The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein

Fertilization, the union of sperm and egg to form a new organism, is a critical process that bridges generations. Although the cytological and physiological aspects of fertilization are relatively well understood, little is known about the molecular interactions that occur between gametes. C. elegans has emerged as a powerful system for the identification of genes that are necessary for fertilization. C. elegans spe-42 mutants are sterile, producing cytologically normal spermatozoa that fail to fertilize oocytes. Indeed, male mating behavior, sperm transfer to hermaphrodites, sperm migration to the spermatheca, which is the site of fertilization and sperm competition are normal in spe-42 mutants. spe-42 mutant sperm make direct contact with oocytes in the spermatheca, suggesting that SPE-42 plays a role during sperm-egg interactions just prior to fertilization. No other obvious defects were observed in spe-42 mutant worms. Cloning and sequence analysis revealed that SPE-42 is a novel predicted 7-pass integral membrane protein with homologs in many metazoan species, suggesting that its mechanism of action could be conserved.     

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 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.     

The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments

Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491--502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.     

GTSF‐1 is required for formation of a functional RNA‐dependent RNA Polymerase complex in Caenorhabditis elegans

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte‐specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C‐terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf‐1 and characterized it in the context of the sRNA pathways of C. elegans. We report that GTSF‐1 is not required for Piwi‐mediated gene silencing. Instead, gtsf‐1 mutants show a striking depletion of 26G‐RNAs, a class of endogenous sRNAs, fully phenocopying rrf‐3 mutants. We show, both in vivo and in vitro, that GTSF‐1 interacts with RRF‐3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF‐1 is required for the assembly of a larger RRF‐3 and DCR‐1‐containing complex (ERIC), thereby allowing for 26G‐RNA generation. We propose that GTSF‐1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA‐mediated silencing activities.     

AMPK‐mediated formation of stress granules is required for dietary restriction‐induced longevity in Caenorhabditis elegans

Stress granules (SGs) are nonmembranous organelles that are dynamically assembled and disassembled in response to various stressors. Under stressed conditions, polyadenylated mRNAs and translation factors are sequestrated in SGs to promote global repression of protein synthesis. It has been previously demonstrated that SG formation enhances cell survival and stress resistance. However, the physiological role of SGs in organismal aging and longevity regulation remains unclear. In this study, we used TIAR‐1::GFP and GTBP‐1::GFP as markers to monitor the formation of SGs in Caenorhabditis elegans. We found that, in addition to acute heat stress, SG formation could also be triggered by dietary changes, such as starvation and dietary restriction (DR). We found that HSF‐1 is required for the SG formation in response to acute heat shock and starvation but not DR, whereas the AMPK‐eEF2K signaling is required for starvation and DR‐induced SG formation but not heat shock. Moreover, our data suggest that this AMPK‐eEF2K pathway‐mediated SG formation is required for lifespan extension by DR, but dispensable for the longevity by reduced insulin/IGF‐1 signaling. Collectively, our findings unveil a novel role of SG formation in DR‐induced longevity.     

The Caenorhabditis elegans par-5 gene encodes a 14-3-3 protein required for cellular asymmetry in the early embryo.

The establishment of anterior-posterior polarity in the Caenorhabditis elegans embryo requires the activity of the maternally expressed par genes. We report the identification and analysis of a new par gene, par-5. We show that par-5 is required for asynchrony and asymmetry in the first embryonic cell divisions, normal pseudocleavage, normal cleavage spindle orientation at the two-cell stage, and localization of P granules and MEX-5 during the first and subsequent cell cycles. Furthermore, par-5 activity is required in the first cell cycle for the asymmetric cortical localization of PAR-1 and PAR-2 to the posterior, and PAR-3, PAR-6, and PKC-3 to the anterior. When PAR-5 is reduced by mutation or by RNA interference, these proteins spread around the cortex of the one-cell embryo and partially overlap. We have shown by sequence analysis of par-5 mutants and by RNA interference that the par-5 gene is the same as the ftt-1 gene, and encodes a 14-3-3 protein. The PAR-5 14-3-3 protein is present in gonads, oocytes, and early embryos, but is not asymmetrically distributed. Our analysis indicates that the par-5 14-3-3 gene plays a crucial role in the early events leading to polarization of the C. elegans zygote.     

The C. elegans nck-1 gene encodes two isoforms and is required for neuronal guidance

The NCK adaptor proteins are composed entirely of SH3 and SH2 domains and serve as protein interaction bridges for several receptors during signal transduction events. Here we report the molecular and genetic analysis of the Caenorhabditis elegans nck-1 gene. C. elegans nck-1 encodes two isoforms: NCK-1A and a shorter isoform that lacks the first SH3 domain, NCK-1B. C. elegans nck-1 mutants exhibit defects in axon guidance and neuronal cell position, as well as defects in the excretory canal cell, gonad, and male mating. NCK-1 is broadly expressed in neurons and epithelial cells with NCK-1B being the most abundant isoform. NCK-1A and NCK-1B share a similar expression pattern in parts of the nervous system, but also have independent expression patterns in other tissues. Interestingly, NCK-1B is localized to the nuclei of many cells. Genetic rescue experiments show that NCK-1 functions cell autonomously and, in general, either NCK-1A or NCK-1B is sufficient to function in axon guidance. However, there appears to be specific roles for each isoform, for example NCK-1B is required for HSN cell migration while NCK-1A is required for efficient male mating. Genetic epistasis experiments show that NCK-1 functions redundantly with the LAR Receptor Tyrosine Phosphatase, PTP-3, and the Netrin receptor UNC-40.     

A functional NR4A nuclear receptor DNA‐binding domain is required for organ development in Caenorhabditis elegans

NR4A nuclear receptors are a diverse group of orphan nuclear receptors with critical roles in regulating cell proliferation and cell differentiation. The ortholog of the NR4A nuclear receptor in Caenorhabditis elegans, NHR‐6, also has a role in cell proliferation and cell differentiation during organogenesis of the spermatheca. Here we show that NHR‐6 is able to bind the canonical NR4A monomer response element and can transactivate from this site in mammalian HEK293 cells. Using a functional GFP‐tagged NHR‐6 fusion, we also demonstrate that NHR‐6 is nuclear localized during development of the spermatheca. Mutation of the DNA‐binding domain of NHR‐6 abolishes its activity in genetic rescue assays, demonstrating a requirement for the DNA‐binding domain. This study represents the first genetic demonstration of an in vivo requirement for an NR4A nuclear receptor DNA‐binding domain in a whole organism. genesis 48:485–491, 2010. © 2010 Wiley‐Liss, Inc.     

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line.     

The C. elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length

The Caenorhabditis elegans maternal-effect clk genes are involved in the temporal control of development and behavior. We report the genetic and molecular characterization of clk-2. A temperature-sensitive mutation in the gene clk-2 affects embryonic and post-embryonic development, reproduction, and rhythmic behaviors. Yet, virtually all phenotypes are fully maternally rescued. Embryonic development strictly requires the activity of maternal clk-2 during a narrow time window between oocyte maturation and the two- to four-cell embryonic stage. Positional cloning of clk-2 reveals that it encodes a protein homologous to S. cerevisiae Tel2p. In yeast, the gene TEL2 regulates telomere length and participates in gene silencing at subtelomeric regions. In C. elegans, clk-2 mutants have elongated telomeres, and clk-2 overexpression can lead to telomere shortening. Tel2p has been reported to bind to telomeric DNA repeats in vitro. However, we find that a functional CLK-2::GFP fusion protein is cytoplasmic in worms. We discuss how the phenotype of clk-2 mutants could be the result of altered patterns of gene expression.     

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/beta-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.