RESUMEN

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Heparitina Sulfato/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Caenorhabditis elegans , Estabilidad Proteica , Sindecanos/metabolismo

RESUMEN

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.

Asunto(s)

Caenorhabditis elegans/genética , Elementos Transponibles de ADN , Proteínas de Unión al ADN , Ingeniería Genética/métodos , Genoma/genética , Recombinación Genética , Transposasas , Animales , Animales Modificados Genéticamente , Recombinación Homóloga , Mutagénesis Insercional , Investigación

RESUMEN

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Músculos/metabolismo , Animales , Caenorhabditis elegans/citología , Clonación Molecular , Genoma/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Mitocondriales/metabolismo , Músculos/citología , Sistemas de Lectura Abierta/genética , Fenotipo , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo

RESUMEN

To identify the exact spot position of human, rat and chicken ribosomal proteins (RP) separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), a 2-DE system was designed to separate RP with a pI>8.6 according to their charge in the first dimension and to their molecular mass in the second dimension. Individual proteins were excised from the gels and identified by mass spectrometry after digestion by trypsin. In addition, a mixture of purified RP from these three species was also analyzed by tandem mass tag spectrometry. By combining those two methods 74 RP from human, 76 from rat and 67 from chicken were identified according to the nomenclature initially defined for rat liver RP and by using the Swiss-Prot/trEMBL databases. Whereas human and rat RP were well described, most of RP from chicken were not characterized in databases, since 35 out of 67 chicken RP identified in this study were not listed yet. We propose here the first comprehensive description of chicken RP and their comparison to those from human and rat.

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Proteínas Ribosómicas/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Pollos , Bases de Datos Factuales , Células HeLa , Humanos , Ratas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Tripsina/metabolismo , Células Tumorales Cultivadas

RESUMEN

In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Estructuras Citoplasmáticas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Distrofina/metabolismo , Proteínas Musculares/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Secuencia Conservada , Estructuras Citoplasmáticas/ultraestructura , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Proteínas con Dominio LIM , Microscopía Inmunoelectrónica , Modelos Biológicos , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Mutación/genética , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Zixina

RESUMEN

Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.

Asunto(s)

Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Expresión Génica , Receptores Acoplados a Proteínas G/genética , Receptores de Serotonina/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Células COS , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/metabolismo , Conducta Reproductiva , Homología de Secuencia de Aminoácido

RESUMEN

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.

Asunto(s)

Caenorhabditis elegans/genética , Elementos Transponibles de ADN , Genómica/métodos , Mutagénesis Insercional/métodos , Animales , Mapeo Cromosómico , Proyectos Piloto , Análisis de Secuencia de ADN , Transcripción Genética