Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-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.     

Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans

BACKGROUND: Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration.RESULTS: We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm. CONCLUSIONS: As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model.     

Mutations in the dystrophin-like dys-1 gene of Caenorhabditis elegans result in reduced acetylcholinesterase activity

Mutations of the Caenorhabditis elegans dystrophin/utrophin-like dys-1 gene lead to hyperactivity and hypercontraction of the animals. In addition dys-1 mutants are hypersensitive to acetylcholine and acetylcholinesterase inhibitors. We investigated this phenotype further by assaying acetylcholinesterase activity. Total extracts from three different dys-1 alleles showed significantly less acetylcholinesterase-specific activity than wild-type controls. In addition, double mutants carrying a mutation in the dys-1 gene plus a mutation in either of the two major acetylcholinesterase genes (ace-1 and ace-2) display locomotor defects consistent with a strong reduction of acetylcholinesterases, whereas none of the single mutants does. Therefore, in C. elegans, disruption of the dystrophin/utrophin-like dys-1 gene affects acetylcholinesterase activity.     

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

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

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.     

Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3

Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C.     

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

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.     

The third human FER-1-like protein is highly similar to dysferlin

Dysferlin, the protein product of the gene mutated in patients with an autosomal recessive limb-girdle muscular dystrophy type 2B (LGMD2B) and a distal muscular dystrophy, Miyoshi myopathy, is homologous to a Caenorhabditis elegans spermatogenesis factor, FER-1. Analysis of fer-1 mutants and of sequence predictions of the FER-1 and dysferlin ORFs has predicted a role in membrane fusion. Otoferlin, another human FER-1-like protein (ferlin), has recently been shown to be responsible for autosomal recessive nonsyndromic deafness (DFNB9). In this report we describe the third human ferlin gene, FER1L3, which maps to chromosome 10q23.3. Expression analysis of the orthologous mouse gene shows ubiquitous expression but predominant expression in the eye, esophagus, and salivary gland. All the ferlins are characterized by sequences corresponding to multiple C2 domains that share the highest level of homology with the C2A domain of rat synaptotagmin III. They are predicted to be Type II transmembrane proteins, with the majority of the protein facing the cytoplasm anchored by the C-terminal transmembrane domain. Sequence and predicted structural comparisons have highlighted the high degree of similarity of dysferlin and FER1L3, which have sequences corresponding to six C2 domains and which share more than 60% amino acid sequence identity.     

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.     

Mislocalization of fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of α-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of α-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and α-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of α-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.     

eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.     

unc-119 homolog required for normal development of the zebrafish nervous system

The UNC-119 proteins, found in all metazoans examined, are highly conserved at both the sequence and functional levels. In the invertebrates Caenorhabditis elegans and Drosophila melanogaster, unc-119 genes are expressed pan-neurally. Loss of function of the unc-119 gene in C. elegans results in a disorganized neural architecture and paralysis. The function of UNC-119 proteins has been conserved throughout evolution, as transgenic expression of the human UNC119 gene in C. elegans unc-119 mutants restores a wild-type phenotype. However, the nature of the conserved molecular function of UNC-119 proteins is poorly understood. Although unc-119 genes are expressed throughout the nervous system of the worm and fly, the analysis of these genes in vertebrates has focused on their function in the photoreceptor cells of the retina. Here we report the characterization of an unc-119 homolog in the zebrafish. The Unc119 protein is expressed in various neural tissues in the developing zebrafish embryo and larva. Morpholino oligonucleotide (MO)-mediated knockdown of Unc119 protein results in a "curly tail down" phenotype. Examination of neural patterning demonstrates that these "curly tail down" zebrafish experience a constellation of neuronal defects similar to those seen in C. elegans unc-119 mutants: missing or misplaced cell bodies, process defasciculation, axon pathfinding errors, and aberrant axonal branching. These findings suggest that UNC-119 proteins may play an important role in the development and/or function of the vertebrate nervous system.     

Nuclear envelope defects in muscular dystrophy

Muscular dystrophies are a heterogeneous group of disorders linked to defects in 20-30 different genes. Mutations in the genes encoding a pair of nuclear envelope proteins, emerin and lamin A/C, have been shown to cause the X-linked and autosomal forms respectively of Emery-Dreifuss muscular dystrophy. A third form of muscular dystrophy, limb girdle muscular dystrophy 1b, has also been linked to mutations in the lamin A/C gene. Given that these two genes are ubiquitously expressed, a major goal is to determine how they can be associated with tissue specific diseases. Recent results suggest that lamin A/C and emerin contribute to the maintenance of nuclear envelope structure and at the same time may modulate the expression patterns of certain mechanosensitive and stress induced genes. Both emerin and lamin A/C may play an important role in the response of cells to mechanical stress and in this way may help to maintain muscle cell integrity.     

Reduced expression of neuropeptide genes in a genome-wide screen of a secretion-deficient mouse

Activity-dependent changes in synapses rely on functional changes in resident proteins and on gene expression. We addressed the relationship between synapse activity and the expression of synaptic genes by comparing RNA levels in the neocortex of normal mice versus secretion-deficient and therefore synaptically silent munc18-1 (mammalian homologue of Caenorhabditis elegans uncoordinated locomotion-18) null mutants, using microarray expression analysis, real-time quantitative PCR and northern blotting. We hypothesized that genes under the control of synaptic activity would be differentially expressed between mutants and controls. We found that few synaptic genes were differentially expressed. However, most neuropeptide genes with detectable expression on the microarray were differentially expressed, being expressed 3-20-fold higher in control cortex. Several other secreted proteins were also differentially expressed, but genes encoding their receptors and many other synaptic components were not. Differential expression was confirmed by real-time quantitative PCR analysis. In situ hybridization indicated that the difference in neuropeptide expression was uniform and not due to the loss of specific cells in the mutant. In primary sensory neurons, which do not depend on synaptic activity for their input, the differential expression of neuropeptides was not observed. These data argue against a general relationship between the activity of synapses and the expression of their resident proteins, but suggest a link between secretion and the expression of genes encoding the secreted products.     

ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1

Intersectins (Itsn) are conserved EH and SH3 domain containing adaptor proteins. In Drosophila melanogaster, ITSN is required to regulate synaptic morphology, to facilitate efficient synaptic vesicle recycling and for viability. Here, we report our genetic analysis of Caenorhabditis elegans intersectin. In contrast to Drosophila , C. elegans itsn-1 protein null mutants are viable and display grossly normal locomotion and development. However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membrane-associated vesicles at putative endocytic hotspots, approximately 300 nm from the presynaptic density. This defect occurs precisely where endogenous ITSN-1 protein localizes in wild-type animals and is associated with a significant reduction in synaptic vesicle number and reduced frequency of endogenous synaptic events at neuromuscular junctions (NMJs). ITSN-1 forms a stable complex with EHS-1 (Eps15) and is expressed at reduced levels in ehs-1 mutants. Thus, ITSN-1 together with EHS-1, coordinate vesicle recycling at C. elegans NMJs. We also found that both itsn-1 and ehs-1 mutants show poor viability and growth in a Disabled (dab-1) null mutant background. These results show for the first time that intersectin and Eps15 proteins function in the same genetic pathway, and appear to function synergistically with the clathrin-coat-associated sorting protein, Disabled, for viability.     

Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family

We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.     

Hyperactive neuroendocrine secretion causes size, feeding, and metabolic defects of C. elegans Bardet-Biedl syndrome mutants

Bardet-Biedl syndrome, BBS, is a rare autosomal recessive disorder with clinical presentations including polydactyly, retinopathy, hyperphagia, obesity, short stature, cognitive impairment, and developmental delays. Disruptions of BBS proteins in a variety of organisms impair cilia formation and function and the multi-organ defects of BBS have been attributed to deficiencies in various cilia-associated signaling pathways. In C. elegans, bbs genes are expressed exclusively in the sixty ciliated sensory neurons of these animals and bbs mutants exhibit sensory defects as well as body size, feeding, and metabolic abnormalities. Here we show that in contrast to many other cilia-defective mutants, C. elegans bbs mutants exhibit increased release of dense-core vesicles and organism-wide phenotypes associated with enhanced activities of insulin, neuropeptide, and biogenic amine signaling pathways. We show that the altered body size, feeding, and metabolic abnormalities of bbs mutants can be corrected to wild-type levels by abrogating the enhanced secretion of dense-core vesicles without concomitant correction of ciliary defects. These findings expand the role of BBS proteins to the regulation of dense-core-vesicle exocytosis and suggest that some features of Bardet-Biedl Syndrome may be caused by excessive neuroendocrine secretion.     

Conservation rules, their breakdown, and optimality in Caenorhabditis sinusoidal locomotion

Undulatory locomotion is common to nematodes as well as to limbless vertebrates, but its control is not understood in spite of the identification of hundred of genes involved in Caenorhabditis elegans locomotion. To reveal the mechanisms of nematode undulatory locomotion, we quantitatively analysed the movement of C. elegans with genetic perturbations to neurons, muscles, and skeleton (cuticle). We also compared locomotion of different Caenorhabditis species. We constructed a theoretical model that combines mechanics and biophysics, and that is constrained by the observations of propulsion and muscular velocities, as well as wavelength and amplitude of undulations. We find that normalized wavelength is a conserved quantity among wild-type C. elegans individuals, across mutants, and across different species. The velocity of forward propulsion scales linearly with the velocity of the muscular wave and the corresponding slope is also a conserved quantity and almost optimal; the exceptions are in some mutants affecting cuticle structure. In theoretical terms, the optimality of the slope is equivalent to the exact balance between muscular and visco-elastic body reaction bending moments. We find that the amplitude and frequency of undulations are inversely correlated and provide a theoretical explanation for this fact. These experimental results are valid both for young adults and for all larval stages of wild-type C. elegans. In particular, during development, the amplitude scales linearly with the wavelength, consistent with our theory. We also investigated the influence of substrate firmness on motion parameters, and found that it does not affect the above invariants. In general, our biomechanical model can explain the observed robustness of the mechanisms controlling nematode undulatory locomotion.