UNC-52/perlecan isoform diversity and function in Caenorhabditis elegans.

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulphate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin and neural cell-adhesion molecules. Three major classes of UNC-52/perlecan isoforms are produced through alternative splicing, and these distinct proteins exhibit complex spatial and temporal expression patterns throughout development. The unc-52 gene plays an essential role in myofilament assembly in body-wall muscle during embryonic development.

The let-268 locus of Caenorhabditis elegans encodes a procollagen lysyl hydroxylase that is essential for type IV collagen secretion.

Basement membranes are thin sheets of specialized extracellular matrix molecules that are important for supplying mechanical support and for providing an interactive surface for cell morphology. Prior to secretion and assembly, basement membrane molecules undergo intracellular processing, which is essential for their function. We have identified several mutations in a procollagen processing enzyme, lysyl hydroxylase (let-268). The Caenorhabditis elegans lysyl hydroxylase is highly similar to the vertebrate lysyl hydroxylase, containing all essential motifs required for enzymatic activity, and is the only lysyl hydroxylase found in the C. elegans sequenced genome. In the absence of C. elegans lysyl hydroxylase, type IV collagen is expressed; however, it is retained within the type IV collagen-producing cells. This observation indicates that in let-268 mutants the processing and secretion of type IV collagen is disrupted. Our examination of the body wall muscle in these mutant animals reveals normal myofilament assembly prior to contraction. However, once body wall muscle contraction commences the muscle cells separate from the underlying epidermal layer (the hypodermis) and the myofilaments become disorganized. These observations indicate that type IV collagen is required in the basement membrane for mechanical support and not for organogenesis of the body wall muscle.

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.

Complex patterns of alternative splicing mediate the spatial and temporal distribution of perlecan/UNC-52 in Caenorhabditis elegans.

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan. Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

A metalloprotease prepares the way.

In the nematode Caenorhabditis elegans gonad shape and size is determined by the migration of a leader cell, which is at the tip of the growing gonad arm. A metalloprotease secreted by the leader cell has recently been found to play an essential role in this process, preparing the way ahead for the cell's migration.

A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans.

We describe here the molecular and functional characterization of the Caenorhabditis elegans unc-97 gene, whose gene product constitutes a novel component of muscular adherens junctions. UNC-97 and homologues from several other species define the PINCH family, a family of LIM proteins whose modular composition of five LIM domains implicates them as potential adapter molecules. unc-97 expression is restricted to tissue types that attach to the hypodermis, specifically body wall muscles, vulval muscles, and mechanosensory neurons. In body wall muscles, the UNC-97 protein colocalizes with the beta-integrin PAT-3 to the focal adhesion-like attachment sites of muscles. Partial and complete loss-of-function studies demonstrate that UNC-97 affects the structural integrity of the integrin containing muscle adherens junctions and contributes to the mechanosensory functions of touch neurons. The expression of a Drosophila homologue of unc-97 in two integrin containing cell types, muscles, and muscle-attached epidermal cells, suggests that unc-97 function in adherens junction assembly and stability has been conserved across phylogeny. In addition to its localization to adherens junctions UNC-97 can also be detected in the nucleus, suggesting multiple functions for this LIM domain protein.

The mec-8 gene of C. elegans encodes a protein with two RNA recognition motifs and regulates alternative splicing of unc-52 transcripts.

Mutations in the mec-8 gene of Caenorhabditis elegans were previously shown to affect the functions of body wall muscle and mechanosensory and chemosensory neurons. Mutations in mec-8 also strongly enhance the mutant phenotype of specific mutations in unc-52, a gene that encodes, via alternative splicing of pre-mRNA, a set of basement membrane proteins, homologs of perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis and cuticle. We have cloned mec-8 and found that it encodes a protein with two RNA recognition motifs, characteristic of RNA binding proteins. We have used reverse transcription-PCR and RNase protection experiments to show that mec-8 regulates the accumulation of a specific subset of alternatively spliced unc-52 transcripts. We have also shown with antibodies to UNC-52 that mec-8 affects the abundance of a subset of UNC-52 isoforms. We propose that mec-8 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and one or more additional genes that affect mechanosensory and chemosensory neuron function.

Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of Caenorhabditis elegans.

Perlecan, a component of the extracellular matrix (ECM), is essential for myofilament formation and muscle attachment in Caenorhabditis elegans. We show here that perlecan is a product of muscle and that it behaves in a cell autonomous fashion. That is, perlecan expressed in an individual muscle cell does not spread beyond the borders of the ECM underlying that cell. Using a polyclonal antibody that recognizes all isoforms of perlecan, we demonstrate that this protein first appears extracellularly at the comma stage (approx. 350 min) of development. We also show that during morphogenesis muscle cells have a heretofore undescribed plasticity of shape. This ability to regulate cell shape allows cells within a muscle quadrant to compensate for missing cells and to form a functional quadrant. A dramatic example of this morphological flexibility can be observed in animals in which the D blastomere has been removed by laser ablation. Such animals, lacking 20 of the 81 embryonic body wall muscle cells, can survive to become viable adult animals indistinguishable from wildtype animals. This demonstrates that the assembly of an embryo via a stereotypic lineage does not preclude a more general regulation during morphogenesis. It appears that embryos are flexible enough to immediately compensate for drastic alterations in tissue composition, a feature of development that may be of general importance during evolution.

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.

Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan.

Mutations in the unc-52 gene of Caenorhabditis elegans affect attachment of the myofilament lattice to the muscle cell membrane. Here, we demonstrate that the unc-52 gene encodes a nematode homolog of perlecan, the mammalian basement membrane heparan sulfate proteoglycan. The longest potential open reading frame of this gene encodes a 2482-amino-acid protein with a signal peptide and four domains. The first domain is unique to the unc-52 polypeptide, whereas the three remaining domains contain sequences found in the LDL receptor (domain II) laminin (domain III) and N-CAM (domain IV). We have identified three alternatively spliced transcripts that encode different carboxy-terminal sequences. The two larger transcripts encode proteins containing all or part of domain IV, whereas the smaller transcript encodes a shortened polypeptide that completely lacks domain IV. We have determined that the disorganized muscle phenotype observed in unc-52(st196) animals is caused by the insertion of a Tc1 transposon into domain IV. Two monoclonal antibodies that recognize an extracellular component of all contractile tissues in C. elegans fail to stain embryos homozygous for a lethal unc-52 allele. We have mapped the epitopes recognized by both monoclonal antibodies to a region of domain IV in the unc-52-encoded protein sequence.

Mutations in the sup-38 gene of Caenorhabditis elegans suppress muscle-attachment defects in unc-52 mutants.

Mutations in the unc-52 locus of Caenorhabditis elegans have been classified into three different groups based on their complex pattern of complementation. These mutations result in progressive paralysis (class 1 mutations) or in lethality (class 2 and 3 mutations). The paralysis exhibited by animals carrying class 1 mutations is caused by disruption of the myofilaments at their points of attachment to the cell membrane in the body wall muscle cells. We have determined that mutations of this class also have an effect on the somatic gonad, and this may be due to a similar disruption in the myoepithelial sheath cells of the uterus, or in the uterine muscle cells. Mutations that suppress the body wall muscle defects of the class 1 unc-52 mutations have been isolated, and they define a new locus, sup-38. Only the muscle disorganization of the Unc-52 mutants is suppressed; the gonad abnormalities are not, and the suppressors do not rescue the lethal phenotype of the class 2 and class 3 mutations. The suppressor mutations on their own exhibit a variable degree of gonad and muscle disorganization. Putative null sup-38 mutations cause maternal-effect lethality which is rescued by a wild-type copy of the locus in the zygote. These loss-of-function mutations have no effect on the body wall muscle structure.

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.

Germline excision of the transposable element Tc1 in C. elegans.

We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans.

We report here an unusual activation of the Tc1 transposable element system in Caenorhabditis elegans. Germline Tc1 activity, as measured by reversion of unc-22::Tc1 alleles, is elevated 50- to 100-fold by certain crosses. For example, unc-22::Tc1 reversion is 1 x 10(-3) in a mut-6 IV strain and less than 1 x 10(-6) in a non-mutator strain, but in the unc-22::Tc1 progeny of a cross between mut-6 hermaphrodites and non-mutator males, reversion is 10(-1). The reciprocal cross does not induce this enhancement of reversion. Results similar to those for mut-6 were obtained using a mut-5 II strain. The mutator hermaphrodite by nonmutator male cross per se is not required for the enhancement of reversion, as mut-5 hermaphrodites x mut-6/+ males also induce unc-22 revertants at an elevated frequency. This reversion enhancement appears to depend on a maternal component inherited from a mutator strain, suggesting that the regulation of Tc1 activity may be complex.

Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans.

The Caenorhabditis elegans gene unc-22 encodes a very large muscle protein, called twitchin, which consists of a protein kinase domain and several copies of two short motifs. The sequence of twitchin has unexpected similarities to the sequences of proteins of the immunoglobulin superfamily, cell adhesion molecules and vertebrate muscle proteins, including myosin light-chain kinase. These homologies, together with results from earlier genetic and molecular analyses, indicate that twitchin is involved in a novel mechanism of myosin regulation.

Analysis of a mutator activity necessary for germline transposition and excision of Tc1 transposable elements in Caenorhabditis elegans.

The Tc1 transposable element family of the nematode Caenorhabditis elegans consists primarily of 1.6-kb size elements. This uniformity of size is in contrast to P in Drosophila and Ac/Ds in maize. Germline transposition and excision of Tc1 are detectable in the Bergerac (BO) strain, but not in the commonly used Bristol (N2) strain. A previous study suggested that multiple genetic components are responsible for the germline Tc1 activity of the BO strain. To analyze further this mutator activity, we derived hybrid strains between the BO strain and the N2 strain. One of the hybrid strains exhibits a single locus of mutator activity, designated mut-4, which maps to LGI. Two additional mutators, mut-5 II and mut-6 IV, arose spontaneously in mut-4 harboring strains. This spontaneous appearance of mutator activity at new sites suggests that the mutator itself transposes. The single mutator-harboring strains with low Tc1 copy number generated in this study should be useful in investigations of the molecular basis of mutator activity. As a first step toward this goal, we examined the Tc1 elements in these low copy number strains for elements consistently co-segregating with mutator activity. Three possible candidates were identified: none was larger than 1.6 kb.

Transposon-induced deletions in unc-22 of C. elegans associated with almost normal gene activity.

The unc-22 gene of Caenorhabolitis elegans encodes a protein which is a component of the myosin-containing A-band of the worm's striated body-wall muscle. Among 51 revertants of a transposon-induced mutant, we have identified four which retain a barely detectable mutant phenotype. Molecular analysis shows that three of these have in-frame deletions of 1.0, 1.3 and 2.0 kilobases, whereas the fourth partial revertant and two other apparently complete revertants have small insertions. All these rearrangements involve coding sequence and, in the case of the deletions, result in polypeptides that are shorter than the wild-type protein. The region of the gene containing these rearrangements contains 10 copies of a motif recognized in other regions of the gene (our unpublished data). We suggest that one explanation for the minimally mutant phenotype associated with the deletions is that the size and the repeated nature of the unc-22 protein structure make it relatively tolerant of substitutions or deletions involving one or a small number of repeated motifs. These results could explain why in some human genetic diseases, such as Duchenne's muscular dystrophy, deletions can be associated with only mild forms of the disease.

Transposable element Tc1 of Caenorhabditis elegans recognizes specific target sequences for integration.

The frequency of movement of Tc1, a 1.6-kilobase transposable element in the nematode Caenorhabditis elegans, is under genetic control, and Tc1 insertion sites are widely but nonrandomly distributed. The usually high frequency of insertions at multiple sites in the gene unc-22 suggested that this gene might be particularly rich in preferred target sites. To discover the features of Tc1 target sites, we have sequenced the sites of seven independent Tc1 transpositions into unc-22 and three other sites. Our comparison of these and two other sites from the literature indicates that in all cases Tc1 integrates at the dinucleotide T-A when it is flanked both 5' and 3' by particular preferred nucleotides. Our analysis revealed the following consensus target for Tc1 integration: G-A-K-A-T-A-T-G-T, in which K = G or T. This target site sequence specificity has implications both for the mechanism of Tc1 transposition and the use of Tc1 in cloning genes by transposon-tagging.