Aamna Kaul et al. (2003) International Worm Meeting "Hyperactivation of glutamate-gated chloride channels with ivermectin results in necrosis."

Joseph Dent, Aamna Kaul

[International Worm Meeting, 2003]

Ivermectin is a widely used antiparasitic drug. It kills worms by activating glutamate-gated chloride channels (GluCls), which belong to the family of ligand-gated anion channels that includes the GABA and glutamate receptors (Cully et al., 1994; Dent et al., 2000). The chloride permeability that ivermectin induces in excitable cells tends to prevent excitation. For example, ivermectin targets a GluCl expressed in the pharyngeal muscle to inhibit muscle contraction and prevent eating (Dent et al., 1997). The worms linger for several days in the presence of ivermectin before they starve to death. However, we have found that the lethal effects of ivermectin on C. elegans become irreversible after only a few hours of exposure. When L1 worms were exposed to 20ng/ml for 5 hours and then washed, they gradually developed large vacuoles in their pharyngeal muscle over the next several days. A mutant strain that lacks ivermectin receptors shows little or no necrosis when treated. Ivermectin is hydrophobic and it irreversibly opens GluCls expressed in Xenopus oocytes. So it is possible that ivermectin persists in membranes and continues to activate GluCls. Furthermore, it has been shown that hyperactive cation channels can induce excitotoxic necrosis (Driscoll and Chalfie, 1991). Why, though, would an inhibitory channel have a similar effect when hyperactivated? We are trying to address this question by looking at whether mutations known to inhibit excitotoxicity also inhibit the necrotic effects of ivermectin. Cully DF, Vassilatis DK, Liu KK, Paress PS, Van der Ploeg LHT, Schaeffer JM, Arena JP. Nature 371: 707-711 1994 Dent JA, Smith MM, Vassilatis DK, Avery L. PNAS USA 97: 2674-2679 2000 Dent JA, Davis MW, Avery L. EMBO Journal 16: 5867-5879 1997 Driscoll, M and Chalfie, M. Nature 349: 588-593 1991

Carta L (1998) East Coast Worm Meeting "TOXICITY OF 4 SERRATIA MARCESCENS BACTERIAL STRAINS TOWARD C. ELGANS N2, PGP-3 AND OTHER NEMATODES IS INVERSELY RELATED TO PIGMENT"

Carta L

[East Coast Worm Meeting, 1998]

We are testing a number of biocontrol bacteria (eg. Burkholderia, Pseudomonas and Stenotrophomonas) for toxicity to bacterial feeding nematodes including C. elegans N2 and pgp-3. Serratia marcescens in particular had a measurable effect on number of eggs over 24 hours and relative motility in microtiter wells. Serratia marcescens strains with increasing amounts of red pigmentation (BF1-5@ (albino), D1, N4-5, NIMA) were tested for relative motility to C. elegans N2, pgp3, Oscheius myriophila DF5020, Panagrellus redivivus PS 1163, Mesorhabditis sp. PS1170 and Zeldia punctata PS 1153. Toxicity directly increases with decreasing amount of pigment in Caenorhabditis, Oscheius and Panagrellus. The order of relative toxicity is reversed for the 2 highly pigmented strains and the 2 least pigmented strains (2143 vs. 1234) in Mesorhabditis and Zeldia. These nematodes are phylogenetically distant from C. elegans. Bacterial strains were provided by Phyllis Martin and Dan Roberts, USDA.

Zohar Bromberg et al. (2003) International Worm Meeting "Heat acclimation: Do reactive oxygen species play a role?"

Millet Treinin, Zohar Bromberg, Michal Horowitz, Judith Shlaier

[International Worm Meeting, 2003]

Persistent exposure to environmental heat stress upregulates protective mechanisms, leading to enhanced endurance during exposure to severe heat stress. This process is defined as 'heat acclimation". Unfortunately, our knowledge of the "architecture" of acclimatory signaling in mammals is limited. C. elegans, like mammals, acclimates to heat. Thus, it was possible to use this genetically tractable organism to investigate the mechanisms of heat acclimation. Based on this analysis we have previously shown that hif-1 knockout worm hif-1(ia4), can not heat acclimate despite the presence of elevated levels of HSP 70 which is usually linked with enhanced thermotolerance. To further characterize candidate cues for heat acclimation, mutants known for enhanced thermotolerance, those affecting sensory transduction and mitochondrial function (as likely candidates for regulators of hif-1 elevation) were studied for their ability to acclimate. Heat acclimation was attained by exposure of age-synchronized groups to 25C for 18 hrs whereas control worms were grown at 20C. Heat endurance during subjection to heat stress at 35oC (displayed as survivorship curves) was served as a marker for heat acclimation. In this presentation we report results obtained for 1.the stress resistent (daf-2(e1370)], 2) isp-1 which shows inability to produce mitochondrial mediated reactive oxygen species (ROS) and 3) isp-1(qm150)daf-2(e1370). Heat-acclimated wild-type C. elegans showed significantly increased heat endurance during exposure to heat stress at 35C. daf-2(e1370) mutant had markedly enhanced thermotolerance at both normothermic and 35C ambient temperatures and environmental stress of 37C was required to show acclimation effect. Acclimated worms of this mutant had elevated HIF-1 level (145% 25, Western blot, ab contributed by PJ. Ratckliffe). isp-1 mutant showed only weak ability to acclimate compared to the wild type. In contrast, the double mutant isp-1(qm150)daf-2(e1370), showed enhanced ability to acclimate. Given that complex III originated mitochondrial ROS production may trigger a signal transduction pathway leading to HIF-1 stabilization (Chandel et al.,2000), we may suggest that isp-1 mutation which results in low O2 consumption and low ROS production may attenuate HIF production and in turn heat acclimation. Chandel, NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, and Schumacker PT. Reactive oxygen species generated at mitochondrial complex III stabilize HIF-1- during hypoxia: a mechanism of O2 sensing. J Biol Chem 275: 25130-25138, 2000

Jiang H et al. (2000) Midwest Worm Meeting "AHA! bHLH-PAS proteins may mediate response to hypoxia in C. elegans"

Jiang H, Guo R, Powell-Coffman JA

[Midwest Worm Meeting, 2000]

O2 homeostasis is essential for the survival of all organisms. From bacteria to mammals, cellular O2 levels are carefully regulated. Low O2 (hypoxia) will endanger ATP generation by shutting down the oxidative phosphorylation. In mammals hypoxia-inducible factor (HIF-1) plays a central role in regulation of responses to low O2 levels. It consists of two bHLH-PAS transcriptional subunits, HIF-1a and HIF-1b. HIF-1b is better known as aryl hydrocarbon receptor nuclear translocator (ARNT). Under hypoxic conditions, HIF-1a protein is stabilized and dimerizes with ARNT to form an active transcription heterodimer (HIF-1), which in turn activates the transcription of downstream targets. The target gene products either promote O2 delivery or switch metabolism to low O2 consumption. Multiple mechanisms of O2 sensing have been proposed, but none have been vigorously tested in vivo (1). Here, we report that bHLH-PAS genes may mediate response to hypoxia in C. elegans. The C. elegans genome encodes 5 putative transcription factors that contain basic-helix-loop-helix and PAS domain motifs. Two of these genes, ahr-1 and aha-1, encode the orthologs of the aryl hydrocarbon receptor and ARNT, respectively (2). Genetic analysis of these two genes has demonstrated that aha-1 is essential for viability, while ahr-1 is not (3,4). This indicates that AHA-1 may dimerize with other bHLH-PAS proteins to regulate developmentally important processes. Four lines of evidence suggest that AHA-1 and F38A6.3a may form hypoxia-responsive complex in C. elegans. First, AHA-1 and the F38A6.3a gene product can be co-immunoprecipitated when expressed in vitro. Second, F38A6.3a mRNA levels increase in hypoxic conditions. Third, we have identified potential target genes that are induced by hypoxia in N2, but not in animals homozygous for a deletion in F38A6.3a. Fourth, AHA-1 is expressed in many tissues (3, 4), and F38A6.3a:GFP is ubiquitously expressed. Further characterization of the mutant phenotype will be presented at the meeting. References: 1. Semenza GL. Perspectives on Oxygen Sensing. Cell (1999) 98(3): 281-4. 2. Powell-Coffman JA, Bradfield CA, Wood WB. Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner with the aryl hydrocarbon receptor nuclear translocator. PNAS (1998), 95: 2844-2849. 3. Jiang H, Guo R, Powell-Coffman JA. An essential role for aha-1, a PAS-domain-containing regulatory gene in C. elegans. 1999 International Worm Meeting, 438. 4. See other Powell-Coffman lab abstracts for this meeting.

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

Wang X et al. (1998) East Coast Worm Meeting "A MOLECULE CONTAINING A HEDGEHOG-LIKE AUTOPROCESSING DOMAIN IS REQUIRED FOR MOLTING IN C. ELEGANS."

Beachy P, Seydoux GC, Wang X

[East Coast Worm Meeting, 1998]

The Hedgehog family of signaling molecules controls many aspects of animal development, from embryonic patterning to limb formation. Recently, these proteins have been shown to rely on a novel mechanism to regulate their spatial distribution (1,2). Hedgehog proteins undergo an autoprocessing reaction to yield two distinct products, an amino-terminal fragment (Hh-N), and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms may be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a set of eight C. elegans proteins with sequence similarity to the Hh-C domain of Hedgehog (2,3). In these proteins, as in the Hedgehog family, the Hh-C-like domain is located in the carboxy-teminal end, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). This structure is consistent with the possibility that these proteins are secreted and undergo processing in a manner similar to that described for Hedgehog. To test this possibility we have fused the Hh-C-like domain of one of these proteins to a His tag, and purified the fusion protein from E. coli. We find that such a fusion can undergo the processing reaction in vitro, suggesting that the Hh-C-like domain is functionally active in the nematode proteins. We have begun to characterize the functions of Hh-C-related proteins using RNA-mediated interference. Our initial results suggest that one of these proteins, T05C12, is essential for molting. Injection of T05C12 double-stranded RNA results in 90% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. In many animals, a cuticular plug can be seen obstructing the mouth opening where it presumably interferes with feeding. The amino-terminal domain of T05C12 contains several sequence motifs similar to those found in collagens and other extracellular matrix proteins, raising the possibility that the T05C12 product is a cuticle component. Consistent with this possibility, a T05C12:GFP fusion is expressed in hypodermal cells during larval and adult stages. We are currently determining the effects of expressing mutant forms of T05C12 to test the function of its Hh-C-like domain. 1. Porter, JA. et al. (1996). Cell 86, 21-34. 2. Porter, JA. et al. (1996). Science 274, 255-259. 3. Burglin, TR. (1996). Current Biology 6, 1047-1050.

Adam W Tearle et al. (2004) West Coast Worm Meeting "LEV-9 encodes a putative protein in the sarcoglycan complex"

Adam W Tearle, William R Schafer, Suchitra Jagannathan

[West Coast Worm Meeting, 2004]

The levamisole activated acetylcholine receptor in C.elegans is now known to constitute four major subunits- LEV-1, UNC-29, UNC-38 and UNC-63. Mutants for these genes are strongly resistant to the anthelminthic, levamisole. However, regulators of receptor subunit function have not all been fully characterised. Mutations in lev-9 have been shown to confer partial resistance to both levamisole and nicotine, but do not affect levamisole binding in vitro (Lewis et al ., 1987). Thus, it has been suggested that lev-9 is a possible modulator of levamisole receptor function. We have cloned lev-9 and shown that it encodes a molecule homologous to the transmembrane glycoprotein, alpha sarcoglycan. In humans, the sarcoglycans are a major component of the dystrophin glycoprotein complex (DGC) and are associated with autosomal recessive forms of limb girdle muscular dystrophy. LEV-9 is mainly expressed in the muscles of the head, pharynx, body and vulva. Using fluorescently labeled a -bungarotoxin, we have shown that lev-9 exhibits decreased surface receptor expression compared to wild type. Preliminary results using in vivo calcium imaging from vulval muscles in dissected worm preparations also suggest a difference between lev-9 and wild type in the rate of calcium influx, in response to nicotine. Further experiments using fluorescent labeling to determine how LEV-9 affects the levamisole receptor surface expression and electron microscopic comparison of lev-9 and wild types are in progress. P> P> Lewis JA, et al . (1987) J. Neurosci . 7(10), 3059-71

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Martin Gutternigg, Matthias Hackl, Ute Stemmer, Iain B. H. Wilson, Petra Geier, Gunter Lochnit, Ramona Ranftl, Katharina Paschinger, Verena Jantsch, Dorothea Lubich

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.

Anthony S Rogers (2005) International Worm Meeting "Full Length Transcripts in WormBase"

Anthony S Rogers

[International Worm Meeting, 2005]

UnTranslated Regions (UTRs) of messenger RNAs have been shown to be important in the regulation of several C.elegans genes such as tra-2(1) and fem-3(2). To give an improved representation of UTR regions in WormBase predicted full length transcripts are now generated each release. These are built up automatically from the available transcript data and the current CDS structure. Non-genomic transcript sequence such as Trans-spliced leaders and polyA tails are masked before aligning the transcript to the genome using BLAT(3). Identifying TSL and polyA sequences prior to genome alignment helps to prevent misalignments and means individual features can be made to represent these sequences. UTR structures are built from the subset of transcripts which have been computationally shown to agree with the CDS prediction. This new method of representing full length transcripts allows us to build objects that more closely resemble their biological reality with trans-spliced leader sequence and polyadenylation information included where applicable. It is also possible to show where there is evidence of multiple UTRs for a single CDS.As of WS140 there are 25869 transcripts derived from 22410 CDSs representing the 19735 protein coding genes. 1) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C.elegans Goodwin EB, Okkema PG, Evans TC and Kimble J. Cell, 1993 75:329-339 2) Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region. Ahringer JA, Kimble JE Nature, 1991 349:346-348 3) BLAT The BLAST-Like Alignment Tool W. James Kent. Genome Research (2002) Vol. 12, Issue 4, 656-664

Vallin E. et al. (2006) European Worm Meeting "Molecular Characterization of MOS Transposon-Tag Strains Produced by the NemaGENETAG Project"

Vallin E., Granger L., Segalat L., Martin E.

[European Worm Meeting, 2006]

Vallin E., Granger L., Martin E. & Segalat L.. A collection of Caenorhabditis elegans insertion mutants is being created as part of the NemaGENETAG project. The aim is to produce characterized transposon-tagged mutants for the European and international scientific community. We use the Drosophila mariner element Mos-1, which has been shown to be active in C.elegans. The J. Ewbank laboratory (Marseille) generates and provides us with Mos-positive strains, which we molecular characterize. The localization of the insertion is determined by inverse PCR and sequencing. The sequence is compared with the C. elegans genome sequence to determine the insertion sites using the blast program. At the beginning of 2006, we have characterized approximately 2000 strains. Strains are currently analyzed sequentially with enzymes Hae III and Mbo I. Approximately 70% of the strains received from Marseille give a workable PCR band in these conditions. The percentage of PCR bands that give an unambiguous genome localization is approximately 66%. These strains are frozen in triplicate in both -80C freezer and liquid nitrogen. In case of request or for quality control, strains are thawed and PCR-tested with a Mos primer and an insertion-specific primer to check the presence on the insertion. On 30 strains tested in recovery tests, 28 were positive. These results prove that the insertions are stable and can be easily recovered from frozen samples. The aim of the project is to generate and provide strains for the worm community. The list of the insertions can be found on a database available on the web.