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 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 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.     

spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.     

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.     

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.     

flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.     

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.     

mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.     

FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.