell is harvested for its RNA. In both methods, the RNA from a single cell is reverse-transcribed into cDNA by a reverse transcriptase and subsequently amplified by the polymerase chain reaction, i.e. by the so-called single-cell/reverse transcription/polymerase chain reaction method (SC-RT-PCR). This review presents an analysis of the results of work obtained by using a combination of whole-cell patch-clamp recording or two-electrode voltage clamp and SC-RT-PCR with emphasis on its potential and limitations for quantitative analysis.
Asunto(s)
Canales Iónicos/genética , Técnicas de Placa-Clamp/métodos , Reacción en Cadena de la Polimerasa/métodos , Activación Transcripcional , Animales , Cartilla de ADN , Canales Iónicos/metabolismo , Neuronas/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Procesamiento Postranscripcional del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defect in these mutants, we have examined conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline could increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner, and the rate of fusion approached 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants could be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persisted in the absence of this protein. Tetanic stimulation could also increase the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants did not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP.
Asunto(s)
Calcio/farmacología , AMP Cíclico/farmacología , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas , Neurotransmisores/metabolismo , Cadmio/farmacología , Potenciales Evocados/efectos de los fármacos , Movimiento Fetal/efectos de los fármacos , Mutación , Unión Neuromuscular/efectos de los fármacos , Proteínas R-SNARE , Venenos de Araña/farmacología , Transmisión Sináptica/efectos de los fármacos , Factores de TiempoRESUMEN
Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, we have examined the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion. We generated a series of mutations, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous "minis" that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response.
Asunto(s)
Drosophila/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación/genética , Neuronas/metabolismo , Neurotransmisores/metabolismo , Animales , Femenino , Masculino , Proteínas de la Membrana/fisiología , Unión Neuromuscular/fisiología , Parálisis/genética , Proteínas R-SNARE , Sinapsis/metabolismoRESUMEN
Syntaxins are membrane proteins involved in vesicle trafficking and are required for the release of neurotransmitter at nerve terminals. The presence of syntaxins on target membranes has been hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins, the synaptobrevins or VAMPS. We have mutagenized syntaxin1 in Drosophila and have found that it links the mechanism of synaptic transmission to a distinct cell biological process: the cellularization of early embryos. This specialized form of cell division separates the 6,000 nuclei of the syncytial blastoderm into separate cells through the invagination of the surface membrane of the embryo. During this process, syntaxin1 protein is present on the newly forming lateral cell surfaces and invaginating cleavage furrows. This protein is derived both from maternal deposition of mRNA and protein and from early zygotic transcription. To analyze syntaxin1's role in early development, female germ line mosaics mutant for syntaxin1 expression were generated by mitotic recombination to reduce the maternal contribution. Visualizing the actin cytoskeleton and glycosylated surface proteins reveals that embryos with insufficient syntaxin1 have large acellular patches. The patches do not appear until cellularization begins, and the process fails entirely within these regions. These results provide genetic evidence that membrane trafficking is required for the cellularization of the syncytial blastoderm. We propose that the invagination of the surface membrane proceeds by the fusion of intracellular membrane vesicles with the surface. This reaction uses the same syntaxin1 protein as is required for neurotransmitter secretion at synapses. Thus, a single syntaxin can participate in trafficking steps that are functionally as distinct as synaptic transmission and cell division.
Asunto(s)
Ciclo Celular , Drosophila melanogaster/embriología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Ciclo Celular/genética , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Gástrula/citología , Gástrula/fisiología , Genes Letales , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE , Cigoto/citología , Cigoto/crecimiento & desarrolloRESUMEN
The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)) is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. We report here that the SNAP-25 gene (Snap) in the fruit fly Drosophila melanogaster has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene (Bark, 1993). Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on.
Asunto(s)
Drosophila melanogaster/genética , Proteínas de Insectos/genética , Proteínas de la Membrana , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Exones , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Proteína 25 Asociada a SinaptosomasRESUMEN
A novel NMDA receptor-like (NMDAR-L) cDNA was isolated that contained an open reading frame coding for a predicted polypeptide of 1115 amino acids that shares approximately 27% identity with NMDA receptor subunits. In situ hybridization experiments indicated that NMDAR-L mRNA was expressed in the developing rodent CNS. On postnatal day 1 (P1), NMDAR-L mRNA expression was pronounced in the entorhinal cortex, the subiculum and the thalamus, in layer V of the developing neocortex, in the superior and inferior colliculi, and various regions of the hindbrain, excluding the cerebellum. On P5, NMDAR-L mRNA was expressed in layer V of the neocortex, in the entorhinal cortex, in the subiculum, and in the thalamus. On P14, NMDAR-L mRNA was expressed in layers II-VI of the neocortex, in the entorhinal and piriform cortex, in the subiculum and CA1 field, and in the nucleus of the lateral olfactory tract. In the adult brain, NMDAR-L mRNA was detected predominately in the nucleus of the lateral olfactory tract. Injection of NMDAR-L cRNA into Xenopus oocytes did not lead to the expression of homomeric glutamate-activated channels. However, coinjection of the triple combination of NMDAR-L with NMDAR1 and NMDAR2B cRNAs led to a striking decrease in the current magnitude compared to currents obtained after coexpression of the double combination of NMDAR1 with NMDAR2B. While the function of NMDAR-L remains to be established, its developmental and regional expression pattern suggests that NMDAR-L may influence axonal outgrowth and synaptogenesis during brain development.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Roedores/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ratones , Sondas Moleculares/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Ratas , Distribución Tisular , XenopusRESUMEN
The combination of patch-clamp and molecular biology techniques has made it possible to characterize the pharmacological and biophysical properties of ion channels in single neurons and to screen for expression of specific mRNAs in the same cell. Following whole-cell recording, the cytoplasm of the cell is harvested, and RNA is reverse transcribed into cDNA and amplified in PCR with primers specific for individual ion channel subunits. Additional experiments can then be designed to relate structure and function at the protein level more directly, since cells appear to regulate the composition of ion channels at least partly at the posttranscriptional stage.
Asunto(s)
Canales Iónicos/genética , Canales Iónicos/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Cartilla de ADN , ARN/aislamiento & purificación , ARN/metabolismo , ADN Polimerasa Dirigida por ARN , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
A novel homeobox gene, SOHo-1, was isolated from embryonic chicken retina. On embryonic day 2 (E2), SOHo-1 is expressed in the retina, posterolateral otic pit, and neural tube anterior to the spinal cord. On E4, SOHo-1 is expressed at high levels in anterior retina and low levels in posterior retina, suggestive of a role in patterning the anterior-posterior axis. It is also expressed on E4 in the otocyst, the dorsal root ganglia, some cranial ganglia, and the second branchial arch. SOHo-1 expression in the otic pit and otocyst is restricted to regions that will give rise to the nonsensory tissues of the inner ear. SOHo-1 is not closely related to any identified vertebrate or Drosophila homeobox-containing genes. Since it is expressed in sensory-related structures and does not fit into existing classes of homeobox genes, we propose the name SOHo-1, for sensory organ homeobox-1.
Asunto(s)
Proteínas Aviares , Ganglios/metabolismo , Expresión Génica , Genes Homeobox , Proteínas del Tejido Nervioso/biosíntesis , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Embrión de Pollo , Cartilla de ADN , Drosophila/genética , Oído/embriología , Desarrollo Embrionario y Fetal , Ganglios/embriología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Biblioteca de Genes , Hibridación in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Retina/embriología , Homología de Secuencia de AminoácidoRESUMEN
Proteins associated with synaptic vesicles are likely to control the release of neurotransmitter. Because synaptic transmission is fundamentally similar between vertebrates and invertebrates, vesicle proteins from vertebrates that are important for synaptic transmission should be present in Drosophila as well. This investigation describes Drosophila homologs of vamp, synaptotagmin, and rab3 that are expressed in a pattern consistent with a function in Drosophila neurotransmission. One previously reported candidate (syb), a Drosophila homolog of the vamp or synaptobrevin proteins, has been shown to be expressed at very low levels in neurons and is most abundant in the gut. A neuronal Drosophila vamp (n-syb) is described here and is localized to chromosome band 62A. Northern analysis and in situ hybridizations to mRNA indicate that the novel vamp, as well as the genes for synaptotagmin (syt) and rab3 (drab3), is expressed in the Drosophila nervous system. These genes are widely (perhaps ubiquitously) expressed in the nervous system and we have no evidence of additional neuronal isoforms of synaptotagmin, vamp, or rab3. Immunoreactivity for synaptotagmin and vamp is located in synaptic regions of the nervous system. This distribution suggests that these molecules are components of synaptic vesicles in Drosophila. The conserved structure and neuronal expression pattern of these genes indicate that they may function in processes that are required for both vertebrate and invertebrate synaptic transmission. Because of their distribution in the nervous system and because n-syb, synaptotagmin, and drab3 do not appear to be in a family of functionally redundant homologs, we predict that mutation of these genes will have a profound neurological phenotype and that they are therefore good candidates for a genetic dissection in Drosophila.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Hormonas de Insectos/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuropéptidos/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Hormonas de Insectos/biosíntesis , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Neuropéptidos/biosíntesis , Sistemas de Lectura Abierta , Proteínas R-SNARE , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genética , Empalme Alternativo , Animales , Astrocitos/metabolismo , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Cartilla de ADN , Yoduro de Dimetilfenilpiperazina/farmacología , Expresión Génica , Variación Genética , Hipocampo/metabolismo , Humanos , Riñón , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Peso Molecular , N-Metilaspartato/farmacología , Factores de Crecimiento Nervioso/farmacología , Células PC12 , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Membranas Sinápticas/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.
Asunto(s)
Trasplante de Tejido Encefálico/fisiología , Cerebelo/trasplante , Neuronas/trasplante , Envejecimiento , Animales , Biomarcadores , Diferenciación Celular , Línea Celular , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Técnicas de Cultivo/métodos , Genes myc , Ratones , Ratones Endogámicos , Microscopía Electrónica , Neuronas/citología , Neuronas/ultraestructura , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
We deleted the cytoplasmic domain of the polymeric immunoglobulin receptor. When expressed in fibroblasts, the truncated receptor, like the wild-type, reaches the cell surface, can bind ligand, and is cleaved to secretory component. Unlike the wild-type, it is not endocytosed. When expressed in polarized Madin-Darby canine kidney cells, the mutant receptor is transported from the Golgi apparatus directly to the apical surface and cleaved to secretory component. In contrast, the wild-type receptor travels from the Golgi to the basolateral surface and is then endocytosed and sent to the apical surface. These results suggest that the cytoplasmic domain of the receptor is necessary for both basolateral localization and endocytosis.
Asunto(s)
Endocitosis , Inmunoglobulinas/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Citoplasma/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina A/metabolismo , Riñón/metabolismo , Cinética , Mutación , Polímeros , Receptores Inmunológicos/genéticaRESUMEN
We expressed cDNA for the rabbit polymeric immunoglobulin receptor in polarized Madin-Darby Canine Kidney epithelial cells, which normally do not produce this receptor. The receptor appeared to function as in vivo; dimeric IgA was transported from the basolateral to the apical surface and released into the apical medium, together with the cleaved fragment of the receptor, known as secretory component. This system enabled us, for the first time, to study quantitatively IgA transcytosis in vitro and thus make the following observations. First, greater than 90% of the newly made receptor that is ultimately cleaved to secretory component and released into the apical medium goes first to the basolateral surface. Second, transport of the receptor does not depend on ligand binding. Third, transcytosis of bound ligand has a t 1/2 of 30 min.
Asunto(s)
Inmunoglobulina A/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Transporte Biológico , Compartimento Celular , Clonación Molecular , Perros , Endocitosis , Epitelio/metabolismo , Técnica del Anticuerpo Fluorescente , Ratones , Componente Secretorio/metabolismoRESUMEN
Rabbits have a minimum of two polymeric immunoglobulin receptor primary translation products. A cDNA clone of the smaller product lacked two of the five receptor domains. These two domains were on a single exon. As there was one receptor gene, we suggest that this exon can be spliced in or out.
Asunto(s)
Empalme del ARN , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/análisis , ConejosRESUMEN
The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.