Toshiaki Suzuki et al. (2003) International Worm Meeting "Analysis of Ce-PARKIN in C. elegans"

Nobutaka Hattori, Toshiaki Suzuki, Keiji Tanaka, Yoshikuni Mizuno, Shohei MItani, Noriyuki Matsuda, Hideki Yashiroda, Toshiaki Kitami, Keiko Gengyo-Ando

[International Worm Meeting, 2003]

Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial parkinsons disease characterized by selective loss of dopaminergic neurons in substantia nigra and the locus coeruleus. parkin is the causative gene of AR-JP. The human parkin gene encodes 465 amino acids with a ubiquitin-like domain in the amino-terminus and two RING finger motifs in the carboxy terminus. So far, various deletion mutations and point mutations have been discovered in patients of AR-JP, suggesting that the loss of function of Parkin is the cause of AR-JP. Recently we and others showed that Parkin has a ubiquitin-protein ligase activity and therefore suggested that the defect of protein degradation in the neurons of AR-JP patients (Shimura H. et al. Nat. Genet. 25, 302-5, 2000). To investigate the function of Parkin in vivo, we began to analyze the Ce-PARKIN of C. elegans. Two deletion mutations in parkin genes show no defect in their viabilities. The expression of Ce-PARKIN seems to be specific to subset of neurons. Therefore, we focused on the function of Ce-PARKIN in the neurons and the analysis is underway.

Kimura, Yoshishige et al. (2017) International Worm Meeting "Imaging mass spectrometry for C. elegans."

Kaneko, Tomomi, Kimura, Yoshishige, Aoyagi, Satoka

[International Worm Meeting, 2017]

Imaging mass spectrometry (IMS) is a two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules. The free-living soil nematode C. elegans is a common model organism, extensively used in life science research. Though, various investigations have been performed for metabolomic profiling of worms, the information of a single worm has been lost by? the conventional mass spectrometry (MS) techniques. Thus, the development of a label-free, non-targeted MS technique for molecular mapping in C. elegans has been required. We have previously performed MALDI imaging of C. elegans. However, the resolution was not enough to analyze cellular or subcellular level of biomoleculer distribution. Thus, we next tried the application of TOF-SIMS (Time-of-Flight Secondary Mass Spectrometry) system for C. elegans, which enables us to obtain subcellular distribution of metabolites. By comparison of several sample preparation methods, the frozen sections of C. elegans fixed by paraformaldehyde (PFA) were suitable for TOF-SIMS analysis. By sputtering of Ar gas cluster ion beam (Ar-GCIB), the sensitivity to fatty acids (e.g. stearic acid (SA), oleic acid (OA), and eicosapentaenoic acid (EPA)) was significantly enhanced, and high-resolution images of biomolecules were acquired. Further modification to prepare C. elegans samples for TOF-SIMS imaging is in progress. This is promising to obtain the cellular and subcellular distributions of the various biomolecules easily and efficiently.

Tod Thiele et al. (2002) West Coast Worm Meeting "The step response of spatial orientation behaviors in C. elegans."

Tod Thiele, Hatim Zariwala, Shawn R Lockery, Adam C Miller, Serge Faumont

[West Coast Worm Meeting, 2002]

C. elegans exhibits two forms of spatial orientation behavior: migration to the peak of a chemical gradient (chemotaxis) and migration to cultivation temperature in a thermal gradient (thermotaxis). Both behaviors involve successive comparisons of stimulus strength through time, a process analogous to computing the time derivative of the stimulus. To investigate the neuronal basis of this computation, we devised a means of delivering rapid, step-like changes in chemical concentration or temperature to freely moving worms.

Kwang-Zin Lee et al. (2003) International Worm Meeting "Operon structure in Pristionchus pacificus"

Kwang-Zin Lee, Ralf J Sommer

[International Worm Meeting, 2003]

Trans-splicing and gene organization in operons are two peculiar features of C. elegans. Whereas trans-splicing has been shown to be general features of nematodes (1), operons have only been observed in Caenorhabditis and Dolichorhabditis, a genus now called Oscheius(2). Here we describe evidence for the first operon in Pristionchus pacificus. P. pacificus has been established as a satellite organism for comparing developmental processes to Caenorhabditis elegans. These species share a last common ancestor some 100-200 mya years ago and P. pacificus is more distantly related to C. elegans than Oscheius. The genetic and the molecular analysis of developmental processes, such as vulva development, have recently been complemented by a genomic approach (3). We have been studying programmed cell death in P. pacificus and have isolated mutations in two complementation groups, namely ipa-1 and ipa-2. Molecular analysis indicated that ipa-1 corresponds to Ppa-ced-3 (4). To clone Ppa-ced-4, we wanted to make use of the fact that Cel-ced-4 is a member of the operon CEOP3224. In addition to ced-4, there are four other genes in this operon, with the aldose reductase (AR), C35D10.6 being the most 3' gene in the operon. The orthologue of C35D10.6 was cloned in P. pacificus and placed at the same genetic linkage map position as ipa-2. Further investigation of the aldose reductase region indicates that the other genes of the C. elegans operon CEOP3224 are not linked to Ppa-AR. However Ppa-AR is located 100 bp downstream of the putative ortholog of ZK1128.4, a gene that is located on the same chromosome in C. elegans. A third, more upstream gene, R144.2, in C. elegans is also located on Chromosome III. Ppa-ZK1128.4 is SL2 spliced, providing further evidence that this group of genes is organized in an operon. The SL2 sequence in P. pacificus differs from the one described in C. elegans and Oscheius. Further analysis is being undertaken to give a representative display of the operon organization in P. pacificus and on operon structure in general. 1.T. Blumenthal, Trends Genet 11, 132-6 (Apr, 1995). 2.D. Evans et al., Proc Natl Acad Sci U S A 94, 9751-6 (Sep 2, 1997). 3.J. Srinivasan et al., Genetics 162, 129-34 (Sep, 2002). 4.R. J. Sommer et al., Development 125, 3865-73. (1998).

Urquiza, S. et al. (2019) International Worm Meeting "Natural microbiome of Chilean C. elegans isolates and their relationship with neuroprotection."

Calixto, A., Urrutia, A., Urquiza, S.

[International Worm Meeting, 2019]

In nature Caenorhabditis elegans eats bacteria from rotting fruits and vegetation. The natural microbiome of C. elegans influences growth, lifespan and stress (Dirksen et al., 2016. Zhang et al,. 2017. Samuel et al., 2017). We are interested in understanding how the relationship between C. elegans and its intestinal bacteria influences behavior and phenotype. We showed that specific metabolites from laboratory bacteria affect the rate at which mechanosensory neurons (MN) degenerate in a genetic model of neuronal necrosis (Urrutia et al., under review). The bacterial enzyme involved in MN neuroprotection is the glutamate decarboxylase enzyme (GAD) and the protective metabolite, its product g-amino butyric acid (GABA). In this work we studied the impact of a natural microbiome in neurodegeneration of the MN. The natural microbiome we studied was isolated from wild C. elegans obtained from the Universidad Mayor Campus in Huechuraba, Chile. This microbiome was composed of three different culturable bacteria identified by means of ribosomal RNA 16S marker sequencing. Isolate 1 has 100% identify with Brucellaceae bacterium bfzh12, Pseudochrobactrum sp. AR-333 and Pseudochrobactrum kiredjianiae. Isolate 2 has 100% identity with Stenotrophomonas humi and Stenotrophomonas sp. Isolate 3 has 100% identity with Bacillus pumilus and Bacillus altitudinis. GAD enzymatic activity provided information about the bacteria neuroprotective potential because all isolates displayed higher levels than E. coli OP50 (10%, 15% and 40% respectively). Functional morphological categories of the MN were significantly higher in animals feeding natural isolates than on E. coli OP50, suggesting a correlation with GABA production. Ongoing and future work include dissecting the contribution in abundance of each bacterium to the final intestinal microbiome as well as the metabolite production of individual bacteria and in conjunction.

Paulini, Michael (2011) International Worm Meeting "A can of worms - challenges and strategies for next generation nematode genomics."

Paulini, Michael

[International Worm Meeting, 2011]

Next generation sequencing has provided many laboratories with the means to sequence whole nematode genomes. At WormBase we have created standards to streamline the integration and presentation of this data. This enables small nematode communities to use the comprehensive information available for Caenorhabditids in context of their research species. Using comparative genomics with/against highly curated nematodes, sparse and rough genomes can have interesting features annotated. I will outline the integration process using the examples of Trichinella spiralis and Strongyloides ratti and describe some possible use cases, such as creating list of potential anti-helminthic drug targets.

Penkov, Sider et al. (2013) International Worm Meeting "Nematoil - a novel secreted lipid that coats the outer surface of the dauer larva of Pristionchus pacificus."

Kurzchalia, Teymuras, Gruner, Margit, Knolker, Hans-Joachim, Penkov, Sider, Ogawa, Akira, Sommer, Ralf, Passler, Ulrike

[International Worm Meeting, 2013]

The dauer larva formed by multiple nematodes is a specialized developmentally arrested stage for survival and dispersal from unfavorable environment. The survival abilities of dauer larvae are determined by their specific morphology, metabolism, and enhanced stress resistance. A major morphological feature of dauer larvae is the remodeled body surface - they are effectively sealed off by a dauer-specific cuticle that restricts the chemical exchange with the environment. Although the dauer formation is genetically well investigated in several nematode models, there is no much information about the chemical means by which dauer larvae resist to the various kinds of environmental stress. We have found that the nematode Pristionchus pacificus synthesizes dauer stage specific lipids that form a hydrophobic film covering the entire outer surface of the animal. Detailed observation showed that the synthesis and the secretion of the lipids are simultaneously executed late in dauer differentiation, shortly preceded by the molt to dauer larva. The hydrophobic film is a complex mixture of several lipids and advanced chemical analysis revealed that its major component is a very long-chain polyunsaturated wax ester that we name Nematoil. The lipid coat alters the surface properties of the animals - they tend to congregate in tight "dauer clumps" consisting of up to hundreds of individuals, which supposedly enhances their impermeability. Thus, P. pacificus dauer larvae have the biochemical means to enhance their stress response by counteracting collectively.

Martyn Quinn et al. (2001) International C. elegans Meeting "The structural role and interactions of the DPY-7 cuticular collagen in the exoskeleton of Caenorhabditis elegans."

Martyn Quinn, Iain Johnstone

[International C. elegans Meeting, 2001]

The C.elegans cuticle (exoskeleton) is a multi-layered extracellular matrix consisting predominantly of small collagen-like proteins that are extensively cross-linked. The cuticle is synthesised by the underlying hypodermis 5 times throughout the lifecycle of the animal. Previous investigations have indicated that the cuticular collagen DPY-7 has an important structural role within the complex architecture of the exoskeleton. Using immuno-fluorescence we have shown that this collagen locates to circumferential stripes within the cuticle. There is one DPY-7 containing stripe per cuticular annulus. We are utilizing a range of techniques (including immuno-electron microscopy and scanning electron microscopy) to further elucidate the precise localisation of this protein within the cuticular matrix. Using a mono-clonal antibody specific for DPY-7, we can detect the protein on western blots from cuticle extracts. The DPY-7 detected is much larger than monomer. Cuticle collagens are crosslinked by non-reducible bonds during formation of the cuticle. The various high molecular weight species we detect will be cross-linked forms of DPY-7. By a combination of genetic and immuno-fluorescent means we have shown that DPY-7 physically interacts with at least three other cuticle collagens. We are interested to determine if the cross-linking of DPY-7 we detect by westerns is with the same collagen molecules we know by other means DPY-7 interacts with. We are attempting to purify the cross-linked species with the intention of identifying cross-linked partners.

JA Waddle et al. (1999) International C. elegans Meeting "Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis"

JA Waddle, RH Waterston

[International C. elegans Meeting, 1999]

A powerful feature of C. elegans research is the ability to determine mutant phenotypes at the resolution of single cells. The advent of green fluorescent protein (GFP) provides means to carry the single cell analysis one step further, that is, to characterize critical events that take place during a short interval within a cell cycle. Improvements in computers and imaging devices motivated us to develop a 3-dimensional image acquisition system capable of merging traditional embryonic cell lineage analysis with the latest GFP technology. To gauge the utility of our microscope setup, we imaged a transgenic line carrying a fusion between GFP and an isoform of histone H1 (kindly provided by Melanie Dunn and Geraldine Seydoux). The most important outcome of our efforts is the ability to non-destructively (e.g the embryos hatch and are fertile) collect a 3-dimensional time lapse fluorescence image sequence starting at first cleavage and spanning more than 8 hours of development. The histone H1::GFP images greatly facilitate the tracking of nuclear movements and cell divisions in the bottom half of later stage embryos. The application of computational methods, to reduce the effects of out of focus light, greatly improves image detail and the signal-to-noise ratio. Current efforts are focused on custom software capable of tracking the 3-dimensional distribution and behavior of the histone H1::GFP labeled nuclei, that is, automated embryonic cell lineage analysis. In summary, several technologies have coalesced to provide non-damaging means to track the 3-dimensional distribution of important molecules during embryogenesis. Such technology will complement existing methods for phenotypic analysis in an era when both the embryonic cell lineage and the genome descriptions are complete.

Lee, Bo Yun et al. (2011) International Worm Meeting "ABI-1 controls acetylcholine receptor clustering and rpy-1 expression level."

Nam, Seugnhee, Lee, Bo Yun, Lee, Junho

[International Worm Meeting, 2011]

Rapsyn has a role in acetylcholine receptor clustering at neuromuscular junction. In mammalian cells, rapsyn interacts with actin cytoskeleton through utrophin. Although there might be many components in acetylcholine receptor (AChR) clustering, most of them are unrevealed. In this research, we tried to find the partners of rapsyn for acetylcholine receptor clustering using C. elegans. rpy-1, rapsyn homologue of C. elegans, interacts with unc-29, an AChR b subunit homologue of C. elegans, and controls AChR clustering. We conducted yeast two hybrid screenings and immunoprecipitation experiments, and found that ABI-1 binds to RPY-1. Abi, abi-1 homologue of mammals, is a component of the SCAR/WAVE complexes which trigger actin polymerization at filopodia tips. Also, in C. elegans, SCAR/WAVE proteins are involved in apical F-actin enrichment. Furthermore, ABI-1 interacts with ARP2/3 complex to regulate actin cytoskeleton remodeling. To identify where abi-1 works in C. elegans, we checked the expression pattern of abi-1 using Pabi-1::GFP construct. abi-1 was expressed in muscles, neurons, hypodermis and many other cells. To verify that abi-1 is related to AChR clustering, we treated abi-1 mutant with 25 mM levamisole. While rpy-1 or unc-29 mutants were resistant to levamisole, abi-1 mutant showed severer hypercontraction than wild type, N2. Also, when wild type ABI-1 protein was expressed in abi-1 mutant, the phenotype was rescued. It means that abi-1 would be involved in AChR clustering. Next, we tested rpy-1 expression in abi-1 mutant. Unexpectedly, the level of rpy-1 expression was decreased in abi-1 mutants. It means that abi-1 is involved in the regulation of rpy-1 expression level. Our results suggest that abi-1 has a role in AChR clustering and in the control of rpy-1 expression.