unc-94 encodes a tropomodulin in Caenorhabditis elegans

unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries.     

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 Caenorhabditis elegans odr-2 gene encodes a novel Ly-6-related protein required for olfaction

Caenorhabditis elegans odr-2 mutants are defective in the ability to chemotax to odorants that are recognized by the two AWC olfactory neurons. Like many other olfactory mutants, they retain responses to high concentrations of AWC-sensed odors; we show here that these residual responses are caused by the ability of other olfactory neurons (the AWA neurons) to be recruited at high odor concentrations. odr-2 encodes a membrane-associated protein related to the Ly-6 superfamily of GPI-linked signaling proteins and is the founding member of a C. elegans gene family with at least seven other members. Alternative splicing of odr-2 yields three predicted proteins that differ only at the extreme amino terminus. The three isoforms have different promoters, and one isoform may have a unique role in olfaction. An epitope-tagged ODR-2 protein is expressed at high levels in sensory neurons, motor neurons, and interneurons and is enriched in axons. The AWC neurons are superficially normal in their development and structure in odr-2 mutants, but their function is impaired. Our results suggest that ODR-2 may regulate AWC signaling within the neuronal network required for chemotaxis.     

The C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein.

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. Transposon insertions in three unc-104 alleles (e2184, rh1016, and rh1017) were used as physical markers to clone the unc-104 gene. DNA sequence analysis of unc-104 cDNAs revealed an open reading frame capable of encoding a 1584 amino acid protein with similarities to kinesin heavy chain. The similarities are greatest in the amino-terminal ATPase and microtubule-binding domains. Although the primary sequence relatedness to kinesin is weak in the remainder of the molecule, the predicted secondary structure and regional isoelectric points are similar to kinesin heavy chain.     

The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization

A mutation in the Caenorhabditis elegans spe-38 gene results in a sperm-specific fertility defect. spe-38 sperm are indistinguishable from wild-type sperm with regards to their morphology, motility and migratory behavior. spe-38 sperm make close contact with oocytes but fail to fertilize them. spe-38 sperm can also stimulate ovulation and engage in sperm competition. The spe-38 gene is predicted to encode a novel four-pass (tetraspan) integral membrane protein. Structurally similar tetraspan molecules have been implicated in processes such as gamete adhesion/fusion in mammals, membrane adhesion/fusion during yeast mating, and the formation/function of tight-junctions in metazoa. In antibody localization experiments, SPE-38 was found to concentrate on the pseudopod of mature sperm, consistent with it playing a direct role in gamete interactions.     

The Caenorhabditis elegans polarity gene ooc-5 encodes a Torsin-related protein of the AAA ATPase superfamily.

The PAR proteins are required for polarity and asymmetric localization of cell fate determinants in C. elegans embryos. In addition, several of the PAR proteins are conserved and localized asymmetrically in polarized cells in Drosophila, Xenopus and mammals. We have previously shown that ooc-5 and ooc-3 mutations result in defects in spindle orientation and polarity in early C. elegans embryos. In particular, mutations in these genes affect the re-establishment of PAR protein asymmetry in the P(1) cell of two-cell embryos. We now report that ooc-5 encodes a putative ATPase of the Clp/Hsp100 and AAA superfamilies of proteins, with highest sequence similarity to Torsin proteins; the gene for human Torsin A is mutated in individuals with early-onset torsion dystonia, a neuromuscular disease. Although Clp/Hsp100 and AAA family proteins have roles in diverse cellular activities, many are involved in the assembly or disassembly of proteins or protein complexes; thus, OOC-5 may function as a chaperone. OOC-5 protein co-localizes with a marker of the endoplasmic reticulum in all blastomeres of the early C. elegans embryo, in a pattern indistinguishable from that of OOC-3 protein. Furthermore, OOC-5 localization depends on the normal function of the ooc-3 gene. These results suggest that OOC-3 and OOC-5 function in the secretion of proteins required for the localization of PAR proteins in the P(1) cell, and may have implications for the study of torsion dystonia.     

Neuron navigator: a human gene family with homology to unc-53, a cell guidance gene from Caenorhabditis elegans

We have cloned the gene neuron navigator-1 (NAV1), a human homolog of unc-53, a gene involved in axon guidance in Caenorhabditis elegans. Duplications during evolution gave rise to three human homologs located on chromosomes 1q32.1, 11p15.1, and 12q21.1. NAV1 and NAV2 are expressed in the developing brain. NAV1, NAV2, and NAV3 expression is detected in adult heart, kidney, and brain, respectively. NAV1 encodes a protein lacking, in the aminoterminal part, a CH domain present in the other NAV genes. The first exon of NAV1 arose through an ancient internal duplication of sequences that also gave rise to exon 8 of NAV3 and exon 7 of NAV2. A detailed study of the NAV environment on the different chromosomes reveals incomplete micro-syntheny between the three regions. Through analysis of the phylogenetic relationships for three different gene families in the NAV environment, we reconstructed part of the events that formed these regions.     

mua-3, a gene required for mechanical tissue integrity in Caenorhabditis elegans, encodes a novel transmembrane protein of epithelial attachment complexes

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force through a series of mechanical linkages from the myofibrillar lattice of the body wall muscles, across an intervening extracellular matrix and epithelium (the hypodermis) to the cuticle. Mutations in mua-3 cause a separation of the hypodermis from the cuticle, suggesting this gene is required for maintaining hypodermal-cuticle attachment as the animal grows in size postembryonically. mua-3 encodes a predicted 3,767 amino acid protein with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein modules: 5 low density lipoprotein type A, 52 epidermal growth factor, 1 von Willebrand factor A, and 2 sea urchin-enterokinase-agrin modules. MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments, including the rectum, vulva, mechanosensory neurons, and excretory duct/pore. In addition, it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments (IFs) at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress.     

sel-7, a positive regulator of lin-12 activity, encodes a novel nuclear protein in Caenorhabditis elegans

Suppressor genetics in C. elegans has identified key components of the LIN-12/Notch signaling pathway. Here, we describe a genetic and molecular characterization of the suppressor gene sel-7. We show that reducing or eliminating sel-7 activity suppresses the effects of constitutive lin-12 activity, enhances the effects of partially reduced lin-12 activity, and causes a synthetic Lin-12(0) phenotype when combined with a null mutation in the sel-12 presenilin gene. These observations suggest that sel-7 is a positive regulator of lin-12 activity. We also show that SEL-7 encodes a novel nuclear protein. Through yeast two-hybrid screening, we identified an apparent interaction partner, K08E3.8, that also interacts with SEL-8, a known component of the nuclear complex that forms upon LIN-12 activation. Our data suggest potential roles for SEL-7 in the assembly or function of this nuclear complex.     

The Caenorhabditis elegans K10C2.4 gene encodes a member of the fumarylacetoacetate hydrolase family: a Caenorhabditis elegans model of type I tyrosinemia

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.