RESUMEN
In the past few years, yeast and cultured cells have been the model systems of choice for the study of protein sorting and transport. Recently, there has been a surge in research in these areas in Caenorhabditis elegans, with advances in experimental techniques and genomics. New in vivo assays that monitor endocytosis and neuronal transport have been used to delineate roles for several genes in these processes.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Genes de Helminto , Animales , Transporte Biológico/genética , Membrana Celular/metabolismo , Dendritas/metabolismo , Dineínas/metabolismo , Endocitosis/fisiología , Cinesinas/metabolismo , Modelos Genéticos , Neuronas Aferentes/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. We measured evoked postsynaptic currents at the neuromuscular junction in both wild-type and mutants and observed that the removal of SLO-1 greatly increased quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release.
Asunto(s)
Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , Potenciales Postsinápticos Excitadores/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio , Datos de Secuencia Molecular , Mutación/genética , Unión Neuromuscular/genética , Neurotransmisores/genética , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Terminales Presinápticos/metabolismo , XenopusRESUMEN
Little is known of mechanisms regulating presynaptic differentiation. We identified rpm-1 in a screen for mutants with defects in patterning of a presynaptic marker at certain interneuronal synapses. The predicted RPM-1 protein contains zinc binding, RCC1, and other conserved motifs. In rpm-1 mutants, mechanosensory neurons fail to accumulate tagged vesicles, retract synaptic branches, and ectopically extend axons. Some motor neurons branch and overgrow; others show altered synaptic organization. Expression of RPM-1 in the presynaptic mechanosensory neurons is sufficient to rescue phenotypes in these cells. Certain rpm-1 phenotypes are temperature sensitive, revealing that RPM-1 function can be bypassed by maintaining mutants at the permissive temperature at stages commensurate with synapse formation in wild-type animals. These results indicate that RPM-1 functions cell autonomously during synaptogenesis to regulate neuronal morphology.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Marcación de Gen , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Caenorhabditis elegans/anatomía & histología , Secuencia Conservada/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mecanorreceptores/fisiología , Datos de Secuencia Molecular , Neuronas Motoras/ultraestructura , Mutación/fisiología , Neuronas/metabolismo , Sinapsis/ultraestructura , Vesículas Sinápticas/ultraestructura , TemperaturaRESUMEN
Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas Portadoras/metabolismo , Genes de Helminto , Proteínas del Helminto/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Electrofisiología , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido , Proteínas del Helminto/genética , Locomoción/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/fisiología , Unión Neuromuscular/ultraestructura , Estructura Terciaria de Proteína , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular , Dedos de Zinc , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Rabfilina-3ARESUMEN
We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-tagged Caenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , Vesículas Sinápticas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Evolución Biológica , Caenorhabditis elegans/metabolismo , Células Cultivadas , Genes Reporteros , Hipocampo/citología , Inmunohistoquímica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , SinaptogirinasRESUMEN
We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63-64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.
Asunto(s)
Antígenos de Superficie/genética , Caenorhabditis elegans/genética , Proteínas del Helminto/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans , ADN Complementario , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas R-SNARE , Homología de Secuencia de Aminoácido , Transmisión Sináptica/fisiología , Sintaxina 1RESUMEN
The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Clatrina/biosíntesis , Endocitosis , Homocigoto , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Sistema Nervioso/metabolismo , Neurotransmisores/metabolismo , Fosfoproteínas/genética , Isoformas de Proteínas , Proteínas R-SNARE , Homología de Secuencia de Aminoácido , Vesículas Sinápticas/ultraestructura , VertebradosRESUMEN
Little is known about the development of presynaptic specializations. Recent studies that visualize tagged synaptic components in cultured cells and in vivo have identified molecular participants and reveal common features in cellular processes of presynaptic assembly.
Asunto(s)
Sistema Nervioso/crecimiento & desarrollo , Transducción de Señal/fisiología , Sinapsis/fisiología , Animales , Citoesqueleto/genética , Citoesqueleto/fisiología , Humanos , Sistema Nervioso/metabolismo , Transducción de Señal/genéticaRESUMEN
Rabphilin, a putative rab effector, interacts specifically with the GTP-bound form of the synaptic vesicle-associated protein rab3a. In this study, we define in vivo functions for rabphilin through the characterization of mutants that disrupt the Caenorhabditis elegans rabphilin homolog. The mutants do not display the general synaptic defects associated with rab3 lesions, as assayed at the pharmacological, physiological, and ultrastructural level. However, rabphilin mutants exhibit severe lethargy in the absence of mechanical stimulation. Furthermore, rabphilin mutations display strong synergistic interactions with hypomorphic lesions in the syntaxin, synaptosomal-associated protein of 25 kDa, and synaptobrevin soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) genes; double mutants were nonresponsive to mechanical stimulation. These synergistic interactions were independent of rab3 function and were not observed in rab3-SNARE double mutants. Our data reveal rab3-independent functions for rabphilin in the potentiation of SNARE function.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Conducta Animal/fisiología , Biomarcadores , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Electrofisiología , Guanosina Trifosfato/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Locomoción/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Fenotipo , Estimulación Física , Estructura Terciaria de Proteína/fisiología , Proteínas SNARE , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Fases del Sueño/genética , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab3/genética , Rabfilina-3ARESUMEN
The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2 delta 10) was constructed. The srb2 delta 10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II.
Asunto(s)
ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Aminoácidos , Deleción Cromosómica , Clonación Molecular , Análisis Mutacional de ADN , Inositol/metabolismo , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , Supresión Genética , Factores de Transcripción/fisiologíaRESUMEN
Synaptic specializations are difficult to visualize at the light microscope level in living preparations. To circumvent this problem, synaptic vesicle proteins were fused to green fluorescent protein (GFP) and expressed in C. elegans neurons. C. elegans synaptobrevin-GFP and synaptogyrin-GFP fusion proteins were observed to target to synaptic sites. This localization allowed the visualization of synaptic specializations in living animals with light microscopy. Restricted expression of synaptobrevin-GFP fusions in subsets of neurons enables the visualization of individual presynaptic varicosities. The cell biology underlying the sorting of synaptic vesicle proteins, trafficking of vesicles to terminals, and the development of presynaptic specializations is now more amenable to forward genetic analysis using these synaptic markers.
Asunto(s)
Proteínas Luminiscentes/análisis , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructura , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Técnicas de Transferencia de Gen , Genes de Helminto , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Reacción en Cadena de la Polimerasa/métodos , Proteínas R-SNARE , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , SinaptogirinasRESUMEN
The sdc-1 gene acts at an early step in the regulatory hierarchy that controls the choice of sexual fate in Caenorhabditis elegans. It functions at a point before the control of sex determination and X-chromosome dosage compensation diverge. Here we report that sdc-1 encodes a protein of 1,203 amino acids containing seven zinc fingers. This protein motif in combination with other genetic and molecular information suggests that sdc-1 is likely to function as an embryonic transcription factor regulating downstream genes involved specifically in the sex determination and dosage compensation pathways, or regulating other genes involved in the coordinate control of both processes. These results enhance our general understanding of sex determination strategies, which are already known to involve transcriptional regulation and alternative RNA splicing in Drosophila melanogaster, DNA rearrangements in Saccharomyces cerevisiae, and transcriptional regulation in mammals.
Asunto(s)
Caenorhabditis/genética , Análisis para Determinación del Sexo , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis/fisiología , Femenino , Biblioteca de Genes , Intrones , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
We have characterized how perturbations of normal synaptic activity influence the morphology of cholinergic SAB motor neurons that innervate head muscle in C. elegans. Mutations disrupting components of the presynaptic release apparatus, acetylcholine (ACh) synthesis or ACh loading into synaptic vesicles each induced sprouting of SAB axonal processes. These sprouts usually arose in the middle of the normal innervation zone and terminated with a single presynaptic varicosity. Sprouting SAB neurons with a similar morphology were also observed upon reducing activity in muscle, either by using mutants lacking a functional nicotinic ACh receptor subunit or through muscle-specific expression of a gain-of-function potassium channel. Analysis of temperature-sensitive mutants in the choline acetyltransferase gene revealed that the sprouting response to inactivity was developmentally regulated; reduction of synaptic activity in early larval stages, but not in late larval stages, induced both sprouting and addition of varicosities. Our results indicate that activity levels regulate the structure of certain synaptic connections between nerve and muscle in C. elegans. One component of this regulatory machinery is a retrograde signal from the postsynaptic cell that mediates the formation of synaptic connections.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Unión Neuromuscular/crecimiento & desarrollo , Acetilcolina/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Colina O-Acetiltransferasa/genética , Genes de Helminto , Larva/crecimiento & desarrollo , Larva/fisiología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Mutación , Unión Neuromuscular/fisiología , Transducción de Señal , Transmisión Sináptica/genéticaRESUMEN
A 9.7-kilobase pair segment of the Escherichia coli chromosome spanning the hisT and purF loci has been characterized. Six genes were identified in this region by complete DNA sequence analysis, in vivo expression in maxicells, and RNA transcript analysis. S1 nuclease analysis has demonstrated that some of these genes are part of the hisT or purF operons. Two of the newly identified genes, dedA and dedB, were localized immediately downstream of hisT in the hisT operon. Two other genes, denoted dedC and dedD, have been localized between the hisT and purF operons. The other two genes, dedE and dedF flank the purF gene. dedE has been previously described as the first gene in the purF operon (Makaroff, C.A., and Zalkin, H. (1985) J. Biol. Chem. 260, 10378-10387). dedF was localized downstream from purF and is part of the purF operon. In addition, dedF is homologous to the ubiX gene of Salmonella typhimurium. Adjacent to dedF is the E. coli homologue of the S. typhimurium argT locus encoding the lysine/arginine/ornithine-binding protein. All of the genes in this region of the chromosome were found to be transcribed in a counter-clockwise direction on the E. coli map which revises the direction of purF transcription.
Asunto(s)
Cromosomas/análisis , Escherichia coli/genética , Genes Bacterianos , Operón , Secuencia de Bases , Codón , Enzimas de Restricción del ADN/metabolismo , Procesamiento Automatizado de Datos , Regulación de la Expresión Génica , PlásmidosRESUMEN
Synaptotagmin is an abundant synaptic vesicle-associated protein proposed to be involved in calcium-mediated neurotransmitter release. Our molecular and genetic results demonstrate that, although synaptotagmin is required for the proper function of the presynaptic nerve terminal in C. elegans, some neurotransmitter release persists in synaptogamin mutants. In C. elegans neurons, synaptotagmin is localized to regions known to be rich in synapses and appears to be associated with synaptic vesicles. Mutants defective in the synaptotagmin gene, called snt-1, exhibit severe behavioral abnormalities that are characteristic of deficiencies in synaptic function, including severe locomotion, feeding, and defecation defects. The mutants are defective in exocytosis, since they accumulate acetylcholine, and are resistant to cholinesterase inhibitors, but they nevertheless remain sensitive to cholinergic receptor agonists. In spite of these exocytic defects, snt-1 mutants are capable of coordinated motor movements, indicating that the mutants do not have a complete block of neurotransmitter release.
Asunto(s)
Caenorhabditis elegans/genética , Proteínas de Unión al Calcio , Glicoproteínas de Membrana/genética , Proteínas del Tejido Nervioso/genética , Sinapsis/fisiología , Transmisión Sináptica/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Secuencia de Consenso , Defecación , Electrofisiología , Exocitosis/genética , Conducta Alimentaria , Eliminación de Gen , Expresión Génica , Locomoción , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/aislamiento & purificación , Alineación de Secuencia , Vesículas Sinápticas/química , SinaptotagminasRESUMEN
Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these models in vivo, we have isolated and characterized the first synaptobrevin mutants in metazoans and show that neurotransmission is severely disrupted in mutant animals. Mutants lacking snb-1 die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. We also have isolated and characterized several hypomorphic snb-1 mutants. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to the acetylcholinesterase inhibitor aldicarb, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic-helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Finally, we demonstrate that double mutants lacking both the v-SNAREs synaptotagmin and snb-1 are phenotypically similar to snb-1 mutants and less severe than syntaxin mutants. Our work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, our analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs.
Asunto(s)
Caenorhabditis elegans/genética , Proteínas de la Membrana/genética , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular , Animales , Conducta Animal/fisiología , Exocitosis/fisiología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Movimiento/fisiología , Mutagénesis/fisiología , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/química , Faringe/fisiología , Fenotipo , Estructura Terciaria de Proteína , Proteínas R-SNARE , Proteínas SNARE , Homología de Secuencia de AminoácidoRESUMEN
Synaptotagmin, an integral membrane protein of the synaptic vesicle, binds calcium and interacts with proteins of the plasma membrane. These observations suggest several possible functions for synaptotagmin in synaptic vesicle dynamics: it could facilitate exocytosis by promoting calcium-dependent fusion, inhibit exocytosis by preventing fusion, or facilitate endocytosis of synaptic vesicles from the plasma membrane by acting as a receptor for the endocytotic proteins of the clathrin AP2 complex. Here we show that synaptic vesicles are depleted at synaptic terminals in synaptotagmin mutants of the nematode Caenorhabditis elegans. This depletion is not caused by a defect in transport or by increased synaptic vesicle release, but rather by a defect in retrieval or synaptic vesicles from the plasma membrane. Thus we propose that, as well as being involved in exocytosis, synaptotagmin functions in vesicular recycling.
Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Aldicarb/farmacología , Animales , Caenorhabditis elegans/genética , Inhibidores de la Colinesterasa/farmacología , Exocitosis , Proteínas de Unión al GTP/metabolismo , Glicoproteínas de Membrana/genética , Mutación , Proteínas del Tejido Nervioso/genética , Membranas Sinápticas/metabolismo , Sinaptotagminas , Proteínas de Unión al GTP rab3RESUMEN
The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.
Asunto(s)
Anestésicos por Inhalación/farmacología , Caenorhabditis/genética , Proteínas de la Membrana/genética , Mutación , Aldicarb/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis/efectos de los fármacos , Cruzamientos Genéticos , Trastornos del Desarrollo Sexual , Genes Dominantes , Genes de Helminto , Halotano/farmacología , Isoflurano/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Fenotipo , Proteínas Qa-SNARE , Análisis de Regresión , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transmisión Sináptica/fisiología , Sinaptofisina/genética , Sinaptofisina/fisiología , Proteína 25 Asociada a SinaptosomasRESUMEN
GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.