Bessou C et al. (1997) International C. elegans Meeting "SEROTONIN-CONTROLLED BEHAVIORS IN C. elegans"

Segalat LS, Bessou C

[International C. elegans Meeting, 1997]

Serotonin is a neuromodulator which acts mainly through seven transmembrane receptors (7-TMRs) coupled to heterotrimeric G-proteins. Upon ligand binding, G-proteins modulate the level of cytoplasmic second messengers, which in turns alter the activity of ion channels. Despite its importance in human physiology, there is still little known about the ion channels whose activity is regulated by serotonin. We are taking advantage of the fact that serotonin modulates locomotion and foraging, two behaviors of C.elegans easy to follow, to screen for mutants with altered behaviors. In the past, such screens have led to the identification of Go as an component of the serotonin signaling cascade in the worm (1). However, Go-deficient mutants are still partly responsive to serotonin, suggesting that serotonin might signal through other pathways as well. We have now screened for EMS-mutagenized animals that behave like wild-type animals exposed to ketanserin, a 5HT2 antagonist. We isolated 10 mutants displaying the same ketanserin-like phenotype, and representing 4 to 5 genes. Interestingly, none of the other serotonin controlled behaviors of the worm (i.e. eating, egg-laying, mating) seem affected by the mutations. We are now testing the response of these mutants to various drugs including serotonin and we have started to map them. (1) Segalat et al. Science, 267, 1648.

Bessou C et al. (1998) Neurogenetics "Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission."

Bessou C, Franks CJ, Giugia JB, Holden-Dye L, Segalat LS

[Neurogenetics, 1998]

Mutations in the human dystrophin gene cause Duchenne muscular dystrophy, a common neuromuscular disease leading to a progressive necrosis of muscle cells. The etiology of this necrosis has not been clearly established, and the cellular function of the dystrophin protein is still unknown. We report here the identification of a dystrophin-like gene (named dys-1) in the nematode Caenorhabditis elegans. Loss-of-function mutations of the dys-1 gene make animals hyperactive and slightly hypercontracted. Surprisingly, the dys-1 mutants have apparently normal muscle cells. Based on reporter gene analysis and heterologous promoter expression, the site of action of the dys-1 gene seems to be in muscles. A chimeric transgene in which the C-terminal end of the protein has been replaced by the human dystrophin sequence is able to partly suppress the phenotype of the dys-1 mutants, showing that both proteins share some functional similarity. Finally, the dys-1 mutants are hypersensitive to acetylcholine and to the acetylcholinesterase inhibitor aldicarb, suggesting that dys-1 mutations affect cholinergic transmission. This study provides the first functional link between the dystrophin family of proteins and cholinergic transmission.

Gieseler K et al. (1999) Neurogenetics "Dystrobrevin- and dystrophin-like mutants display similar phenotypes in the nematode Caenorhabditis elegans."

Segalat LS, Bessou C, Gieseler K

[Neurogenetics, 1999]

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, forms a transmembrane complex with dystrophin-associated proteins. Dystrobrevins, proteins showing homology to the C-terminal end of dystrophin, and whose function is unknown, are part of the dystrophin complex. We report there that, in the nematode Caenorhabditis elegans, animals carrying mutations in either the dystrophin-like gene dys-1 or the dystrobrevin-like gene dyb-1 display similar behavioral and pharmacological phenotypes consistent with an alteration of cholinergic signalling. These findings suggest that: (1) dystrobrevin and dystrophin are functionaly related and (2) their disruption impairs cholinergic signalling.

Gieseler K et al. (2001) J Mol Biol "Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans."

Holden-Dye L, Franks CJ, Segalat L, Mariol MC, Bessou C, Gieseler K, Migaud M

[J Mol Biol, 2001]

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dtb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.

Martin E et al. (2002) Genetics "Identification of 1088 new transposon insertions of Caenorhabditis elegans: a pilot study toward large-scale screens."

Bessou C, Alvarez T, Sookhareea S, Segalat L, Laloux H, Labouesse M, Hauser O, Couette G, Martin E

[Genetics, 2002]

We explored the feasibility of a strategy based on transposons to generate identified mutants of most Caenorhabditis elegans genes. A total of 1088 random new insertions of C. elegans transposons Tc1, Tc3, and Tc5 were identified by anchored PCR, some of which result in a mutant phenotype.

CJ Franks et al. (1999) International C. elegans Meeting "Evaluation of Excitation-Contraction Coupling in C. elegans Muscle."

L Holden-Dye, C Rogers, ET Claverol, JE Chad, RJ Walker, CJ Franks

[International C. elegans Meeting, 1999]

Electrophysiology is a vital technique for the phenotypic analysis of C. elegans muscle (Bessou et al . 1998). However, excitation-contraction coupling consists of a series of phenomena, only some of which manifest themselves as measureable change in the electrical properties of the cells. For example, systems that modulate realease of calcium from intracellular compartments, or calcium sensitivity of the contractile apparatus, can profoundly affect muscle function. We are currently developing an automated system for the correlation of electrophysiological and imaging data that provides a robust measure of the latency between action potentials and the muscle contractions with which they are associated. Video images of the muscle preparation are acquired using a standard black and white CCD camera and the data are recorded to hard disk via a proprietary frame grabber. Single electrode, membrane potential recordings are made from pharyngeal muscle in the manner described by Franks et al . (1997) and data are recorded using an Axon instruments Digidata multichannel acquisition system. One channel is used to record the electrophysiological data while the second records the output from a circuit designed and built 'in-house'. This circuit is connected between the CCD and the frame grabber. It essentially splits the video signal in two. The first output from the circuit, contains the full video signal and is recorded by the frame grabber. The second output contains only the vertical sync signal, stripped from the original video feed. This timing signal is recorded on channel two of the acquisition system producing a timestamp for each frame of the video. We have also designed an image processing algorithm that makes it possible to quantify the degree of muscle contraction in each video frame. Thus we have a standardised and automated system that can correlate electrophysiological and video data with a temporal resolution limited only by that of the CCD. We are using the system to analyse disruption of E-C coupling by pharmacological agents and to assess phenotypic variation between wild-type and mutant C. elegans . We will report on our progress at the meeting. Bessou, et al . (1998). Neurogenetics . In Press. Franks et al . (1997) J. Physiol. Lond. 504P: 15.

Hongkyun Kim et al. (2003) International Worm Meeting "A Novel Acetylcholine Transporter Interacts with the Dystrophin-Glycoprotein Complex (DGC): Evidence for Altered Acetylcholine Transport in a C. elegans Model of Muscular Dystrophy."

Steven L McIntire, Matthew Rogers, Hongkyun Kim

[International Worm Meeting, 2003]

Mutations in dystrophin and other components of the DGC cause muscular dystrophies in humans. Despite extensive analysis of the DGC, the exact pathogenesis of the disease is unclear. The expression of the DGC at many brain synapses and the NMJ as well as the association with syntrophins (PDZ containing proteins) has led to the suggestion that the DGC could function as a local membrane organizer at synaptic sites. Segalat and his colleagues demonstrated that mutations in the C. elegans homologue of the dystrophin gene, dys-1 and other DGC components (such as dyb-1) cause an unusual exaggerated bending of the anterior body and head during locomotion, hypersensitivity to aldicarb and acetylcholine, and muscle degeneration in a sensitized genetic background (1, 2). We have previously described a genetic screen based on altered ethanol sensitivity that led to the identification of 12 mutants with a dys-1-like phenotype, including five alleles (eg28, eg114, eg115, eg121, eg137) of a previously unidentified gene (2000 wcm abstract 170). To gain insight into the role of this gene in the function of the DGC, we cloned the gene based on the locomotory phenotype. The gene encodes a transporter consisting of multiple transmembrane domains. Analysis of transgenic animals expressing a GFP reporter construct reveals strong GFP expression in muscles. Increased expression is observed along the ventral and dorsal midlines where NMJs are located. Muscle-specific expression of the gene rescues all of the phenotypes. In uptake assays with a stable cell line expressing the transporter, we observe consistent transport of 3H-acetylcholine (3H-Ach) (and some variable transport of 3H-choline). The transporter may function at NMJs in C. elegans to take up released Ach. In the absence of Ach transport, delayed clearance of Ach from the synaptic cleft may cause a prolonged excitation of muscles and the characteristic locomotory phenotype. Examination of double mutants, dys-1(cx18);eg28, dyb-1(cx36) ;eg28 and stn-1(ok292); eg28 (stn-1 is a syntrophin homologue), indicates that the transporter may function in the same pathway as other DGC genes. Furthermore, co-immunoprecipitation experiments provide evidence for a physical interaction with the DGC.This study suggests that a novel Ach transporter functions in muscle cells and may be localized through an interaction with the DGC. Defective Ach transport may play a role in the pathogenesis of human muscular dystrophies. 1. Bessou, C. et al.(1998) Neurogentics 2, 61. 2. Gieseler, K. et al. (2000) Curr. Biol. 10, 1092.

Sushant Khandekar et al. (2001) International C. elegans Meeting "A role of sarcoglycans in C. elegans"

John Plenefisch, Sushant Khandekar

[International C. elegans Meeting, 2001]

In vertebrates, muscle-wasting disorders arise from mutation in dystrophin and dystrophin associated proteins such as the sarcoglycans. Sarcoglycans are trans-membrane proteins that are part of the dystroglycan complex that links dystrophin to the plasma membrane in muscle cells (1). In humans, limb girdle muscular dystrophy arises from mutation of sarcoglycan proteins, which leads to sarcolemma dysfunction and ultimately necrosis of the skeletal muscle cells (1). In C elegans many of the components of the dystrophin/dystroglycan complex have been conserved, including dystrophin, dystroglycan and three sarcoglycans (alpha, beta, gamma) (2). Surprisingly, the phenotype of mutated dystrophin in C. elegans is mild; mutants display a hyperactive phenotype but no muscle degeneration (3), whether mutating other genes within the C elegans dystrophin/dystroglycan complex has a more severe effect remains to be determined. We are investigating the functional role of the sarcoglycans using RNAi in variety of different muscle mutant background. Moreover, antibody staining and/or GFP reporter gene analysis will examine the distribution and expression of all three sarcoglycans. In addition to examining the functions of the C elegans homologs of classic vertebrate muscular dystrophy genes, we are interested in molecular characterization of genes in C elegans initially identified through a dystrophic paralytic phenotype. These genes include the mua-1 (for m uscle a ttachment-1) gene. MUA-1 is a zinc finger protein of the EKLF family and is required for normal muscle attachment to hypodermis. Mutations in the mua-1 results in postembryonic paralysis and progressive detachment of body wall muscles from the hypodermis. We are determming the molecular lesions of mua-1 mutants alleles as well as using RNAi to help define the null phenotype of this gene. Culligan et.al. International Journal of Molecular Medicine (1998) 2:639-648. Hutter et.al. Science (2000) 287:989-994 Bessou et.al. Neurogenetics (1998) 2:61-72

Kathrin Gieseler et al. (2003) International Worm Meeting "DYSTROPHIN AND ASSOCIATED PROTEINS IN C. elegans"

Marie-Christine Mariol, Kathrin Gieseler, Karine Grisoni, Maite Carre-Pierrat, Laurent Segalat, Edwige Martin

[International Worm Meeting, 2003]

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy, a neuromuscular disease leading to muscle necrosis. The function of the dystrophin protein is not known. In mammals, dystrophin is located under the muscle plasma membrane, and is associated with a protein complex spanning the membrane. The C. elegans genome contains a dystrophin homologue named dys-1. Our goal is to understand dystrophin function in C. elegans. Loss-of-function mutations in the dys-1 gene do not alter the muscle structure, but make animals hyperactive and slightly hypercontracted. In a forward genetics approach, we isolated mutations with dys-1-like phenotypes affecting four additional genes: dyb-1 (Gieseler et al., 2001), dyc-1 (Gieseler et al. 2000), dys-4 and slo-1. dyb-1 is the C. elegans homologue of dystrobrevins, a family of proteins belonging to the dystrophin complex and known to bind dystrophin directly through coiled-coil domains present on each protein The DYC-1 protein is not homologous to any known mammalian proteins, but comprises two regions of similarity to CAPON, a nNOS-binding protein. Like DYS-1 and DYB-1, DYC-1 contains a putative coiled-coil domain. A search for protein motifs with the Prosite program reveals a high number of putative PKA- and PKC-dependent phosphorylation sites in DYC-1. Since C. elegans possesses a dystrophin-like and a dystrobrevin gene, we investigated whether other homologues of dystrophin associated proteins known in mammals could also be found in the C. elegans genome. Our results strongly suggest that a protein complex comprising functional analogies with that of mammals exists in C. elegans (Grisoni et al. 2002). The DYC-1, SLO-1 and DYS-4 proteins may be part of this complex in C. elegans. In order to verify this hypothesis and to identify other members of the dystrophin associated protein complex , we are using the yeast two hybrid system. Bessou et al. (1998) Neurogenetics 2: 61-72. Gieseler et al. (2000) Current biology 10:1092-7. Gieseler et al. (2001) J Mol Biol.307 :107-17. Grisoni et al. (2002) Gene 294 : 77-84.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.