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[
European Worm Meeting,
1998]
The C. elegans genome sequencing project revealed at the moment 65 cytochromes P450 putative coding sequences (about 0.5% of the estimated total coding sequences). Cytochromes P450 (P450) are NADPH dependent monooxygenases involved in endogenous (fatty acids, steroids ...) and exogenous (xenobiotics) compounds metabolism. These enzymes are virtually present in all organisms (bacteria, plants, animals). Cytochromes P450 gene expression can be modulated by the presence of xenobiotic presence in the media. In order to study P450 expression in C. elegans, we choosed to identify xenobiotic inductible P450 genes. This work was performed by Northern blot analysis with total RNAs extracted from C. elegans mixed stages exposed for 24 to 48 hours to various xenobiotics. This allowed to identify P450 genes inducible by polycyclic aromatic hydrocarbons (PAH) and weedkillers. We have also studied in vivo and in vitro xenobiotic related enzymatic activities. We are able to trace PAH metabolites in parts of the pharynx. In vitro experiments were made to demonstrate P450 involvement in these activities. The P450 expression induction and enzymatic activities studies in the major model C. elegans would certainly provide interesting elements to understand the invertebrates P450 expression and induction mecanisms. C. elegans would also be useful in order to develop pollution biomonitoring tools.
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[
International C. elegans Meeting,
1991]
We are interested in the structure of the different molecular forms of acetylcholinesterase (AChE) with a particular interest for the association of AChE to membranes, and in cloning the three ace genes of C.elegans. Preliminary studies have been conducted on the nematode Steinernema carpocapsae since it shows a much higher AChE activity than C. elegans. Sucrose gradient centrifugation analysis of crude extracts shows four AChE peaks sedimenting at 4S, 6S, 12S and 14S, a situation similar to that found in C. elegans wild type strains ( Johnson and Russell, 1983). Further analysis of gradient fractions by non denaturing electrophoresis reveals the existence of both hydrophilic and hydrophobic components in 6S and 14S forms. The hydrophobic 6S form is completely converted into its hydrophilic counterpart by phosphatidylinositol-specific-phospholipase C (PIPLC), indicating that this molecule possesses a glycolipid anchor close to the type already described in insect and vertebrate dimeric AChEs. The hydrophobic 14S component however is not susceptible to PIPLC, and the nature of the hydrophobic domain is still under investigation, as well as the molecular organization of all forms. We started the cloning of C. elegans ace 3 gene. For this purpose we improved the genetic map at the tip of chromosome II. ace 3 maps 0.4 map unit from unc 52, which has been cloned by D. Moerman, and lies in the center of a 600 kb contig. We are trying to clone ace 3 by transformation rescue.
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[
International C. elegans Meeting,
2001]
The C. elegans intestine is a bilaterally symmetric tube of 20 polarized epithelial cells. Cell polarity in the intestine involves a reorganization of the microtubule cytoskeleton, followed by migration of the intestinal nuclei apically, and other organelles basally. Small apical membrane separations coalesce into a continuous compartment to form the lumen of the intestine. Cell ablation experiments suggest that morphogenesis of the intestine requires interactions between intestinal cells and interactions between intestinal and non-intestinal cells surrounding the intestine 1,2 . We have been conducting screens for embryonically-expressed genes required for intestinal morphogenesis, and are currently characterizing one mutant,
zu450 , in more detail. The intestines of
zu450 homozygotes appear slightly disorganized, and have abnormally wide intestinal lumens. Analysis of
zu450 intestines using two indicators of epithelial polarity, the adherens junction marker MH27 and the lumenal marker MH33, reveal defects in the attachment of the intestine to the pharynx and rectum as well as discontinuities in the intestinal lumens. Preliminary analysis of the mutation
zu450 suggests that it may play a role in multiple epithelia;
zu450 homozygotes have elongation and enclosure defects in addition to intestinal morphogenesis defects. 1 Leung, B., G. J. Hermann, and J. R. Priess (1999). Developmental Biology 216 : 114-134. 2 Hermann, G. J., B. Leung, and J. R. Priess (2000). Development 127 : 3429-3440.
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[
International C. elegans Meeting,
1997]
We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant
fem-3 3'UTR at the 3' end. The
fem-3(gf) mutations map to the
fem-3 3'UTR and release
fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a
lag-2 promoter::GFP construct that carries either a strong or a weak
fem-3(gf) 3'UTR. The
lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a
fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the
lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.
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[
International C. elegans Meeting,
2001]
Heparan sulfate binds and activates a large variety of growth factors, enzymes and extracellular matrix proteins. These interactions largely depend on the specific arrangement of sulfated moieties and uronic acid epimers within the chains. These oligosaccharide sequences are generated in a step-wise manner, initiated by the formation of a linkage tetrasaccharide which is then extended by copolymerization of alternating
a1,4GlcNAc and
b1,4GlcA residues. As the chains polymerize, they undergo a series of sulfation and epimerization reactions. The first set of modifications involves the removal of acetyl units from subsets of GlcNAc residues, and the addition of sulfate groups to the resulting free amino groups. These reactions are catalyzed by a family of enzymes designated as GlcNAc N-deacetylase/N-sulfotransferases (NDST), since they simultaneously. Four members of the family have been identified in vertebrates, with single orthologs present in Drosophila and C. elegans. We have revealed tissue-specific expression pattern and unique enzymatic properties of these four isozymes1,2). In fly, loss of NDST (sulfateless) results in unsulfated chains and defective signaling by multiple growth factors and morphogens. I reconstituted cDNA for worm NDST from EST clones and 5' RACE products. Enzymatic activities will be discussed. 1) Aikawa, J. & Esko, J. D J. Biol. Chem. 274, 2690-2695 (1999) 2) Aikawa, J., Grobe, K., Tsujimoto, M. & Esko, J. D J. Biol. Chem. 276, 5876-5882 (2001)
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[
European Worm Meeting,
2004]
Caenorhabditis elegans has been found to be good model system for parasitic nematodes, drug screening and developmental studies. Like the respective parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins, carrying, in part, phosphorylcholine (PC) substitutents, which might play important roles in nematode development, fertility and, at least in the case of parasites, the survival within the host (1). With the exception of a major secretory/ excretory product from Achanthocheilonema viteae (ES-62) (2) and the aspartyl-protease ASP-6 (3), no other proteins carrying this epitope has been identified and studied in detail yet. For C. elegans two N-linked PC-epitopes have been reported so far: (I) a pentamannosyl-core structure carrying three PC-residues (4) and (II) a trimannosyl-core species elongated by a N-acetylglucosamine substituted at C-6 with PC (5). Furthermore, in Dauer larvae of C. elegans there was evidence for the presence of glycans with the composition PC1Hex3HexNAc3 to PC2dHex2Hex4HexNAc7 (6). Here we present the 2D-electrophoretic separation of C. elegans proteins, the comparison of the PC-substitution pattern in distinct developmental stages and the mass spectrometric identification of PC-modified proteins. References: 1.Lochnit, G., Dennis, R. D., and Geyer, R. (2000) Biol Chem 381, 839-847 2.Harnett, W., Harnett, M. M., and Byron, O. (2003) Curr Protein Pept Sci 4, 59-71 3.Lochnit, G., Grabitzki, J., Henkel, B., and Geyer, R. (2003) Biochemical Journal submitted 4.Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002) J Biol Chem 277, 49143-49157 5.Haslam, S. M., Gems, D., Morris, H. R., and Dell, A. (2002) Biochem. Soc. Symp. 69, 117-134 6.Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004) Proc Natl Acad Sci U S A 101, 3404-3408
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Guan, A., Meng, J., Zhen, M., Witvliet, D., Mulcahy, B., Lu, Y., Samuel, A., Wen, Q.
[
International Worm Meeting,
2017]
Throughout development, C. elegans maintains serpentine-like dorsal/ventral bending waves for locomotion. In the adult motor circuit, the cholinergic A- and B-type motor neurons innervate and stimulate dorsal and ventral body wall muscles, whereas the D-type GABAergic motor neurons, receiving inputs from A- and B-type motor neurons, innervate and inhibit muscles on the opposing side. Together, they constitute symmetric motor circuit input to the dorsal and ventral body muscle walls that generates balanced muscle activity (1). The cellular components of motor circuit, however, differ drastically in younger animals. All ventral muscle innervating motor neurons are not born until the mid-first larval stage (L1). Partial EM reconstruction in a previous study (2) showed that in L1 stage, the A- and B-type motor neurons only innervate dorsal body wall muscles, and D-type motor neurons innervate only ventral muscles. It remains unclear when the transition to the adult motor circuit synaptic wiring completes. Regardless, it is difficult to explain how such a circuit can produce the symmetric ventral/dorsal bending that constitutes the undulatory motor behavior. We have undertaken anatomical and functional analyses of the L1 motor circuit. I will present studies that lead to the exclusion of multiple potential mechanisms, and the potential involvement of additional, uncharacterized cells in facilitating ventral muscle contraction in the L1 stage animals. References 1. J. G. White, E. Southgate, J. N. Thomson, S. Brenner, Philos Trans R Soc Lond B Biol Sci 314, 1 (Nov 12, 1986). 2. J. G. White, D. G. Albertson, M. A. Anness, Nature 271, 764 (Feb 23, 1978).
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[
International C. elegans Meeting,
1993]
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[
International Worm Meeting,
2009]
How are polarized epithelia established and maintained? This question is of critical importance, as the loss of epithelial polarity is associated with metastasis(1). There are many well-studied protein complexes that lie in specific membrane compartments with roles integral to the epithelial cell. The E-cadherin-containing adherens junction serves to link neighboring epithelial cells together while the more basal tight junction functions to separate the apical and basolateral surfaces. For some cells, E-cadherin is the major initiator of cell polarity and epithelium formation via cell-cell adhesion(2). However, recent studies have discovered E-cadherin independent polarity pathways(3-6). C. elegans offers a powerful system to study this cadherin-independent mechanism, as E-cadherin is dispensible for the initiation of epithelial polarity in nematodes(4). We study cadherin-independent epithelium formation during pharynx development. Nine pharyngeal arcade cells undergo a mesenchymal-to-epithelial transition to link the pharynx to the outer epidermis(7). Ablation of the arcade cells results in a Pharynx unattached (Pun) phenotype, in which the pharynx fails to connect to the epidermis(7). Pun animals die as they are unable to eat. Our lab has undertaken a genetic screen for Pun mutants that fail to form the arcade cell epithelium (Portereiko and Mango, unpublished). This screen revealed that loss of the central-spindlin component ZEN-4/MKLP1 induces a Pun phenotype because the arcade cells fail to polarize(8). We are currently studying where and when ZEN-4 is needed for arcade cell polarization. We have also undertaken a structure/function analysis of this mitotic kinesin in order to elucidate its role in epithelialization. In addition, we are in the process of cloning several mutants that were isolated in the Pun mutagenesis screen. (1). J. M. Lee, S. Dedhar, R. Kalluri, E. W. Thompson, J Cell Biol 172, 973 (Mar 27, 2006). (2). L. N. Nejsum, W. J. Nelson, J Cell Biol 178, 323 (Jul 16, 2007). (3). A. F. Baas et al., Cell 116, 457 (Feb 6, 2004). (4). M. Costa et al., J Cell Biol 141, 297 (Apr 6, 1998). (5). T. J. Harris, M. Peifer, J Cell Biol 167, 135 (Oct 11, 2004). (6). W. B. Raich, C. Agbunag, J. Hardin, Curr Biol 9, 1139 (Oct 21, 1999). (7). M. F. Portereiko, S. E. Mango, Dev Biol 233, 482 (May 15, 2001). (8). M. F. Portereiko, J. Saam, S. E. Mango, Curr Biol 14, 932 (Jun 8, 2004).
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Landuyt, Bart, Schoofs, Liliane, Gottschalk, Alexander, Horvitz, H.Robert, Temmerman, Liesbet, Husson, Steven J., Ringstad, Niels, Meelkop, Ellen
[
International Worm Meeting,
2011]
Egg laying has mainly been studied at the behavioral, neuronal and neurochemical levels, but little is known about the biochemical control of the relevant neuropeptidergic signaling systems. Biosynthesis of endogenous peptides requires processing enzymes, such as proprotein convertase 2, which is encoded by
egl-3 (1, 2), and a carboxypeptidase encoded by
egl-21 (3, 4). Mutants defective in these genes have egg-laying defects, consistent with the finding that FMRFamide-like peptides (FLPs) have been linked to egg laying behavior. C. elegans enzymes that carry out the last step in the production of biologically active peptides, the carboxy-terminal amidation reaction, have not been characterized. This multistep reaction involves hydroxylation of the glycine a-carbon by a peptidyl-a-hydroxylating monooxygenase (PHM), followed by a cleavage reaction performed by peptidyl a-hydroxyglycine a-amidating lyase (PAL) to generate a glyoxylate molecule and the a-amidated peptide. In vertebrates, both enzymatic activities responsible for the carboxyterminal amidation reaction are contained in one bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM). By contrast, invertebrates generally express two separate enzymes encoded by two different genes. Here we report the identification and characterization of C. elegans amidating enzymes using bioinformatics to identify candidate genes and mass spectrometry to compare the neuropeptides in wild-type and newly generated mutants. Mutants lacking a functional PHM displayed an altered neuropeptide profile, showed impaired egg laying behavior and had a decreased brood size. Interestingly, PHM mutants still displayed fully processed amidated neuropeptides, probably as a result of the presence of a bifunctional PAM, the main amidating enzyme in vertebrates. Our data indicate the existence of a robust complementation system for the amidation reaction of neuropeptides in nematodes and suggest the involvement of amidated neuropeptides in egg laying. (1) S. J. Husson et al., J. Neurochem. 98, 1999 (2006); (2) J. Kass et al., J. Neurosci. 21, 9265 (2001); (3) S. J. Husson et al., J. Neurochem. 102, 246 (2007); (4) T. C. Jacob, J. M. Kaplan, J. Neurosci. 23, 2122 (2003).