Eric Miska et al. (2002) European Worm Meeting "Functional analysis of the micro RNA genes of C. elegans"

Ezequiel Alvarez-Saavedra, David P Bartel, Eric Miska, Nelson C Lau, Bob Horvitz, Victor Ambros, Allison Abbott

[European Worm Meeting, 2002]

* HHMI and Dept. Biology, MIT, Cambridge, MA 02139, USA Dept. Genetics, Dartmouth Medical School, Hanover, NH, 03755, USA # Whitehead Institute for Biomedical Research and Dept. Biology, MIT, Cambridge, MA 02142, USA

PR Stancombe et al. (1999) International C. elegans Meeting "Characterisation of neprilysin (NEP) and endothelin-converting enzyme (ECE) from C. elegans"

PR Stancombe, D Coates, M Sajid, RE Isaac

[International C. elegans Meeting, 1999]

Cell-surface peptidases participate in the postsecretory processing and metabolism of neuropeptides and peptide hormones. Neutral endopeptidase-24.11 (NEP) is the prototype of a mammalian subfamily of zinc metallopeptidases that also includes endothelin-converting enzyme (ECE), the product of the PEX gene and KELL, an erythrocyte cell-surface antigen. These enzymes have central roles in a variety of physiological and disease processes, including cardiovascular function, cartilage and bone metabolism, inflammation and embryogenesis. Human zinc metalloproteinases belonging to this subfamily are important targets for therapeutic drugs. C. elegans has in the region of 90 genes with the zinc-binding consensus motif HEXXH of the superfamily of zinc metalloproteinases. Cluster analysis of predicted amino acid sequences reveals a group of 8 genes with closer similarity to mammalian NEPs/ECEs than the others. Hydrophobicity plots of the predicted protein sequences for these 8 genes revealed 5 that gave the expected domain structure seen in mammalian homologues; a short NH 2 -terminus intracellular region, a single transmembrane domain and a large extracellular region with a COOH-terminus active site. Expression patterns of beta-galactosidase and GFP have been obtained for 4 of these genes. T16A9.4 has a largely neuronal expression pattern with a pharyngeal and vulva muscle component; ZK970.1 and T05A8.4 are expressed in body wall muscle cells; ZK20.6 is pharynx specific. RNA interference experiments are being conducted with these genes to determine any null phenotypes.

Gert Jansen et al. (2001) International C. elegans Meeting "The C. elegans G-protein gamma subunit gpc-1 is involved in adaptation to water-soluble attractants."

David Weinkove, Gert Jansen, Ronald H A Plasterk

[International C. elegans Meeting, 2001]

Heterotrimeric G proteins form a first intracellular step in many signal transduction cascades. C. elegans has 21 Galpha, 2 Gbeta and 2 Ggamma subunits. Based on functional analyses the Galpha subunits can be divided into 2 groups. The conserved alpha subunits are ubiquitously expressed and regulate muscle and neuron activity. At least 14 other Galpha's play a role in sensory transduction. A similar discrimination can be made between the 2 G-protein gamma subunits, gpc-1 and gpc-2 . This latter gamma subunit is quite ubiquitously expressed. By contrast, gpc-1 is specifically expressed in only 6 pairs of amphid neurons and 1 pair of phasmid neurons, suggesting a function in sensory signaling. However, gpc-1 is not essential for the detection of various environmental cues, such as odorants or salts. To test if GPC-1 is involved in other types of sensory plasticity we developed a water-soluble compound adaptation assay. The behavior of wild-type animals in this assay confirms the idea that prolonged exposure to water-soluble compounds can abolish chemo-attraction to these same water-soluble compounds. This decrease in chemotaxis is time and concentration dependent, salt specific and reversible. gpc-1 mutant animals show clear deficits in their ability to adapt to three water-soluble compounds, NaAc, NaCl and NH 4 Cl. Also adp-1 and osm-9 , two loci previously implicated in odorant adaptation, are involved in adaptation to these salts. Our findings clearly indicate that G proteins are involved in the perception of salts. It is still unclear where in the signaling cascade these molecules function.

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

K Eto, H Tokumitsu, Y Kimura, M Muramatsu

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

H Qadota et al. (1999) International C. elegans Meeting "Functional analysis of C. elegans PKN homologue"

T Miyauchi, H Qadota, K Kasuya, K Kaibuchi

[International C. elegans Meeting, 1999]

The Rho family GTPases (Rho, Rac, Cdc42) are involved in the regulation of actin reorganization, cell polarity, cell growth, and cell-cell adhesion. The Rho family GTPases have two interconvertible forms; the GTP-bound active and GDP-bound inactive forms. The GTP-bound form of Rho family GTPases interacts with their specific effectors. The GTP-bound form of RhoA binds preferentially to Protein Kinase N (PKN), Rho-kinase (also called ROK), myosin binding subunit of myosin phosphatase (MBS). Besides proceeding of functional analysis of Rho-kinase and MBS, the physiological functions of other effectors of Rho GTPase including PKN remain to be clarified. Here we examined physiological functions of PKN using C. elegans as a genetic model system. We have identified the C. elegans homologue of mammalian PKN in genome sequencing database and Yuji Kohara's EST sequence library. The cDNA clone, yk345c10, which we call pkn-1 ( C. elegans PKN), maps to cosmid F46F6 on the right arm of LGX. The predicted protein shows significant homology to mammalian PKN. E. coli expressed NH 2 -terminus of PKN-1 fused with maltose binding protein binds preferentially to GTP g S •GST-CeRhoA, but not to GDP•GST-CeRhoA, suggesting that the C. elegans PKN homologue also interacts with Rho GTPase in C. elegans . Based on expression studies using the pkn-1::gfp fusion construct, pkn-1 is preferentially expressed in the muscle cells. Overexpression of the catalytic domain of PKN-1 under the control of heat shock promoter caused abnormal movement, suggesting that CeRhoA and PKN-1 are involved in the regulation of the muscle contraction.

Samantha McCavera et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Molecular Pharmacology of an Ivermectin-sensitive Glutamate-gated Chloride Channel"

Debra Woods, Samantha McCavera, Adrian Wolstenholme, Tom Walsh, Darran Yates, Adrian Rogers

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Glutamate-gated chloride channels are ligand-gated ion channels found only in invertebrates. They are the targets of the macrocyclic lactone family of anthelmintics and insecticides, of which ivermectin is probably the best known. The avr-14 gene of C. elegans encodes two GluCl subunits, alpha 3A and alpha 3B, and mutations in this gene contribute to avermectin resistance: an amino-acid substitution (L256F) has also found in the alpha;3B subunit of ivermectin-resistant Cooperia oncophora, a parasite of cattle. We have been comparing the pharmacology of GluClα3B from C. elegans and Haemonchus contortus, a close-related parasite of sheep and goats. When expressed in Xenopus oocytes, the H. contortus receptor is much more sensitive to L-glutamate (the natural ligand), with an EC50 of 27.5 ± 1.1mM compared to that of 2.23 ± 1.2 mM for the C. elegans channel. In addition, L-glutamate activation of the channel exhibits positive co-operativity (nH = 1.89 ± 0.35) which is not observed for the C. elegans channel. Ivermectin opens the HcGluClα3B channels slowly and irreversibly, at concentrations down to 0.1nM, with an EC50 of ~12nM. When we introduced the L256F mutation into the HcGluCl alpha 3B channel, the EC50 for L-glutamate was increased to 92.2 mM and the positive co-operativity was lost. Ivermectin binding to the channel was assessed by expressing wild-type and mutated HcGluClα3B in COS-7 and measuring the specific binding of radiolabelled ivermectin to membrane preparations. The kD for ivermectin binding was not significantly different between wild-type (2.64nM) and L256F mutant (2.96nM) receptors, but again the Hill number was reduced (from 3.2 to 1.8), indicating a reduction in co-operativity in the binding to the mutant channel. These data indicate that the H. contortus GluCl alpha 3B channel has a higher affinity for both L-glutamate and ivermectin than its C. elegans counterpart and that it is possible that the L256F polymorphism may contribute to ivermectin resistance, possibly by affecting the interactions between individual subunits to prevent co-operative binding of agonists. Preliminary attempts to detect this polymorphism in ivermectin-resistant isolates of H. contortus have been unsuccessful.

Kyuhyung Kim et al. (2001) International C. elegans Meeting "The family of genes encoding FMRFamide-related neuropeptides of Caenorhabditis elegans"

Chris Li, Kyuhyung Kim

[International C. elegans Meeting, 2001]

FMRFamide (Phe-Met-Arg-Phe-NH 2 ) and related peptides (FaRPs) are short peptide neurotransmitters that have been found throughout the animal kingdom. FaRPs have been shown to have many general functions, including cardioregulation, muscle control, pain modulation, and learning. Our lab uses Caenorhabditis elegans as a model system to study FaRPs. In C. elegans , at least 50% of the neurons express FaRPs, which are encoded by at least 23 flp ( F MRFamide- l ike p eptides) genes. Expression of most flp genes has been confirmed by the isolation or identification of peptides, cDNAs, or EST sequences. In addition, genetic analysis of flp-1 has shown that at least one of flp genes has unique functions in C. elegans . To determine the cell-specific expression of the flp genes in C. elegans , transgenic animals carrying a gfp (green fluorescent protein) reporter construct under the control of each flp promoter are being generated by germ-line transformation. To date, the cell-specific expression patterns of 18 flp genes have been determined. Each flp gene is expressed in distinct, but sometimes overlapping, sets of cells, suggesting that flp genes function in both unique and overlapping pathways. Furthermore, several genes show stage-specific or sex-specific expression patterns, suggesting that some FLP peptides may function during development and in reproductive behaviors. The cell-specific expression patterns of the remaining flp genes are being determined. To determine the function of the flp genes in C. elegans , several flp deletion mutants were isolated by screening libraries of chemically-mutagenized C. elegans . All deletion mutants are still being characterized. We are continuing the screen for new deletion mutants. To identify genes that may regulate expression of the flp genes, we are using transgenic animals expressing a gfp reporter construct under the control of a flp-1 or flp-12 promoter in genetic screens. These strains are mutagenized, and mutants showing altered expression of these markers are isolated. Several mutants that show no, faint, or strong GFP expression have been isolated. The genes corresponding to these mutations are being mapped, characterized, and cloned.