Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

Alyson Sujkowski et al. (2008) C.elegans Neuronal Development Meeting "Endocytic Trafficking of GABA Receptor in C. elegans"

Bruce Bamber, Brianne Sturt, Andrew Friedmann, Kathleen Davis, Alyson Sujkowski

[C.elegans Neuronal Development Meeting, 2008]

GABA is an important inhibitory neurotransmitter in vertebrates and invertebrates. In C. elegans, GABA neurotransmission at the inhibitory neuromuscular junction is required for coordinated locomotion. We are studying this synapse to better understand how GABA receptor abundance and GABA synapse strength are regulated. This synapse is a useful model because it is simple, containing only a single GABA receptor (encoded by the unc-49 gene). It also displays homeostatic plasticity: prolonged exposure to the GABA agonist muscimol elicits a ten-fold reduction in GABA receptor abundance over ten hours. In cup-5 mutants, which have reduced lysosome function, GABA receptors are still downregulated at the synapse, but they accumulate in the muscle cell bodies during muscimol exposure. This result suggests that downregulation involves receptor trafficking to the lysosome, and subsequent lysosomal degradation, which is blocked in the cup-5 background. Our objective is to better characterize the signaling and trafficking mechanisms that underlie homeostatic GABA receptor regulation. Our approach is to inactivate endocytic proteins by mutation or RNA interference, and observe the effects on GABA receptor downregulation and degradative trafficking. In conjunction, we have generated transgenic worms expressing various GFP-tagged endosome proteins to better characterize the trafficking pathway. First, we found that the intracellular GABA receptor immunofluorescence in cup-5 mutants exposed to muscimol co-localized with RAB-7-GFP, a late endosome protein, confirming the involvement of endocytic trafficking in agonist-dependent downregulation. Next, we used RNAi to knock down the expression of RAB-7, and RAB-5, which is required for the fusion of endocytic vesicles with early sorting endosomes. In these worms, GABA receptor staining was grossly normal before muscimol exposure. After muscimol exposure, GABA receptors were downregulated at the synapse normally, but accumulated in unique intracellular patterns at later time points. Knocking down RAB-11, required for receptor recycling, had little effect before or after muscimol treatment. These results are consistent with the interpretation that GABA receptors are normally stable at the C. elegans neuromuscular junction, but undergo increased endocytosis when chronically activated. They then traffic through early endosomes, then late endosomes, and finally to the lysosome where they are degraded.

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Soltesz, Z., de Bono, M., Bretscher, A. J.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.

Gonzalez-Serricchio A et al. (1996) West Coast Worm Meeting "GREEN FLUORESCENT PROTEIN VISUALIZATION OF EXTRACHROMOSOMAL ARRAYS"

Gonzalez-Serricchio A, Sternberg PW

[West Coast Worm Meeting, 1996]

We are developing a technique using Green Fluorescent Protein (GFP) to visualize extrachromosomal arrays in C.elegans. Our longer term goal is to use fine scale mosaic analysis to test whether the site of action of lin-15A and lin-15B overlap. We have constructed transgenes that express GFP-LacI fusion proteins and that contain multiple copies of the binding site for the LacI protein (LacO ). We typically see a single spot of bright fluorescence in nuclei, presumably the nuclear localized GFP-LacI protein. Cells with additional bright spots or with only the diffuse nuclear fluorescence are seen. We are now directly determining lineages of these transgenes to address the basic questions concerning segregation and stability of transgenes that will determine the utility of this method for mosaic analysis. Andrew Belmont, who developed this technique for mammalian cells, kindly provided us with the DNAs and technical advice.

Dernburg AF et al. (1997) International C. elegans Meeting "Cytological investigation of homologous pairing during meiotic prophase"

Dernburg AF, Villeneuve AM

[International C. elegans Meeting, 1997]

Pairing and recombination between homologous chromosomes are essential for their faithful segregation during the first meiotic division. The mechanisms leading to homologous chromosome pairing and synapsis are still only poorly understood in any experimental organism. C. elegans provides a highly advantageous model system in which genetic and cytological approaches can be combined to study this process. We are developing tools and methods to enable us to examine chromosome pairing in the light microscope in order to address several specific questions: Do special sites on each chromosome (particularly the genetically-defined !pairing centers!) become associated earlier than others? Do chromosome rearrangements that act as crossover suppressors disrupt homologous pairing as dramatically as genetic evidence seems to suggest? Are gene products that are required for meiotic recombination associated with particular regions of the chromosomes? To answer these and other questions, we are using several approaches, including fluorescence in situ hybridization (FISH) methods, and labeling of chromosomes in living worms using the GFP-Lac repressor binding method developed by Aaron Straight, Andrew Murray, and Andy Belmont for other experimental systems. By applying high-resolution 3-dimensional microscopy, we hope to obtain a better understanding of rules governing the organization and interaction of chromosomes in the meiotic prophase nucleus.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

Awani, Awani et al. (2011) International Worm Meeting "Genome wide RNAi screen to study uv1 cell necrosis."

Awani, Awani, Hanna-Rose, Wendy, Crook, Matt

[International Worm Meeting, 2011]

Necrosis is more than a phenomenon of uncontrolled cell death. There is increasing evidence to suggest that necrosis is genetically programmed. We are exploiting a new model to investigate necrotic cell death. In worms that lack pnc-1 nicotinamidase, the four uv1 cells die by necrosis (1). We want to investigate why uv1 cells die in pnc-1 mutants and to identify genes that promote or inhibit uv1 cell necrosis. In pnc-1 mutant animals with a uv1 cell specific GFP transgene, we can screen for rescue of uv1 cell necrosis by screening for animals that retain the uv1 GFP signal. We aim to screen using the genome-wide RNAi library and expect to gain insight into how and why uv1 cells die in pnc-1 mutants. We are using a split GFP system to engineer animals with uv1 cell specific GFP expression for this screen (2), and we will discuss progress in engineering the strain and preparing for the screen. Reference: 1. EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus. Li Huang , Hanna-Rose Wendy 2. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Indraneel Ghosh , Andrew D. Hamilton , Lynne Regan.

Ben Lehner et al. (2006) European Worm Meeting "Systematic Mapping of Genetic Interactions in C. elegans Suggests a New Paradigm for Human Genetic Disease"

Catriona Crombie, Andrew G. Fraser, Ben Lehner, Angelo Fortunato, Julia Tischler

[European Worm Meeting, 2006]

Ben Lehner, Catriona Crombie, Julia Tischler, Angelo Fortunato and Andrew G. Fraser Most heritable traits, including disease susceptibility, are affected by the interactions between multiple genes. However, we still understand very little about how genes interact since only a minute fraction of possible genetic interactions have been explored experimentally. To begin to address this, we are using RNA interference to identify genetic interactions in C. elegans, focussing on genes in signalling pathways that are mutated in human diseases. We tested ~65,000 pairs of genes for possible interactions and identify ~350 genetic interactions. This is the first systematically constructed genetic interaction map for any animal. We successfully rediscover most components of previously known signalling pathways; furthermore, we verify 9 novel modulators of EGF signalling. Crucially, our dataset also provides the first insight into the global structure of animal genetic interaction maps. Most strikingly, we identify a class of highly connected ''hub'' genes: inactivation of these genes greatly enhances phenotypes resulting from mutations in many different pathways. These hub genes all encode chromatin regulators, and their activity as genetic hubs appears conserved across metazoans. We propose that these genes function as general buffers of genetic variation and that these hub genes will act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

James P McCarter et al. (2001) International C. elegans Meeting "Genes from Other"

Michael Dante, James P McCarter, John C Martin, Deana Pape, Brandi Chiapelli, Todd N Wylie, Robert H Waterston, Sandra W Clifton

[International C. elegans Meeting, 2001]

To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.

Noble, M. et al. (2017) International Worm Meeting "The Nucleosome Remodelling Factor subunit pyp-1 negatively regulates the heat shock response upon aging in Caenorhabditis elegans ."

Guisbert, E, Bowie, L, Deonarine, A., Westerheide, S, Lugano, D., Noble, M.

[International Worm Meeting, 2017]

Title: The Nucleosome Remodelling Factor subunit pyp-1 negatively regulates the heat shock response upon aging in Caenorhabditis elegans Mark Noble, Andrew Deonarine, Doreen Lugano, Lori-Ann Bowie and Sandy D. Westerheide The heat shock response is a conserved eukaryotic stress response known to regulate protein homeostasis (proteostasis) and it is regulated by the transcription factor hsf-1. Previous studies have shown tremendous effects of the HSR on life span, health span, and disease models of neurodegenerative diseases such as Alzheimer's, Parkinson and Huntington's disease. In C. elegans, proteostasis has been shown to decline with the onset of adulthood in parallel to the decline of the HSR. Therefore, understanding the molecular mechanisms and regulation of this response is vital in revealing its effects on various stresses. Recent studies have shown a link between chromatin remodelers and regulation of the heat shock response. Thus, utilizing RNAi genetic screen, we were able a uncover pyp-1, a pyrophosphatase and subunit of the Nucleosome-remodeling factor (NURF) which showed tremendous effects on the HSR. From a different screen, pyp-1 was also found to be a negative regulator of the heat shock response in C. elegans. We hypothesize that the age-dependent decrease in HSR induction may be due to repressive chromatin changes conferred by chromatin remodeling complexes. As a result, we identified pyp-1 as a novel negative regulator of the heat shock response and proteostasis upon aging in C.elegans.