Mutants with altered muscle structure in Caenorhabditis elegans

In the small nematode, Caenorhabditis elegans, mutants with a disorganized myofilament lattice structure have been identified by polarized light and electron microscopy. Genetic analysis places the mutations in 12 complementation groups which are distributed over the six linkage groups of C. elegans. The phenotypes are described for the mutants from the 9 complementation groups not previously reported on in detail. Most are paralyzed, but some exhibit essentially normal movement; mutants of two loci show changes only in later larval stages and adulthood. Morphological studies show that, in general, all the members of a complementation group show similar changes in muscle structure and that these changes are distinctive for that group. In mutants of several genes, disorganization of the myofilament lattice is general with no one component of the lattice more obviously altered than others. In mutants of other genes specific structures are prominently altered. In one of the instances where thick filaments appear to be abnormal, double mutants combining mutations in this gene (unc-82 IV) with mutations in the gene for a myosin heavy chain (MacLeod et al., 1977a,b) or paramyosin (Waterston et al., 1977) were used to show that the unc-82 gene product probably affects thick filament assembly through its actions on paramyosin. Some possible implications of the morphological features of the mutants as well as the conclusions derived from the genetic studies are discussed.     

Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat- sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.     

A mutation in CHN-1/CHIP suppresses muscle degeneration in Caenorhabditis elegans

Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin-glycoprotein-complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease.     

Novel insertion mutation in Caenorhabditis elegans.

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.     

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.     

Behavioral degradation under mutation accumulation in Caenorhabditis elegans

Spontaneous mutations play a fundamental role in the maintenance of genetic variation in natural populations, the nature of inbreeding depression, the evolution of sexual reproduction, and the conservation of endangered species. Using long-term mutation-accumulation lines of the nematode Caenorhabditis elegans, we estimate the rate and magnitude of mutational effects for a suite of behaviors characterizing individual chemosensory responses to a repellant stimulus. In accordance with evidence that the vast majority of mutations are deleterious, we find that behavioral responses degrade over time as a result of spontaneous mutation accumulation. The rate of mutation for behavioral traits is roughly of the same order or slightly smaller than those previously estimated for reproductive traits and the average size of the mutational effects is also comparable. These results have important implications for the maintenance of genetic variation for behavior in natural populations as well as for expectations for behavioral change within endangered species and captive populations.     

Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations

By taking advantage of a lethal phenotype characteristic of Caenorhabditis elegans embryos that fail to move, we have identified 13 genes required for muscle assembly and function and discovered a new lethal class of alleles for three previously known muscle-affecting genes. By staining mutant embryos for myosin and actin we have recognized five distinct classes of genes: mutations in four genes disrupt the assembly of thick and thin filaments into the myofilament lattice as well as the polarized location of these components to the sarcolemma. Mutations in another three genes also disrupt thick and thin filament assembly, but allow proper polarization of lattice components based on the myosin heavy chain isoform that we analyzed. Another two classes of genes are defined by mutations with principal effects on thick or thin filament assembly into the lattice, but not both. The final class includes three genes in which mutations cause relatively minor defects in lattice assembly. Failure of certain mutants to stain with antibodies to specific muscle cell antigens suggest that two genes associated with severe disruptions of myofilament lattice assembly may code for components of the basement membrane and the sarcolemma that are concentrated where dense bodies (Z- line analogs) and M-lines attach to the cell membrane. Similar evidence suggests that one of the genes associated with mild effects on lattice assembly may code for tropomyosin. Many of the newly identified genes are likely to play critical roles in muscle development and function.     

The Caenorhabditis elegans presenilin sel-12 is required for mesodermal patterning and muscle function

Mutations in presenilin genes impair Notch signalling and, in humans, have been implicated in the development of familial Alzheimer's disease. We show here that a reduction of the activity of the Caenorhabditis elegans presenilin sel-12 results in a late defect during sex muscle development. The morphological abnormalities and functional deficits in the sex muscles contribute to the egg-laying defects seen in sel-12 hermaphrodites and to the severely reduced mating efficiency of sel-12 males. Both defects can be rescued by expressing sel-12 from the hlh-8 promoter that is active during the development of the sex muscle-specific M lineage, but not by expressing sel-12 from late muscle-specific promoters. Both weak and strong sel-12 mutations cause defects in the sex muscles that resemble the defects we found in lin-12 hypomorphic alleles, suggesting a previously uncharacterised LIN-12 signalling event late in postembryonic mesoderm development. Together with a previous study indicating a role of lin-12 and sel-12 during the specification of the pi cell lineage required for proper vulva-uterine connection, our data suggest that the failure of sel-12 animals to lay eggs properly is caused by defects in at least two independent signalling events in different tissues during development.     

The fine structure of Caenorhabditis elegans N-glycans

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.     

Touch receptor development and function in Caenorhabditis elegans

Mutations causing a touch-insensitive phenotype in the nematode Caenorhabditis elegans have been the basis of studies on the specification of neuronal cell fate, inherited neurodegeneration, and the molecular nature of mechanosensory transduction. © 1993 John Wiley & Sons, Inc.     

Effects of a cardiomyopathy-causing troponin t mutation on thin filament function and structure

Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament.     

Caenorhabditis elegans body mechanics are regulated by body wall muscle tone

Body mechanics in the nematode Caenorhabditis elegans are central to both mechanosensation and locomotion. Previous work revealed that the mechanics of the outer shell, rather than internal hydrostatic pressure, dominates stiffness. This shell is comprised of the cuticle and the body wall muscles, either of which could contribute to the body mechanics. Here, we tested the hypothesis that the muscles are an important contributor by modulating muscle tone using optogenetic and pharmacological tools, and measuring animal stiffness using piezoresistive microcantilevers. As a proxy for muscle tone, we measured changes in animal length under the same treatments. We found that treatments that induce muscle contraction generally resulted in body shortening and stiffening. Conversely, methods to relax the muscles more modestly increased length and decreased stiffness. The results support the idea that body wall muscle activation contributes significantly to and can modulate C. elegans body mechanics. Modulation of body stiffness would enable nematodes to tune locomotion or swimming gaits and may have implications in touch sensation.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

HIM-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes

Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.     

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD⁺ after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD⁺ salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD⁺ salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD⁺ salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD⁺ production or NAM levels. Active NAD⁺ biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD⁺ salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD⁺ biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types.     

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.     

Two homogeneous myosins in body-wall muscle of Caenorhabditis elegans

Myosin purified from the body-wall muscle-defective mutant E675 of the nematode. Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974).When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >80% 210,000 or >80% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains.Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains.E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled E190 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin.     

Fine structure of the Caenorhabditis elegans secretory-excretory system

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.     

The mutational structure of metabolism in Caenorhabditis elegans

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.