Dominant Suppressors of a Muscle Mutant Define an Essential Gene of CAENORHABDITIS ELEGANS

The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.     

Three New Classes of Mutations in the Caenorhabditis Elegans Muscle Gene Sup-9

We are studying five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. A distinctive ``rubber-ban'''' muscle-defective phenotype was previously shown to result from rare altered-function mutations in either of two of these genes, unc-93 and sup-10. Null mutations in sup-9, sup-10, sup-18 or unc-93 act as essentially recessive suppressors of these rubber-band mutations. In this work, we identify three new classes of sup-9 alleles: altered-function rubber-band, partial loss-of-function and dominant-suppressor. The existence of rubber-band mutations in sup-9, sup-10 and unc-93 and the suppression of these mutations by null mutations in any of these three genes suggest that these proteins are required at the same step in muscle contraction. Moreover, allele-specific interactions shown by the partial loss-of-function mutations indicate that the products of these interacting genes may physically contact each other in a multiple subunit protein complex. Finally, the phenotypes of double rubber-band mutant combinations suggest that the rubber-band mutations affect a neurogenic rather than a myogenic input in excitation-contraction coupling in muscle.     

Molecular and Genetic Analysis of Unc-7, a Caenorhabditis Elegans Gene Required for Coordinated Locomotion

Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.     

The Caenorhabditis elegans UNC-87 protein is essential for maintenance, but not assembly, of bodywall muscle

Mutations in the unc-87 gene of Caenorhabditis elegans cause disorganization of the myofilament lattice in adult bodywall muscle. In order to assess the organization of specific bodywall muscle components in the absence of the unc-87 gene product, we examined the bodywall muscles of mutant animals using phalloidin and monoclonal antibodies to various muscle proteins. These studies indicated that the bodywall muscle of unc-87 embryos is initially almost wild type in its organization, but at later stages, the muscle becomes severely disorganized. To address the possibility that this disorganization is due to deterioration of the muscle as a result of contraction, we introduced into the unc-87 mutant background a mutation that decreases myosin heavy chain activity but does not substantially affect muscle structure. The improved muscle structure and motility of the double mutants are consistent with the hypothesis that at least part of the disorganization phenotype of unc-87 mutants is a consequence of the wild-type levels of force generated during muscle contraction. These results imply that the role of the unc-87 gene product is not in specifying organization but rather in serving as a structural component maintaining lattice integrity during and after contraction.     

Characterization of Revertants of Unc-93(e1500) in Caenorhabditis Elegans Induced by N-Ethyl-N-Nitrosourea

Phenotypic reversion of the rubber-band, muscle-defective phenotype conferred by unc-93(e1500) was used to determine the utility of N-ethyl-N-nitrosourea (ENU) as a mutagen for genetic research in Caenorhabditis elegans. In this system, ENU produces revertants at a frequency of 3 X 10(-4), equivalent to that of the commonly used mutagen, EMS. The gene identity of 154 ENU-induced revertants shows that the distribution of alleles between three possible suppressor genes differs from that induced by EMS. A higher percentage of revertants are alleles of unc-93 and many fewer are alleles of sup-9 and sup-10. Three revertants complement the three known suppressor genes; they may therefore identify a new gene product(s) involved in this system of excitation-contraction coupling in C. elegans. Molecular characterization of putative unc-93 null alleles reveals that the base changes induced by ENU are quite different from those induced by EMS; specifically we see an increased frequency of A/T -> G/C transitions. The frequency of ENU-induced intragenic deletions is found to be 13%. We suggest that ENU, at concentrations below 5 mM, will be a superior mutagen for studies of protein function in C. elegans.     

A Visible Allele of the Muscle Gene sup-10 X of C. ELEGANS

In this paper, we extend our previous analyses of a set of genes in Caenorhabditis elegans that are involved in muscle structure and function: unc-93 III, sup-9 II, sup-10 X and sup-11 I. We describe an unusual, visible allele of sup-10, examine how this allele interacts genetically with mutations in other genes of this set and propose that the wild-type products of the unc-93 and sup-10 loci may be components of a protein complex. We also describe a new gene of this set, sup-18 III, and the interaction of sup-18 alleles with mutations in the other genes.     

unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction

Striated muscle contraction is elicited by the release of stored calcium ions through ryanodine receptor channels in the sarcoplasmic reticulum. ryr-1 is a C. elegans ryanodine receptor homologue that is expressed in body-wall muscle cells used for locomotion. Using genetic methods, we show that ryr-1 is the previously identified locus unc-68. First, transposon-induced deletions within ryr-1 are alleles of unc-68. Second, transformation of unc-68 mutants with ryr-1 genomic DNA results in rescue of the Unc phenotype. unc-68 mutants move poorly, exhibiting an incomplete flaccid paralysis, yet have normal muscle ultrastructure. The mutants are insensitive to the paralytic effects of ryanodine, and lack detectable ryanodine-binding activity. The Unc-68 phenotype suggests that ryanodine receptors are not essential for excitation- contraction coupling in nematodes, but act to amplify a (calcium) signal that is sufficient for contraction.     

The Mec-8 Gene of Caenorhabditis Elegans Affects Muscle and Sensory Neuron Function and Interacts with Three Other Genes: Unc-52, Smu-1 and Smu-2

Mutations in the Caenorhabditis elegans gene mec-8 were previously shown to cause defects in mechanosensation and in the structure and dye filling of certain chemosensory neurons. Using noncomplementation screens, we have identified eight new mec-8 alleles and a deficiency that uncovers the locus. Strong mec-8 mutants exhibit an incompletely penetrant cold-sensitive embryonic and larval arrest, which we have correlated with defects in the attachment of body muscle to the hypodermis and cuticle. Mutations in mec-8 strongly enhance the mutant phenotype of unc-52(viable) mutations; double mutants exhibit an unconditional arrest and paralysis at the twofold stage of embryonic elongation, a phenotype characteristic of lethal alleles of unc-52, a gene previously shown to encode a homolog of the core protein of heparan sulfate proteogylcan, found in basement membrane, and to be involved in the anchorage of myofilament lattice to the muscle cell membrane. We have identified and characterized four extragenic recessive suppressors of a mec-8; unc-52(viable) synthetic lethality. The suppressors, which define the genes smu-1 and smu-2, can weakly suppress all mec-8 mutant phenes. They also suppress the muscular dystrophy conferred by an unc-52(viable) mutation.     

The Unc-45 Gene of Caenorhabditis Elegans Is an Essential Muscle-Affecting Gene with Maternal Expression

We have isolated three novel alleles of the unc-45 locus in C. elegans, that are recessive lethals. Two of these alleles, when homozygous, result in a nearly total loss of muscle contraction with a concomitant arrest of development and a displacement of muscle cells. The third allele is similar, but showed maternal rescue by a wild-type allele. All previously identified unc-45 alleles were temperature sensitive and, although they produced paralysis of adult animals, all were homozygous viable. Prior genetic studies with these temperature sensitive alleles had suggested that at least one function of the unc-45 gene product was to interact with the major myosin heavy chain isoform, MHC B, of body wall muscles. Our observations of the lethal alleles suggest that the unc-45 product normally interacts with additional muscle components in both the body wall and pharyngeal muscles. In particular, we suggest that the unc-45 product might interact with all four myosin heavy chains: MHC B; MHC A; and the pharyngeal isoforms, MHC C and MHC D. Maternal rescue of the lethality of the third allele shows that the unc-45 gene product is present in the oocytes, although it may not be necessary until late in development when myofilaments begin to assemble.     

Mutations in the Unc-52 Gene Responsible for Body Wall Muscle Defects in Adult Caenorhabditis Elegans Are Located in Alternatively Spliced Exons

The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping.     

unc-45 gene of Caenorhabditis elegans encodes a muscle-specific tetratricopeptide repeat-containing protein

The unc-45 gene of the nematode, Caenorhabditis elegans, is essential for muscle organization and embryonic development. Genetic evidence suggests the unc-45 gene product controls muscle thick filament assembly. We report here on the determination of the gene's chromosomal location and the isolation and sequencing of its cDNA. The amino terminus of the predicted unc-45 protein contains three tandem repeats that belong in the tetratricopeptide repeat family. Tetratricopeptide motifs have been shown to be involved in protein interactions, and some of the closest homologues have chaperone-like activity. The carboxy terminus of the protein has homology with the related fungal proteins, CRO1 and She4p, which have been postulated to play a role in assembly of or interactions with a cytoplasmic myosin. We have also determined the sequence of the homologous gene from C. briggsae, which demonstrates a high level of conservation. We show that the unc-45 gene promoter can drive reporter gene expression, which is limited to muscle tissues (pharyngeal, body wall, vulval, and anal muscles), consistent with a role for the unc-45 gene in muscle development or function.     

Mutations in the unc-54 myosin heavy chain gene of caenorhabditis elegans that alter contractility but not muscle structure

Reversion analysis of mutants of unc-22 IV, a gene affecting muscle structure and function in Caenorhabditis elegans, led to the isolation of six extragenic dominant suppressors of the “twitching” phenotype of unc-22 mutants. All six suppressors are new alleles of unc-54 I, the major body wall myosin heavy chain gene. Homozygous suppressor strains are slow, stiff and have normal muscle structure, whereas previously identified unc-54 alleles confer flaccid paralysis and drastic reduction in thick filament number and organization. Placement of the three suppressor mutations s74, s77 and s95 on the genetic fine structure map of unc-54 demonstrates that they are clustered near the right end of the map. Since this end of the gene corresponds to the 5′ end of the coding sequence, these suppressor mutations probably result in amino acid substitutions in the globular head of the myosin molecule, and should be of value in studies of myosin force generation.     

Gene Interactions Affecting Muscle Organization in CAENORHABDITIS ELEGANS

Revertants of unc-15(e73)I, a paralyzed mutant with an altered muscle paramyosin, include six dominant and two recessive intragenic unc-15 revertants, two new alleles of the previously identified suppressor gene, sup-3 V, and a new suppressor designated sup-19(m210)V. The recessive intragenic unc-15 revertants exhibit novel alterations in paramyosin paracrystal structure and distribution, and these alterations are modified by interaction with unc-82(e1220)IV, another mutation that affects paramyosin. A strain containing both unc-15 and a mutation in sup-3 V that restores movement was mutagenized, and paralyzed mutants resembling unc-15 were isolated. Twenty mutations that interfere with suppression were divided into three classes (nonmuscle, sus-1, and mutations within sup-3) based on phenotype, genetic map position and dominance. The nonmuscle mutations include dumpy and uncoordinated types that have no obvious direct effect on muscle organization. Two recessive mutations define a new gene, sus-1 III. These mutations modify the unc-15(e73) phenotype to produce a severely paralyzed, dystrophic double mutant that is not suppressed by sup-3. Five semidominant, intragenic sup-3 antisuppressor mutations, one of which occurred spontaneously, restore the wild-type sup-3 phenotype of nonsuppression. However, reversion of these mutants generated no new suppressor alleles of sup-3, suggesting that the sup-3 antisuppressor alleles are not wild type but may be null alleles.     

UNC-87 isoforms,Caenorhabditis elegans calponin-related proteins,interact with both actin and myosin and regulate actomyosin contractility

Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ∼190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.     

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.