a-rapid-protocol-for-integrating-extrachromosomal-arrays-with-high

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the <em>C. elegans</em> Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Université Claude Bernard Lyon

CNRS UMR 5534

Dystrobrevin is one of the intracellular components of the transmembrane dystrophin-glycoprotein complex (DGC). The functional role of this complex in normal and pathological situations has not yet been clearly established. Dystrobrevin disappears from the muscle membrane in Duchenne muscular dystrophy (DMD), which results from dystrophin mutations, as well as in limb girdle muscular dystrophies (LGMD), which results from mutations affecting other members of the DGC complex. These findings therefore suggest that dystrobrevin may play a pivotal role in the progression of these clinically related diseases. In this study, we used the Caenorhabditis elegans model to address the question of the relationship between dystrobrevin binding to dystrophin and dystrobrevin function. Deletions of the dystrobrevin protein were performed and the ability of the mutated forms to bind to dystrophin was tested both in vitro and in a two-hybrid assay, as well as their ability to rescue dystrobrevin (dyb-1) mutations in C. elegans. The deletions affecting the second helix of the Dyb-1 coiled-coil domain abolished the binding of dystrobrevin to dystrophin both in vitro and in the two-hybrid assay. These deletions also abolished the rescuing activity of a functional transgene in vivo. These results are consistent with a model according to which dystrobrevin must bind to dystrophin to be able to function properly.

Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans.

Duchenne muscular dystrophy is one of the most common neuromuscular diseases. It is caused by mutations in the dystrophin gene. Dystrobrevins are dystrophin-associated proteins potentially involved in signal transduction. The nematode Caenorhabditis elegans possesses one dystrophin-like (dys-1) and one dystrobrevin-like (dyb-1) gene. Mutations of dyb-1 and dys-1 lead to similar phenotypes, comprising hyperactivity and a tendency to hypercontract, which suggest that these proteins may participate in a common function. We show here that overexpression of the Dyb-1 protein delays the onset of the myopathy observed in the C. elegans double mutant (dys-1; hlh-1 mutations). This finding indicates that, in C. elegans, (1) the absence of dystrophin can be partly compensated for by extra doses of dystrobrevin, and (2) dystrobrevin is partly functional in absence of dystrophin.

Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans.

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy (DMD). Neither the function of dystrophin nor the physiopathology of the disease have been clearly established so far. In mammals, the dystrophin-glycoprotein complex (DGC) includes dystrophin, as well as transmembrane and cytoplasmic proteins. Since Caenorhabditis elegans possesses a dystrophin-like gene (dys-1), we investigated whether homologues of the DGC members could also be found in the C. elegans genome. Conserved homologues were found for dystroglycan, delta/gamma-sarcoglycan and syntrophin. Divergent but related proteins were found for alpha- and beta-sarcoglycans. No sarcospan counterpart was found. The expression of the conserved homologues was inactivated using the RNA interference technique. Phenotypes similar to that of dys-1 were obtained, both in the wild-type background and in combination with other mutations. These results strongly suggest that a protein complex comprising functional analogies with the DGC exists in C. elegans.

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans.

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.

The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin.

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy.

During evolution, both the architecture and the cellular physiology of muscles have been remarkably maintained. Striated muscles of invertebrates, although less complex, strongly resemble vertebrate skeletal muscles. In particular, the basic contractile unit called the sarcomere is almost identical between vertebrates and invertebrates. In vertebrate muscles, sarcomeric actin filaments are anchored to attachment points called Z-disks, which are linked to the extra-cellular matrix (ECM) by a muscle specific focal adhesion site called the costamere. In this review, we focus on the dense body of the animal model Caenorhabditis elegans. The C. elegans dense body is a structure that performs two in one roles at the same time, that of the Z-disk and of the costamere. The dense body is anchored in the muscle membrane and provides rigidity to the muscle by mechanically linking actin filaments to the ECM. In the last few years, it has become increasingly evident that, in addition to its structural role, the dense body also performs a signaling function in muscle cells. In this paper, we review recent advances in the understanding of the C. elegans dense body composition and function.

The C. elegans dense body: anchoring and signaling structure of the muscle.

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.

DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1.

We previously reported the use of the cheap and fast-growing nematode Caenorhabditis elegans to search for molecules, which reduce muscle degeneration in a model for Duchenne Muscular Dystrophy (DMD). We showed that Prednisone, a steroid that is generally prescribed as a palliative treatment to DMD patients, also reduced muscle degeneration in the C. elegans DMD model. We further showed that this strategy could lead to the discovery of new and unsuspected small molecules, which have been further validated in a mammalian model of DMD, i.e. the mdx mouse model. These proof-of-principles demonstrate that C. elegans can serve as a screening tool to search for drugs against neuromuscular disorders. Here, we report and discuss two methodologies used to screen chemical libraries for drugs against muscle disorders in C. elegans. We first describe a manual method used to find drugs against DMD. We further present a semi-automated method, which is currently in use for the search of drugs against the Schwartz-Jampel Syndrome (SJS). Both assays are simple to implement and can be readily transposed and/or adapted to screens against other muscle/neuromuscular diseases, which can be modeled in the worm. Finally we discuss, with respect to our experience and knowledge, the different parameters that have to be taken into account before choosing one or the other method.

Caenorhabditis elegans as a chemical screening tool for the study o f neuromuscular disorders. Manual and semi-automated methods.

&#x4F7F;&#x7528;&#x540C;&#x6E90;&#x91CD;&#x7EC4;&#x901A;&#x5E38;&#x5DE5;&#x7A0B;&#x5E08;&#x7684;&#x7EBF;&#x866B;&#x57FA;&#x56E0;&#x7EC4;&#x7684;&#x65B9;&#x6CD5;&#x4F7F;&#x7528;&#x643A;&#x5E26;&#x7279;&#x5B9A;&#x63D2;&#x5165;&#x7684;&#x5916;&#x6E90;&#x7684;&#x8F6C;&#x5EA7;&#x5B50; Mos1 &#x83CC;&#x682A;&#x3002;&#x5DF2;&#x77E5; Mos1 &#x63D2;&#x5165;&#x7B49;&#x4F4D;&#x57FA;&#x56E0;&#x7684;&#x5927;&#x96C6;&#x5408;&#xFF0C;&#x56E0;&#x6B64;&#xFF0C;&#x7EBF;&#x866B;&#x7814;&#x7A76;&#x754C;&#x666E;&#x904D;&#x611F;&#x5174;&#x8DA3;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x6B64;&#x5904;&#x63CF;&#x8FF0; Mos1 &#x63D2;&#x5165;&#x7A81;&#x53D8;&#x682A;&#x5927;&#x91CF;&#x9986;&#x85CF;&#x5EFA;&#x8BBE;&#x7684;&#x534A;&#x81EA;&#x52A8;&#x65B9;&#x6CD5;&#x7684;&#x4F18;&#x5316;&#x3002;&#x5728;&#x751F;&#x4EA7;&#x9AD8;&#x5CF0;&#x671F;&#xFF0C;&#x6BCF;&#x6708;&#x751F;&#x6210;&#x4E86;&#x591A;&#x4E2A; 5,000 &#x83CC;&#x682A;&#x3002;&#x8FD9;&#x4E9B;&#x83CC;&#x682A;&#x7136;&#x540E;&#x53D7;&#x5230;&#x5206;&#x5B50;&#x5206;&#x6790;&#x548C;&#x591A;&#x4E2A; 13,300 Mos1 &#x63D2;&#x5165;&#x7684;&#x7279;&#x70B9;&#x3002;&#x9664;&#x4E86;&#x9488;&#x5BF9;&#x76F4;&#x63A5;&#x8D85;&#x8FC7; 4,700 &#x7684;&#x57FA;&#x56E0;&#xFF0C;&#x8FD9;&#x4E9B;&#x57FA;&#x56E0;&#x4EE3;&#x8868;&#x57FA;&#x672C;&#x4E0A;&#x6240;&#x6709;&#x7EBF;&#x866B;&#x57FA;&#x56E0;&#x548C;&#x6539;&#x6027;&#x7684; 40%&#x4EE5;&#x4E0A;&#x7684;&#x5DE5;&#x7A0B;&#x5220;&#x9664;&#x8D77;&#x70B9;&#x7684;&#x6F5C;&#x529B;&#x3002;&#x6B64;&#x96C6;&#x5408;&#x7684;&#x7A81;&#x53D8;&#x4F53;&#xFF0C;&#x751F;&#x6210;&#x7684;&#x6B27;&#x6D32; NEMAGENETAG &#x8D22;&#x56E2;&#xFF0C;&#x4E3B;&#x6301;&#x4E0B;&#x516C;&#x5F00;&#x4E5F;&#x53EF;&#x8868;&#x793A;&#x4E00;&#x79CD;&#x91CD;&#x8981;&#x7684;&#x7814;&#x7A76;&#x8D44;&#x6E90;&#x3002;

&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x96C6;&#x5408;&#x7684; Mos1 &#x8F6C;&#x5EA7;&#x5B50;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x7684;&#x7814;&#x7A76;&#x793E;&#x533A;&#x7EBF;&#x866B;&#x3002;

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.

A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community.

Les m&eacute;thodes qui utilisent la recombinaison &agrave; l&#39;ing&eacute;nieur le g&eacute;nome de c. elegans couramment utilisent souches porteuses des insertions sp&eacute;cifiques du transposon Mos1 h&eacute;t&eacute;rologue. Une grande collection d&#39;all&egrave;les d&#39;insertion Mos1 connus serait donc d&#39;int&eacute;r&ecirc;t g&eacute;n&eacute;ral pour la communaut&eacute; de recherche de c. elegans. Nous d&eacute;crivons ici l&#39;optimisation d&#39;une m&eacute;thode semi-automatique pour la construction d&#39;une importante collection de souches mutantes de Mos1 d&#39;insertion. Au pic de production, plus de 5 000 souches ont &eacute;t&eacute; g&eacute;n&eacute;r&eacute;s par mois. Ces souches ont &eacute;t&eacute; ensuite soumis &agrave; l&#39;analyse mol&eacute;culaire et plus de 13 300 insertions Mos1 caract&eacute;ris&eacute;es. En plus de cibler directement plus de 4 700 g&egrave;nes, ces all&egrave;les repr&eacute;sentent le potentiel point de d&eacute;part pour la suppression machin&eacute;e d&#39;essentiellement tous les g&egrave;nes de c. elegans et la modification de plus de 40 % d&#39;entre eux. Cette collection de mutants, g&eacute;n&eacute;r&eacute; sous les auspices du consortium europ&eacute;en NEMAGENETAG, est accessible au public et repr&eacute;sente une ressource de recherche important.

Un g&eacute;nome-large Collection de Mos1 Transposon Mutants d&#39;Insertion pour le c. elegans, milieu de la recherche.

Methoden, mit denen homologe Rekombination des Genoms von C. Elegans h&auml;ufig Ingenieur verwenden St&auml;mme tragen bestimmte Einf&uuml;gungen von der heterologen Transposon Mos1. Deshalb w&auml;re eine gro&szlig;e Sammlung von bekannten Mos1 Einf&uuml;gung Allele von allgemeinem Interesse f&uuml;r die Forschergemeinschaft C. Elegans. Wir beschreiben hier die Optimierung einer halbautomatischen Methodik f&uuml;r den Bau einer betr&auml;chtlichen Sammlung von Mos1 Einf&uuml;gung mutant St&auml;mmen. Bei Maximalf&ouml;rderung wurden mehr als 5.000 St&auml;mme pro Monat generiert. Diese St&auml;mme wurden dann molekulare Analyse und mehr als 13.300 Mos1 Einf&uuml;gungen charakterisiert. Neben direkt mehr als 4.700 Gene targeting, repr&auml;sentieren diese Allele das Potenzial, die Ausgangspunkt f&uuml;r die ver&auml;nderte L&ouml;schung von im Wesentlichen alle C. Elegans Gene und die &Auml;nderung von mehr als 40 % von ihnen. Diese Sammlung von Mutanten, erzeugt unter der Schirmherrschaft des Europ&auml;ischen NEMAGENETAG Consortium, ist &ouml;ffentlich zug&auml;nglich und stellt eine wichtige Forschung-Ressource dar.

Einer genomweiten Sammlung von Mos1 Transposon Insertion Mutanten f&uuml;r die C. Elegans Forschungsgemeinschaft.

Metodi che utilizzano la ricombinazione omologa all&#39;ingegnere del genoma di c. elegans comunemente utilizzano ceppi che trasportano specifici inserimenti del Trasposone eterologa Mos1. Una grande collezione di alleli di inserimento Mos1 noti sarebbe quindi di interesse generale per la comunit&agrave; di ricerca di c. elegans. Descriviamo qui l&#39;ottimizzazione di una metodologia per la costruzione di una notevole collezione di ceppi mutanti Mos1 inserimento semi-automatica. Con produzione di picco, pi&ugrave; di 5.000 ceppi sono stati generati al mese. Questi ceppi sono stati quindi soggetti ad analisi molecolare e pi&ugrave; di 13.300 inserimenti Mos1 caratterizzati. Oltre a mirare direttamente pi&ugrave; di 4.700 geni, questi alleli rappresentano il punto di partenza per l&#39;eliminazione di ingegneria di sostanzialmente tutti i geni di c. elegans e la modifica di oltre il 40% del loro potenziale. Questa collezione di mutanti, generato sotto l&#39;egida del Consorzio europeo NEMAGENETAG, &egrave; disponibile al pubblico e rappresenta una risorsa importante di ricerca.

Un Genome-Wide collezione di Mos1 Transposon inserimento mutanti per la comunit&agrave; di ricerca di c. elegans.

&#x76F8;&#x540C;&#x7D44;&#x63DB;&#x3048;&#x7DDA;&#x866B;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x3092;&#x3088;&#x304F;&#x30A8;&#x30F3;&#x30B8;&#x30CB;&#x30A2;&#x306B;&#x4F7F;&#x7528;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x7570;&#x7A2E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Mos1 &#x306E;&#x7279;&#x5B9A;&#x306E;&#x633F;&#x5165;&#x3092;&#x904B;&#x3076;&#x7DCA;&#x5F35;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x65E2;&#x77E5; Mos1 &#x633F;&#x5165;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5927;&#x898F;&#x6A21;&#x306A;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x7DDA;&#x866B;&#x306E;&#x7814;&#x7A76;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30C6;&#x30A3;&#x306E;&#x4E00;&#x822C;&#x7684;&#x306A;&#x95A2;&#x5FC3;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3053;&#x3053;&#x3067;&#x534A;&#x81EA;&#x52D5;&#x5316;&#x3055;&#x308C;&#x305F;&#x65B9;&#x6CD5;&#x8AD6;&#x306E;&#x5EFA;&#x8A2D; Mos1 &#x633F;&#x5165;&#x5909;&#x7570;&#x7CFB;&#x7D71;&#x306E;&#x5B9F;&#x8CEA;&#x7684;&#x306A;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x305F;&#x3081;&#x306E;&#x6700;&#x9069;&#x5316;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D4;&#x30FC;&#x30AF;&#x751F;&#x7523;&#x3067;&#x306F; 1 &#x30F6;&#x6708;&#x4EE5;&#x4E0A; 5,000 &#x682A;&#x751F;&#x6210;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x83CC;&#x682A;&#x306F;&#x3001;&#x305D;&#x306E;&#x5206;&#x5B50;&#x89E3;&#x6790;&#x3068;&#x7279;&#x5FB4;&#x4EE5;&#x4E0A; 13,300 Mos1 &#x633F;&#x5165;&#x5BFE;&#x8C61;&#x3067;&#x3057;&#x305F;&#x3002;&#x76F4;&#x63A5;&#x4EE5;&#x4E0A; 4,700 &#x907A;&#x4F1D;&#x5B50;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x5BFE;&#x7ACB;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x307F;&#x63DB;&#x3048;&#x306E;&#x524A;&#x9664;&#x306E;&#x51FA;&#x767A;&#x70B9;&#x306F;&#x672C;&#x8CEA;&#x7684;&#x306B;&#x3059;&#x3079;&#x3066;&#x306E;&#x7DDA;&#x866B;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x3068;&#x305D;&#x308C;&#x3089;&#x306E; 40 &#xFF05; &#x4EE5;&#x4E0A;&#x306E;&#x4FEE;&#x6B63;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x8868;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6B27;&#x5DDE; NEMAGENETAG &#x30B3;&#x30F3;&#x30BD;&#x30FC;&#x30B7;&#x30A2;&#x30E0;&#x306E;&#x5F8C;&#x63F4;&#x306E;&#x4E0B;&#x3092;&#x751F;&#x6210;&#x306E;&#x5909;&#x7570;&#x4F53;&#x306E;&#x3053;&#x306E;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x516C;&#x958B;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;&#x91CD;&#x8981;&#x306A;&#x7814;&#x7A76;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30B2;&#x30CE;&#x30E0;&#x5E83;&#x3044;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306E; Mos1 &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x633F;&#x5165;&#x5909;&#x7570;&#x4F53;&#x7DDA;&#x866B;&#x7814;&#x7A76;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30C6;&#x30A3;&#x306E;&#x305F;&#x3081;&#x3002;

&#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; C. elegans&#xC758; &#xAC8C;&#xB188;&#xC744; &#xAE30;&#xC220;&#xC790;&#xC5D0; &#xAC8C; &#xB3D9;&#xC885; &#xC7AC;&#xC870;&#xD569;&#xC744; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xBA54;&#xC11C;&#xB4DC; &#xC0AC;&#xC6A9; heterologous transposon mos1&#xC758; &#xD2B9;&#xC815; &#xC0BD;&#xC785; &#xB4E4;&#xACE0; &#xAE34;&#xC7A5;. &#xB530;&#xB77C;&#xC11C; &#xC54C;&#xB824;&#xC9C4;&#xB41C; Mos1 &#xC0BD;&#xC785; &#xB300;&#xB9BD;&#xC758; &#xD070; &#xCEEC;&#xB809;&#xC158; C. elegans &#xC5F0;&#xAD6C; &#xCEE4;&#xBBA4;&#xB2C8;&#xD2F0;&#xC5D0; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xAD00;&#xC2EC;&#xC758; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC5EC;&#xAE30; Mos1 &#xC0BD;&#xC785; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC885;&#xC790;&#xC758; &#xC2E4;&#xC9C8;&#xC801;&#xC778; &#xCEEC;&#xB809;&#xC158;&#xC758; &#xAC74;&#xC124;&#xC744; &#xC704;&#xD55C; &#xBC18;&#xC790;&#xB3D9;&#xB41C; &#xBC29;&#xBC95;&#xB860;&#xC758; &#xCD5C;&#xC801;&#xD654;&#xC5D0; &#xC124;&#xBA85;. &#xCD5C;&#xB300; &#xC0DD;&#xC0B0;&#xC5D0; &#xD55C;&#xB2EC;&#xC5D0; 5000 &#xAC1C; &#xC774;&#xC0C1;&#xC758; &#xBCC0;&#xC885;&#xC774; &#xC0DD;&#xC131; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xBCC0;&#xC885; &#xBD84;&#xC790; &#xBD84;&#xC11D; &#xBC0F; &#xD2B9;&#xC9D5; &#xC774;&#xC0C1; 13,300 Mos1 &#xC0BD;&#xC785; &#xD6C4; &#xD588;&#xB2E4;. &#xAC1C; &#xC774;&#xC0C1;&#xC758; &#xC9C1;&#xC811; 4700 &#xC720;&#xC804;&#xC790;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C;, &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC774;&#xB7EC;&#xD55C; &#xB300;&#xB9BD; &#xC870;&#xC791;&#xB41C; &#xC0AD;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xAE30;&#xBCF8;&#xC801;&#xC73C;&#xB85C; &#xBAA8;&#xB4E0; C. elegans &#xC720;&#xC804;&#xC790;&#xC640; &#xADF8;&#xB4E4;&#xC758; 40% &#xC774;&#xC0C1; &#xC218;&#xC815; &#xCD9C;&#xBC1C;&#xC810; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xC720;&#xB7FD; NEMAGENETAG &#xCEE8;&#xC18C;&#xC2DC;&#xC5C4;&#xC758; &#xD6C4;&#xC6D0; &#xD558;&#xC5D0; &#xC0DD;&#xC131; &#xB41C; &#xBBA4;&#xD134;&#xD2B8;&#xC758;&#xC774; &#xCEEC;&#xB809;&#xC158; &#xACF5;&#xAC1C;&#xC801;&#xC73C;&#xB85C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xBC0F; &#xC911;&#xC694; &#xD55C; &#xC5F0;&#xAD6C; &#xB9AC;&#xC18C;&#xC2A4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xAC8C;&#xB188; &#xB113;&#xC740; &#xCEEC;&#xB809;&#xC158;&#xC758; Mos1 Transposon &#xC0BD;&#xC785; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; C. elegans &#xC5F0;&#xAD6C; &#xCEE4;&#xBBA4;&#xB2C8;&#xD2F0;&#xC5D0; &#xB300; &#xD55C;.

M&eacute;todos que utilizan la recombinaci&oacute;n hom&oacute;loga para dise&ntilde;ar el genoma de C. elegans com&uacute;nmente utilizan cepas llevando inserciones espec&iacute;ficas de la heter&oacute;loga transposon Mos1. Una gran colecci&oacute;n de alelos de inserci&oacute;n Mos1 conocidos podr&iacute;a ser de inter&eacute;s general para la comunidad de investigaci&oacute;n de C. elegans. Aqu&iacute; describimos la optimizaci&oacute;n de una metodolog&iacute;a semiautom&aacute;tico para la construcci&oacute;n de una importante colecci&oacute;n de Mos1 estirpes mutantes de inserci&oacute;n. En el pico de producci&oacute;n, se generaron m&aacute;s de 5.000 cepas por mes. Estas cepas fueron luego objeto de an&aacute;lisis molecular y m&aacute;s de 13.300 inserciones de Mos1 caracterizadas. Adem&aacute;s de atacar directamente m&aacute;s de 4.700 genes, estos alelos representan el potencial punto de partida para la supresi&oacute;n de la ingenier&iacute;a de esencialmente todos los genes de C. elegans y la modificaci&oacute;n de m&aacute;s del 40% de ellos. Esta colecci&oacute;n de mutantes, generado bajo los auspicios del consorcio europeo NEMAGENETAG, est&aacute; p&uacute;blicamente disponible y representa un recurso importante de la investigaci&oacute;n.

Un genoma colecci&oacute;n de Mos1 Transposon inserci&oacute;n mutantes para el comunidad investigadora de C. elegans.

In vertebrates, zyxin is a LIM-domain protein belonging to a family composed of seven members. We show that the nematode Caenorhabditis elegans has a unique zyxin-like protein, ZYX-1, which is the orthologue of the vertebrate zyxin subfamily composed of zyxin, migfilin, TRIP6, and LPP. The ZYX-1 protein is expressed in the striated body-wall muscles and localizes at dense bodies/Z-discs and M-lines, as well as in the nucleus. In yeast two-hybrid assays ZYX-1 interacts with several known dense body and M-line proteins, including DEB-1 (vinculin) and ATN-1 (α-actinin). ZYX-1 is mainly localized in the middle region of the dense body/Z-disk, overlapping the apical and basal regions containing, respectively, ATN-1 and DEB-1. The localization and dynamics of ZYX-1 at dense bodies depend on the presence of ATN-1. Fluorescence recovery after photobleaching experiments revealed a high mobility of the ZYX-1 protein within muscle cells, in particular at dense bodies and M-lines, indicating a peripheral and dynamic association of ZYX-1 at these muscle adhesion structures. A portion of the ZYX-1 protein shuttles from the cytoplasm into the nucleus, suggesting a role for ZYX-1 in signal transduction. We provide evidence that the zyx-1 gene encodes two different isoforms, ZYX-1a and ZYX-1b, which exhibit different roles in dystrophin-dependent muscle degeneration occurring in a C. elegans model of Duchenne muscular dystrophy.

ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Article	Year
Dystrobrevin requires a dystrophin-binding domain to function in Caenorhabditis elegans. European journal of biochemistry / FEBS\| PubMed ID: 11895430	2002
Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans. Neuromuscular disorders : NMD\| PubMed ID: 12062255	2002
Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans. Gene\| PubMed ID: 12234669	2002
The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin. Journal of molecular biology\| PubMed ID: 14499607	2003
The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy. Journal of molecular biology\| PubMed ID: 16527307	2006
The C. elegans dense body: anchoring and signaling structure of the muscle. Journal of muscle research and cell motility\| PubMed ID: 17492481	2007
DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1. Molecular biology of the cell\| PubMed ID: 18094057	2008
Caenorhabditis elegans as a chemical screening tool for the study o f neuromuscular disorders. Manual and semi-automated methods. Methods (San Diego, Calif.)\| PubMed ID: 22041718	2012
A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community. PloS one\| PubMed ID: 22347378	2012
ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration. Molecular biology of the cell\| PubMed ID: 23427270	2013
Chemical genetics unveils a key role of mitochondrial dynamics, cytochrome c release and IP3R activity in muscular dystrophy. Human molecular genetics\| PubMed ID: 23804750	2013

Kathrin Gieseler

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