C. ELEGANS unc-105 Mutations Affect Muscle and Are Suppressed by Other Mutations That Affect Muscle

Certain mutations in the unc-105 II gene of the nematode Caenorhabditis elegans have dominant effects on morphology and behavior: animals become small, severely hypercontracted and paralyzed. These unc-105 mutants revert both spontaneously and with mutagens at high frequencies to a wild-type phenotype. Most of the reversion events are intragenic, apparently because the null (loss-of-function) phenotype of unc-105 is wild type. One revertant defined an extragenic suppressor locus, sup-20 X. Such suppressor alleles of sup-20 are rare, and the apparent null phenotype of sup-20 is embryonic lethality. By constructing animals genetically mosaic for sup-20, we have shown that the primary effect of sup-20 is in muscle cells. In addition to mutations in sup-20, other mutations causing muscle defects, such as unc-54 and unc-22 mutations, suppress the hypercontracted phenotype of unc-105. The ease of identifying nonhypercontracted revertants of unc-105 mutants greatly facilitates the isolation of new mutants defective in muscle structure and function.     

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.     

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.     

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.     

Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.     

A Second Informational Suppressor, SUP-7 X, in CAENORHABDITIS ELEGANS

More than 30 independent suppressor mutations have been obtained in the nematode C. elegans through reversion analysis of two unc-13 mutants. Many of the new isolates map to the region of the previously identified informational suppressor, sup-5 III (Waterston and Brenner 1978). Several of the other suppressor mutations map to the left half of the X-linkage group and define a second suppressor gene, sup-7 X. In tests against 40 mutations in six genes, the sup-7(st5) allele was found to suppress to a greater extent the same alleles acted on by sup-5(e1464). Like sup-5(e1464), sup-7(st5) acts on null alleles of the myosin heavy-chain gene unc-54 I (MacLeod et al. 1977; MacLeod, Waterston and Brenner 1977) and the putative paramyosin gene unc-15 I (Waterston et al. 1977). Chemical analysis of unc-15(e1214); sup-7(st5) animals show that paramyosin is restored to more than 30% of the wild-type level.—As was observed for sup-5(e1464), suppression by sup-7(st5) is dose dependent and is greater in animals grown at 15° than at 25°. However, associated with this increased suppression is a decreased viability of sup-7(st5) homozygotes. Reversion of the lethality has resulted in the isolation of deficiency mutations that complement st5 lethality, but lack suppressor function. These properties of sup-7(st5) suggest that it, like sup-5(e1464), is an informational suppressor of null alleles, and its reversion via deficiencies further narrows the possible explanations of its action.     

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.     

The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit

The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.     

The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.     

The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans

Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78-null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1-null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.     

Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans

Over 100 revertants of five different amber mutants were analyzed by Southern blot hybridization using synthetic oligomers as probes to detect a single base change at the anticodon, CCA to CTA (amber), of tRNA(Trp) genes of Caenohrabditis elegans. Of the 12 members of the tRNA(Trp) gene family, a total of eight were converted to amber suppressor alleles. All eight encode identical tRNAs; three of these are new tRNA(Trp) suppressors, sup-21, sup-33 and sup-34. Previous results had suggested that individual suppressor tRNA genes were expressed differentially in a cell-type- or developmental stage-specific manner. To extend these observations to the new genes and to test the specificity of expression against additional genes, cross suppression tests of these eight amber suppressors were carried out against amber mutations in several different genes including genes likely to be expressed in the same cell-type: three nervous system-affecting genes, two muscle structure-affecting genes and two genes presumed to be expressed in hypodermis. Seven out of eight suppressors could be distinguished one from another by the spectrum of their suppression efficiencies. These results also provide further evidence of cell-type-specific patterns of expression in the nervous system, muscle and hypodermis. The suppression pattern of the suppressor against the two muscle-affecting genes, unc-15 and unc-52, suggested that either the suppressors are expressed in a developmental stage-specific manner or that the unc-52 products are expressed in cell-types other than muscle, possibly hypodermis.     

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.     

The Unc-8 and Sup-40 Genes Regulate Ion Channel Function in Caenorhabditis Elegans Motorneurons

Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.     

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.     

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.     

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.