William M Nuttley et al. (2001) International C. elegans Meeting "Characterization of the E. coli-induced suppression of olfactory habituation"

William M Nuttley, Karen Atkinson-Leadbeater, Derek van der Kooy

[International C. elegans Meeting, 2001]

Food is an important motivator for animals. In C. elegans , the presence of E. coli causes changes in locomotion, egg laying, pharyngeal pumping and thermotaxis. We have been investigating the effect that food has on olfactory behavior and found that the presence of E. coli suppresses both the approach to and the habituation to 100% benzaldehyde. Control experiments demonstrated that this suppression is not caused by an inability to sense odorants in the presence of E. coli . Both of these activities (the suppression of chemotaxis and the suppression of habituation) are inactivated by heating the E. coli to 95degC for 15 minutes. As with other food-induced behavioral modulations in C. elegans , these effects can be mimicked by the addition of exogenous serotonin and are dependent on the tph-1 and cat-4 gene products. We will present a further genetic characterization of this behavior. We are also exploring the behavioral characteristics of olfactory habituation. Associative learning involves the association between a conditioned stimulus (CS) and an unconditioned stimulus (US). With food acting as a US while benzaldehyde represents the CS, exposures to benzaldehyde in the absence of food during habituation training would result in the worms learning that benzaldehyde predicts the absence of food, and they no longer approach benzaldehyde on subsequent tests. The lack of habituation observed in the presence of E. coli therefore would occur because these conditions indicate that benzaldehyde is a good predictor of food. Experiments which increase the exposure to benzaldehyde while varying the exposure to food indicate that the degree of habituation is not determined simply by the time of exposure to benzaldehyde, as non-associative (single-stimulus learning) accounts of habituation would predict. Rather, the amount of habituation is dependent on both the duration of the benzaldehyde exposure and the amount of time that food is present during that exposure, revealing an associative component of benzaldehyde habituation. Supported by NSERC.

Roussell DL et al. (1994) Parasitol Today "Germ-line determination in Caenorhabditis and Ascaris: will a helicase begin to unravel the mystery?"

Bennett KL, Roussell DL, Gruidl ME

[Parasitol Today, 1994]

How cell lineages are established during development in higher eukaryotes is being addressed by geneticists and by developmental and molecular biologists. In Drosophila melanogaster, a gene corresponding to a germ-line-specific RNA helicase, vasa, has been shown to be a component o f the posteriorly localized germ granules o f the developing embryo. A putative RNA helicase, glh-I r which appears germ-line specific in its expression, has recently been reported from the free-living nematode Caenorhabditis elegans. Parasitologists studying the nematode Ascaris lumbricoides var. suum have found it to be a useful complement to Caenorhabditis. Deborah Roussell, Michael Gruidl and Karen Bennett predict that Ascaris will be valuable in determining the role played by germ-line helicases in development.

Masler EP et al. (1998) East Coast Worm Meeting "FMRFamide-Like Immunoreactivity in Heterodera glycines: Changes During Development and Comparison with Caenorhabditis elegans and Panagrellus redivivus."

Sardanelli S, Kovaleva ES, Masler EP

[East Coast Worm Meeting, 1998]

A peptide family with a characteristic arginine-phenylalanine-amide C-terminal motif ("FMRFamide") and myoactive properties has been described for both free-living and animal parasitic nematodes. Primary structures reported to date probably represent only a fraction of those present in these animals, and their neuromodulatory nature suggests a potentially extensive involvement of this peptide family in the behavior, development and reproduction of nematodes. Immunocytochemical studies (Atkinson et al. 1988) detected the presence of FMRFamide-like peptides in the nervous system of the plant parasitic nematode Heterodera glycines, but no analyses of these peptides from plant parasitic nematodes have been available. We have developed methods for the collection and extraction of H. glycines and the detection of FMRFamide-like peptides using a competitive ELISA. HPLC-ELISA procedures reveal species-specific differences in the quality and quantity of FMRFamide-like peptides from H. glycines and the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. In addition, changes in FMRFamide-like peptide immunoreactivity were observed during H. glycines development and maturation. The significance of these developmental changes and species differences is discussed, and arguments for the involvement of FMRFamides in parasitic nematode growth and development are presented.

Bird AF (1980) "The nematode cuticle and its surface."

Bird AF

[1980]

A number of review articles on the nematode cuticle have been published in the last decade. The most recent of these are those of Bird and Lee and Atkinson. These authors, while emphasizing the complexity and variability of nematode cuticles, support the use of a simplified nomenclature of cuticle structure which divides the cuticle into three regions or zones-namely, cortical, median, and basal. It is obvious that many exceptions to this fundamental pattern occur, and I shall mention some of these below. However, I think that they are adaptations to survival in changing environments, particularly where parasitism is involved. In particular, I propose to consider the structure and functions of the surface or epicuticle of the cortical zone, for it is here that reactions similar to those occurring at cell surfaces and in cell membranes are thought to occur in a wide range of "helminth" organisms. At the moment, particularly for the Nematoda, these ideas require more experimental evidence to establish them as facts. However, the use of sensitive techniques currently employed by membrane physicists and chemists to isolate, label, analyze, measure, and observe interactions taking place in cell membranes have in many instances yet to be used on the nematode cuticle. There is no doubt that the free-living bacterial-feeding nematodes such as those belonging to the genus Caenorhabditis, and in particular C. elegans, are the experimental models of choice for this purpose.

Prasad SS et al. (1985) Worm Breeder's Gazette "identification and isolation of the coding regions around unc-22."

Baillie DL, Prasad SS

[Worm Breeder's Gazette, 1985]

Our laboratory has been intensively studying the region around unc- 22 gene over the last eight years using conventional genetic analysis. We have obtained cosmids from John Sulston that cover approximately 500 kb. of DNA. These cosmids were identified by means of Tcl RFLD flanking sequences (isolated by Karen Beckenbach) in the region which lies immediately to the left and right of unc-22 gene. We are currently examining the region flanked by dpy-20 on the left and unc-22 on the right. Five overlapping cosmids have been identified which cover this region, (these include D. Moerman's and R. Waterston's unc-22 clones). We are using the method suggested by T. Snutch to identify putative coding regions (Divergent regions between C. elegans and C. briggsae include mostly non-coding DNA sequences whereas the highly conserved regions are likely to code for the proteins essential for nematode growth, development and reproduction). We have restriction mapped and subcloned Hind III and Pst I fragments from these cosmids into pUC19. Nick translated individual fragments have been used to probe Bristol and C. briggsae DNA. This technique helps us to identify the conserved regions or the coding sequences in C. briggsae DNA. We are now in the process of screening B. Meyers's lambda gt10 cDNA library with the probes which show homology to the C. briggsae DNA. We intend to identify and isolate the major coding regions that lie between unc-22 and dpy-20 regions using this approach. The isolation of cDNA clones will be used to study gene expression and regulation during nematode development. The isolated cDNA clones will be sequenced in order to identify the nature of protein coded for by particular gene(s) in this region.

Yook K et al. (1995) Worm Breeder's Gazette "A Screen For Lethal Synaptic Function Mutations."

Yook K, Jorgensen EM

[Worm Breeder's Gazette, 1995]

A screen for lethal synaptic function mutations Karen Yook and Erik Jorgensen Universitv of Utah, Dept. of Biology. UT 84112 Our goal is to identify proteins that function at all synapses. These include proteins that are involved in svnthesis, packaging, transport and the release of neurotransmitters into the synaptic cleft, as well as proteins involved in the recycling of synaptic vesicles. Initially we screened for mutants that behaved as if they were defective for both GABA and acetylcholine function: 11 genes were defined by this approach. One drawback with such a screen is that it will fail to identify lethal mutations. We have developed a screen which can potentially identify lethal mutations that interfere with neurotransmission. Aldicarb is a pesticide that inhibits the breakdown of acetylcholine in the synaptic cleft. The resulting accumulation of acetylcholine in wild-type worms results in paralysis. whereas worms lacking or having reduced acetylcholine transmission are resistant to the drug and continue to move. It was noted that many of the genes uncovered in the behavioral screen conferred resistance to aldicarb as double heterozygotes with n2813. an allele of unc-13 (for example unc-13/+: snt-l/+). Apparently neurosecretion is very sensitive to the dosage of proteins required for this process. This sensitivity allowed us to screen for haploinsufficient mutations in a background sensitized by the unc-13 mutation. We mutagenized wild-type males and crossed them to a strain containing unc-13 (n2813) (see Figure below). In the Fl we picked aldicarb-resistant individuals. Lethal mutations in genes that are essential to synaptic function are maintained as aldicarb resistant heterozygotes. We are interested in two classes of mutations: The first class are lethal mutations, identified as strains that are resistant to aldicarb when unc-13 is heterozygous, and also throw lethal progeny. The second class are homozygous viable aldicarb-resistant mutations that can be separated from the unc-13 mutation. Of 2336 genomes scored. we uncovered 62 possible lethal mutations and 61 homozygous viable aldicarb-resistant mutations. In the homozygous viable class we found both uncoordinated and wild-type mutants. All of these strains have satisfied preliminary criteria for synaptic function mutants and we are confident that our screen will be successfill in uncovering genes of interest. One mutation pulled out of the modified screen has been shown by noncomplementation to be an allele of unc-26, mutations of which are known to be resistant to Aldicarb (J. Rand, personal comm.).

Kreutzer MA et al. (1994) Worm Breeder's Gazette "Expression Studies of the Putative Caenorhabditis elegans cyclin A and B Genes"

Richards JP, Bennett KL, Kreutzer MA

[Worm Breeder's Gazette, 1994]

Expression studies of the putative Caenorhabditis elegans cyclin A and B genes Monique A. Kreutzer, James P. Richards and Karen L. Bennett, University of Missouri-Columbia, Columbia, Missouri 65212. We have cloned putative A, B1 and B2 cyclins from C. elegans. Cyclin B1 and B2 are single copy genes while cyclin A belongs to a multigene family. Cyclin A and B2 each correspond to single transcripts while cyclin B1 recognizes three transcripts. The C. elegans temperature sensitive germline defective mutants fem-1, fem-3 gf) and glp4 were used to analyze cyclin germline expression. When hybridizing C. elegans cyclin A or B2 to the mutant RNAs that produce no oocytes, the cyclin A and B2 messages are significantly reduced. When normal numbers of oocytes are produced, the cyclin A and B2 messages are at wild type levels. Therefore, the cyclin A and B2 transcripts appear to be primarily maternal with some somatic component. Consistent with a somatic component, cyclin A and B2 cDNAs cross-hybridize to Ascans intestinal poly A+ RNA. Hybridization of the cyclin B1 cDNA to these germline defective RNAs suggest that the cyclin B1 transcripts are differentially expressed in the germline. The two larger cyclin B1 transcripts appear to be primarily maternal and the smallest transcript appears to be sperm- specific. The observation that two polyadenylation signals are present in the cyclin B1 3'UTR, as well as results using cyclin B1 probes from specific regions of the B1 3'UTR, suggests that two of the three cyclin B1 transcripts are produced by polyadenylation a~ different sites. There is precedence in mice and flies for producing tissue-specific transcripts by different use of the cyclin B 3'UTRs. It has been shown in mouse that cyclin B1 hybridizes to four differentially expressed transcripts, one of which is testis- specific. Two of these mouse transcripts differ in the choice of polyadenylation sites and therefore in the lengths of their 3'UTRs (Chapman and Wolgemuth (1992) MoL Repro.Dev. 33, 259-269). In Drosophila, the cyclin B cDNA detects two female-specific transcripts that are made by splicing of a region of the cyclin B 3'UTR (Dalby and Glover (1992) Development 115, 989-997). In summary, our initial findings show that the C. elegans cyclin As and Bs are highly expressed in the germline and also suggest that the three cyclin B1 transcripts are differentially expressed in oocytes and sperm.

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Kersey, Paul, Stein, Lincoln, Harris, Todd, Sternberg, Paul, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.

Anthony J Otsuka et al. (2005) International Worm Meeting "Is UNC-44 Involved in Microtubule Capture in Growth Cones?"

Ping Gong, Anthony J Otsuka

[International Worm Meeting, 2005]

Ankyrins are mediators of cytoskeletal interactions. They bind directly to integral membrane proteins and indirectly to the actin cytoskeleton through spectrin and associated proteins. The neural-specific UNC-44 AO13 ankyrin is required for proper axon guidance and outgrowth (Otsuka et al., J. Cell. Biol., 129:1081-1092, 1995; Boontrakulpoontawee and Otsuka, Molec. Genet. Genom., 267:291-302, 2002; Otsuka et al., J. Neurobiol., 50:333-349, 2002). Using a yeast two-hybrid approach, we found interactions between the carboxyl terminal domain (CTD, residues 6510-6800) of AO13 ankyrin and the microtubule-binding protein, CLASP2, encoded by the cls-2 gene, one of three CLASP genes in C. elegans. The interaction was shown to be specific in two-hybrid tests by the lack of reporter gene expression with 1) CLASP:DNA-binding domain clone alone, 2) in combination with the activation domain vector plasmid, and 3) in combination with an unrelated protein in the activation domain plasmid. Consistent with the pattern of in situ RNA hybridization in Kohara's NEXTDB database, affinity purified CLASP2 antibodies (the kind gift of Arshad Desai and Karen Oegema) label the cytoplasm of many cells. In some cases, the cell periphery is labeled. CLASP2 is associated with kinetochores in dividing cells (Desai et al., Genes Develop., 17:2421-2435, 2003). In mammalian fibroblasts, CLASP2 binds to the rapidly growing (plus) ends of microtubules (MTs) at the leading edge of cells in the wound-healing assay (Ahkmanova et al., Cell, 104:923-935, 2001). CLASP binding to microtubules is inhibited by phosphorylation and is under the control of the PI3-kinase and GSK3beta pathways. Further, it has been shown recently that the Drosophila CLASP/Orbit protein is involved in the Slit/Robo repulsive pathway and is downstream of the Abl kinase (Lee et al., Neuron, 42:913-926, 2004). Since Abl also controls actin dynamics, the fly studies link actin filament and MT regulation. In worms, UNC-33 requires UNC-44 for its axonal localization. Recently, it was shown that the UNC-33-related protein, CRMP-2, that has been proposed to be involved in the initiation of cell outgrowth, is phosphorylated by GSK3beta (Yoshimura et al., Cell, 120:137-149, 2005). Similar to CLASP2, phosphorylation of CRMP-2 lowers its ability to interact with MTs, and dephosphorylated CRMP-2 localizes to growth cones. The interaction between UNC-44 and CLASP2, along with the studies of CLASPs in other systems, leads to the hypothesis that one role of UNC-44 may be to act as an adapter protein that captures CLASP-tipped pioneer MTs that are exploring the limits of growth cone filopodia. Clearly, testing this hypothesis will require many additional experiments.

Jean-Claude Labbe et al. (2001) International C. elegans Meeting "Assessing the role of microtubules in the establishment of the first asymmetric cell division of the C. elegans embryo"

Paul S Maddox, Bob Goldstein, Jean-Claude Labbe, E D Salmon

[International C. elegans Meeting, 2001]

We are using the C. elegans 1-cell embryo as a model to understand how cells divide asymmetrically. The first embryonic division of C. elegans gives rise to a larger, anterior cell, termed AB, and a smaller, posterior cell, P 1 . This asymmetry is the result of the position of the first mitotic spindle, which moves from the center to the posterior of the 1-cell embryo during metaphase. Several mutations, including alleles of the par genes, disrupt embryonic polarity and affect the posterior displacement of the spindle, suggesting that components localized at the cell cortex can influence spindle positioning (1). Recent experiments by Grill et al . (2) demonstrated that severing the spindle at the start of anaphase B results in both the anterior and posterior centrosomes migrating toward their respective poles at a faster rate than normal, with the posterior centrosome moving faster than the anterior one. In order to address the role of astral microtubules in spindle positioning, we ablated either the anterior or the posterior centrosome at various phases of the cell cycle. During anaphase B, ablation of the anterior centrosome caused posterior movement of the posterior centrosome and ablation of the posterior centrosome caused anterior movement of the anterior one; these results are similar to those obtained by Grill et al . However, during prophase (before pronuclear envelope breakdown), ablation of the anterior centrosome resulted in posterior displacement of the posterior centrosome, while ablating the posterior centrosome at this time did not cause anterior movement of the anterior centrosome. The anterior centrosome did not move until later, posibly at anaphase onset. This result suggests that a pulling force acts on the posterior aster before anaphase. One posible mechanism for this could be that components of the posterior cortex might modulate the dynamics of microtubules in this region of the embryo, which could asymmetrically modulate pulling forces. PAR-1, a protein with similarity to MARK kinases that localizes to the posterior cortex of the embryo (3), is a good candidate to have a local effect on posterior microtubules. MARK kinases have been implicated in the phosphorylation of microtubule-associated proteins (MAPs) and in the destabilization of microtubules in vivo and in vitro (4). We monitored dynamics of individual microtubules by imaging embryos expressing an alpha-tubulin:gfp fusion (kindly provided by Karen Oogema) with a spinning-disk confocal microscope. Thus far, preliminary quantitative data analysis suggests a similar stability between anterior and posterior astral micrutubules during the time that the spindle becomes asymmetric in wild-type embryos. We will further address this issue by monitoring microtubule dynamics in par mutants. Furthermore, we are developing an in vitro assay to test whether PAR-1 can directly affect microtubules to modulate their stability. To this end, we have isolated C. elegans MAPs and will determine if they can play a role in this process. Progress toward these goals will be presented. 1. Guo, S. and Kemphues, K.J. (1996). Curr. Opin. Genet. Dev. 6, 408-415 2. Grill, S.W. et al. (2001). Nature 409, 630-633 3. Guo, S. and Kemphues, K.J. (1995). Cell 81, 611-620 4. Drewes, G. et al. (1997) Cell 89, 297-308