Hardege, Iris et al. (2021) International Worm Meeting "LGC-50 - a new serotonin receptor involved in aversive olfactory learning that displays regulated plasma membrane trafficking"

Schafer, William, Hardege, Iris, Morud, Julia

[International Worm Meeting, 2021]

Across animal phyla, monoamines signal through both metabotropic and ionotropic receptors. In worms as well as in humans, metabotropic monoamine receptors, which modulate neuronal activity through G-protein-mediated second messenger pathways, have received most attention. However, expression studies have indicated that the vast majority of C. elegans neurons postsynaptic to aminergic neurons do not express metabotropic amine receptors. This implies that synaptic monoamine transmission may be mediated by as yet uncharacterized ionotropic receptors. We have in our recent work identified endogenous ligands for five new amine-gated ion channels (LGC), all of which are found localised postsynaptically to aminergic neurons. In particular we have shown the serotonin-gated receptor LGC-50 to be a cation channel that is localised in the interneuron RIA, which is strongly innervated by the serotonergic neuron ADF. Previous work has indicated a role for ADF and RIA in aversive pathogen learning, through which animals learn to avoid odours released by pathogenic bacteria following infection. We have also shown that lgc-50 mutants show a strong defect in pathogen learning, which can be rescued by expression of LGC-50 in RIA. These results suggest that serotonin may act through LGC-50 to modify the strength of specific synapses in the olfactory navigation circuit. Our recent work also indicated that the plasma membrane localisation of LGC-50 is tightly regulated, and that potential disruption of this trafficking influences memory formation. We have now identified a 17-amino acid long motif in the intracellular M3/4 domain of LGC-50 that conveys this regulated membrane localisation. Further, we have evidence that this motif might act as a binding site for the protein NRA-1 and that this protein-protein interaction might be involved in moving LGC-50 to the plasma membrane during memory formation. LGC-50 thus provides an entry point to define the molecular and neural changes underlying learning and memory in the worm.

Hardege, Iris et al. (2021) International Worm Meeting "Expansion of Cholinergic Signalling Reveals Polymodal and Novel Ligand Gated Ion Channels Involved in Switching Behavioural States"

Morud, Julia, Schafer, William, Hardege, Iris

[International Worm Meeting, 2021]

Ligand-gated ion channels (LGICs) play important roles in synaptic communication and the regulation of behaviours. The cys-loop superfamily of LGICs, which contains mammalian nicotinic acetylcholine and GABA receptors, has undergone vast gene expansion in nematodes and includes channels gated by classical and non-classical neurotransmitters. Yet the majority of C. elegans LGICs remain uncharacterised, with no known ligand or biological function. We have undertaken a deorphanisaiton study of a number of uncharacterised C. elegans LGICs, in particular from a subfamily of 12 channels known as the "diverse" group. Using two-electrode voltage clamp recordings from Xenopus oocytes injected with worm LGICs, we identified ligands for 5 of the 12 channels in the diverse subgroup. All are inhibitory anion-selective channels, yet despite sharing close sequence similarity, they are gated by chemically diverse ligands. We identify three receptors for choline, GGR-1, GGR-2 and LGC-40, which despite binding the same ligand differ in their pharmacological and neuronal expression profiles. This may point towards a potential role for choline in the regulation of the nervous system. Interestingly, we found a single channel, LGC-39 to be gated not only by acetylcholine but also by aminergic ligands, in particular octopamine and tyramine. Thus LGC-39 has the capacity to form a polymodal receptor activated by chemically disparate neurotransmitters. The expression pattern of lgc-39 reveals that it is present in a number of neurons which receive both aminergic and cholinergic input, including AVA, the major synaptic target of the octopamine producing RICs. Finally, we find that LGC-41 is not activated by any classical neurotransmitter, but is activated by betaine, a metabolite chemically related to choline. LGC-41, along with putative betaine synthesis genes, is widely expressed in the nervous system, in particular in a number of neurons associated with regulating search behaviours including ASI. Strikingly, we find that lgc-41 and betaine synthesis mutant worms show defects in transitioning from local to global search behaviour in the absence of food. This implicates betaine and its receptor in regulating complex behaviours which rely on the integration of a number of sensory inputs. Taken together our findings highlight the remarkable functional and behavioural diversification amongst the LGICs in C. elegans.

Courtney, Amy et al. (2021) International Worm Meeting "A Comprehensive Characterisation of C. elegans Neurotransmitter GPCRs Reveals a Novel Adenosine Receptor"

Hardege, Iris, Morud, Julia, Schafer, William, Courtney, Amy

[International Worm Meeting, 2021]

Many G protein coupled receptors (GPCRs) can be activated by neurotransmitters, acting via heteromeric G proteins to control downstream intracellular processes. A GPCR can be simplified as either excitatory (Galphas or Galphaq) or inhibitory (Galphai/o). While ligands for many C. elegans neurotransmitter GPCRs have been identified, many gaps in our knowledge remain. To address this, we expressed C. elegans GPCRs in Xenopus oocytes along with G protein-coupled inwardly rectifying potassium channels (GIRKs) to evaluate ligand specificity and G-protein coupling using two-electrode voltage clamp recordings. Using this approach, we have successfully observed activation of 10 monoamine-activated GPCRs, all with previously identified ligands. Despite this previous knowledge, some results on their G-protein coupling, and the potency of secondary ligands was often conflicting or missing. For example, SER-6 and DOP-4 have previously been shown to couple to Galphas but our experiments indicate Galphaq coupling. Activation by two or more ligands was observed for all the GPCRs tested, but the published data does not reflect this phenomenon. Several additional orphan receptors were evaluated, DOP-5, DOP-6, SER-5 and PCDR-1, and no activation was detected. The reason is unknown, but it could be because they require a GPCR partner to form a functional receptor, because they couple to a non-cannonical G-protein, or because we have not tested the correct ligands. In addition, we have characterized the putative adenosine receptor ADOR-1 and found it to be indeed activated by adenosine. Surprisingly, we obtained evidence suggesting coupling to the excitatory Galphaq protein as well as to Galphai/o or Galphas. Further experiments are ongoing to confirm this dual coupling in vitro and in vivo. Using endogenous fluorescent reporters, we found ador-1 to be broadly expressed in the nervous system as well as in muscle, including MC/I4/pharyngeal muscle and vulval muscle/HSN. However, despite this expression pattern we did not observe any significant differences in pumping rate or egg laying under normal conditions. In other species, intracellular adenosine levels have been shown to increase after injury or stress, and we observed broad expression of ador-1 in multiple 'avoidance' neurons (AWB/ASH/ASK). We hypothesise that adenosine may act via ador-1 to regulate stress related behaviours or lifespan and we plan to investigate this going forward.

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.

Bo Wang et al. (2006) European Worm Meeting "Multi-Parameter Axial Profiling of Transgenic C. elegans Expressing Fluorescent Proteins from Various Cell-Specific, Tissue Specific and Developmentally Regulated Promoters"

Bruce Holcombe, Julia Thompson, Yanping Zhang, Rock Pulak, Bo Wang, Michael Herman, Mariya Lomakina

[European Worm Meeting, 2006]

Bo Wang1, Julia Thompson1, Yanping Zhang2, Michael Herman2, Mariya Lomakina1, Bruce Holcombe1, Rock Pulak1 . The COPAS Biosort instrument automates the analysis, sorting, and dispensing of all stages of C. elegans, measuring the animals size and the intensity of expressed fluorescent markers. Once analyzed, animals can be selected according to user defined criteria, and then dispensed into multi-well plates for high throughput screening or collected in bulk for further analysis. With this technology, time required for large scale screening for certain changes in the optical properties of the animals, such as changes in the levels of expression of a fluorescent protein, can be dramatically reduced and human error minimized. Recent enhancements to an add-on module, called the Profiler II, have been tested for its ability to collect positional information of fluorescent expression. The instrument can simultaneously collect fluorescence information in three separate regions of the spectrum. Here we show that the instrument can analyze multi-colored transgenic animals and can be used to compare the amounts and relative positions of expression of two or three different colors of fluorescence. Furthermore, this technology can be used to screen for independent changes in the intensity or position of each reporter protein. We have tested various transgenic animals expressing green, yellow and/or red fluorescing proteins from a collection of promoters that include myo-2, str-1, egl-17, mab-5, and various others, separately and in certain combinations. We present some proof of principle examples of how these could be used in genetic screens.

Julia Tischler et al. (2006) European Worm Meeting "Analysing the Redundancy of Duplicated Genes and the Evolutionary Conservation of Genetic Interactions Using Combinatorial RNAi in C. elegans"

Ben Lehner, Julia Tischler, Andrew G Fraser

[European Worm Meeting, 2006]

Julia Tischler, Ben Lehner and Andrew G Fraser Systematic analyses of loss-of-function phenotypes have been carried out for almost all genes in S. cerevisiae, C. elegans, and D. melanogaster, and there are major efforts to make a comprehensive collection of mouse knockouts. While such studies greatly expand our knowledge of single gene function, they do not address redundancy in genetic networks, nor do they attempt to identify genetic interactions. Developing tools for the systematic mapping of genetic interactions is thus a key step for exploring the relationship between genotype and phenotype. We thus sought to establish protocols for targeting multiple genes simultaneously by RNA interference (RNAi) in C. elegans to provide a platform for the systematic identification of genetic interactions in this key animal model system.. We set up conditions for RNAi that allow us to target multiple genes in the same animal (combinatorial RNAi) in a high throughput setting and to detect the great majority of previously known synthetic genetic interactions. We then used this assay to test the redundant functions of genes that have been duplicated in the genome of C. elegans since divergence from either S. cerevisiae or D. melanogaster, and identified 16 pairs of duplicated genes that are at least partially functionally redundant. Intriguingly, 14 of these redundant gene pairs were duplicated before the split of C. elegans and C. briggsae 80-110 million years ago. Our data provide the first systematic investigation into the redundancy of duplicated genes in any organism and strongly support population genetics models, which suggest that redundancy can be maintained over substantial periods of evolutionary time.. Furthermore, we set out to test whether systematically compiled yeast genetic interaction data can predict genetic interactions in the worm. We will present these data.

Harnagel, Audrey et al. (2021) International Worm Meeting "Identifying Triggers for Pathogen Learning in C. elegans"

Harnagel, Audrey, Bargmann, Cori

[International Worm Meeting, 2021]

The ability to discriminate between nutritious and harmful food is essential to survival. As a result, learned avoidance to harmful food sources is conserved from invertebrates to humans. The mechanisms enabling the nervous system to associate sensory cues from a food source with an internal state of sickness to trigger aversive memory formation remain elusive. After prolonged exposure to pathogenic food, C. elegans can learn to avoid the pathogen upon subsequent encounter1. This learned aversion requires infection; non virulent forms of bacteria are not sufficient for memory formation. In response to exposure to pathogenic food, serotonin is induced in a pair of sensory neurons called ADF and remodels downstream circuits2. Learned aversion to pathogen requires serotonin signaling from ADF, suggesting that ADF serves as a site of integration for detecting bacterial cues and internal sickness caused by the pathogen. We seek to understand how internal state changes the coupling between sensory activation and serotonin release in ADF neurons. As a first step, we are using calcium imaging to examine ADF responses to bacterial cues in both naive and pathogen-exposed animals. ADF responds robustly to conditioned media from both pathogenic and non-pathogenic bacteria in a dose-dependent fashion, and ADF activity can be modulated by previous odor history. We are screening mutants using these quantitative parameters to assess ADF responses to direct chemosensory stimuli, indirect signaling from other sensory neurons, and signaling from non-neuronal tissues indicating bacterial infection. Our goal is to uncover cell-biological mechanisms through which ADF neurons mediate learned pathogenic behavior in C. elegans. 1. Zhang, Y., Lu, H., & Bargmann, C.I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature, 438(7065), 179. 2. Morud, J., Hardege, I., Liu, H., Wu, T., Basu, S., Zhang, Y., & Schafer, W. (2020). Deoprhanisation of novel biogenic amine-gated ion channels identifies a new serotonin receptor for learning. bioRxiv. doi: https://doi.org/10.1101/2020.09.17.301382.

Brown, Andre et al. (2017) International Worm Meeting "A common format for worm tracking data."

Kerr, Rex, Brown, Andre, Lee, Chee Wai, Hokanson, Jim, Currie, Michael

[International Worm Meeting, 2017]

Many labs, including ours, have built a wide variety of worm trackers. These have a wide range of capabilities, from high-resolution imaging of single animals during calcium imaging, to very low-resolution imaging of animals as points. This diversity of capability enables the C. elegans community to address a wide range of problems at an appropriate scale. Most of these trackers also produce some data that is very similar to that of other trackers: animal position or spine, for example. Unfortunately, each tracker uses its own format to store data, so that any later analysis, despite being general in nature, cannot be performed on data from different machines. As the volume of tracking data grows, and the variety of downstream analysis methods expands, this limitation will pose an increasingly large barrier to replication of and extension of existing work across different labs. To address this issue, we have defined the Worm Common Object Notation, a set of rules for how to write tracking data in the ubiquitous JSON format, so that it can be easily shared between labs. To facilitate easy adoption of WCON, we have further written software in a variety of languages that will read or write data in WCON format. So far, we have implementations in Python, Scala, Matlab, and Julia, and wrapper libraries for Octave, R, and Java to use one of the main implementations. Additionally, the Tracker Commons project of which WCON is a part contains a small but rapidly growing set of pre-packaged analysis tools for routine manipulation of worm tracking data. We will also maintain a list of other WCON-compatible analysis tools as they become available. If you are involved in worm tracking, we invite you to adopt WCON and help make C. elegans behavioral data widely accessible. WCON is developed under the open source Tracker Commons project of the OpenWorm Foundation. We invite contributions and improvements!

Julia Grabitzki et al. (2006) European Worm Meeting "Identification of O-linked N-acetylglucosamine Modified Proteins in Caenorhabditis elegans"

Gunter Lochnit, Rudolf Geyer, Michael Ahrend, Brigitte Schmitz, Julia Grabitzki

[European Worm Meeting, 2006]

Julia Grabitzki1, Michael Ahrend2, Brigitte Schmitz2, Rudolf Geyer1 and Gnter Lochnit1. The posttranslational modification N-acetylglucosamine O-glycosidically linked (O-GlcNAc) to serine and threonine residues of proteins has been shown to be ubiquitous amongst eukaryotic proteins of the nucleus, cytoskeleton, cytoplasm, and has also been detected on cytosolic tails of membrane proteins [1]. O-GlcNAcylated proteins can form reversible multimeric complexes with other polypeptides or structures. The modification is often accompanied by phosphorylation/ dephosphorylation. O-GlcNAc can act either simultaneously or in a reciprocal fashion with phosphorylation. According to the Yin-Yang hypothesis, the phosphorylation/ dephosphorylation regulates O-GlcNAc-modified protein function (z.B. signal transduction and protein-protein interaction) in concert with phosphorylation [2-4]. The addition of O-GlcNAc to and the removal from the protein backbone is dynamic with rapid cycling in response to cellular signals or cellular stages.. Despite the fact, that Caenorhabiditis elegans is the best studied model organism, there have been no studies on O-GlcNAcylation in this organism so far. Therefore, to elucidate the role of O-GlcNAcylation, we investigated the proteome of a C. elegans mixed-stage population by two-dimensional gelelectrophoresis and subsequent western-blotting with the O-GlcNAc-specific antibody CTD 110.6 for the occurrence of this modification and identified the modified proteins by mass-spectrometry. We detected and identify several O-GlcNAc-modified proteins in C. elegans. Most of the identified proteins are involved in metabolic pathways. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic occurrence of the O-GlcNAc modification.. References:. [1]. Rex-Mathes, M., J. Koch, Werner, S., Griffith, L. S and B. Schmitz. 2002. Methods Mol Biol 194: 73-87.. [2] Zachara, N.E. and G.W. Hart, Chem Rev, 2002. 102(2): p.431-8.. [3]. Griffith, L. S. and B. Schmitz. 1999. Eur J Biochem 262(3): 824-31.. [4] Wells, L. and G. W. Hart. 2003. FEBS Lett 546(1): 154-8.

Julia Grabitzki et al. (2006) European Worm Meeting "Phosphorylcholine-Modified Proteins of Caenorhabditis elegans are Specific to Developmental Stages"

Michael Ahrend, Gunter Lochnit, Julia Grabitzki, Rudolf Geyer

[European Worm Meeting, 2006]

Julia Grabitzki, Michael Ahrend, Rudolf Geyer and Gunter Lochnit. The free-living nematode Caenorhabditis elegans has been found to be an excellent model system for developmental studies [1] investigating parasitic nematodes [2] and drug screening [3]. Structural analyses of glycoconjugates derived from this organism revealed the presence of nematode specific glycosphingolipids of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents [2]. PC, a small haptenic molecule, is found in a wide variety of prokaryotic organisms, i. e. bacteria, and in eukaryotic parasites such as nematodes. There is evidence that PC-substituted proteins glycolipids are assumed to be responsible for a variety of immunological effects including invasion mechanisms and long-term persistence of parasites within the host [4]. In contrast to PC-modified glycosphingolipids [5], only a limited number of PC-carrying (glyco)proteins were identified so far [6-9]. We have analysed the expression of PC-modified proteins of C. elegans during developmental stages using two dimensional SDS-Page separation, 2D-Western-blot and MALDI-TOF mass spectrometry. The pattern of PC-modified proteins was found to be stage specific. The PC-modification on proteins was most abundant in the egg and dauer larvae-stages followed by the adult-stage and L4. Only small amounts of the PC-substitution were found in L3 and L2. In L1 we couldnt detect any PC-Modification. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic and mitochondrial occurrence of the PC- modification. Most of the identified proteins are involved in metabolism or in protein synthesis.. 1.. Brenner, S., Genetics, 1974. 77(1): p. 71-94.. 2.. Lochnit, G., R.D. Dennis, and R. Geyer, Biol Chem, 2000. 381(9-10): p. 839-47.. 3.. Lochnit, G., R. Bongaarts, and R. Geyer, Int J Parasitol, 2005. 35(8): p. 911-23.. 4.. Harnett, W. and M.M. Harnett, Mod. Asp. Immunobiol., 2000. 1(2): p. 40-42.. 5.. Friedl, C.H., G. Lochnit, R. Geyer, M. Karas, and U. Bahr, Anal Biochem, 2000. 284(2): p. 279-87.. 6.. Haslam, S.M., H.R. Morris, and A. Dell, Trends Parasitol, 2001. 17(5): p. 231-5.. 7.. Cipollo, J.F., C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2002. 277(51): p. 49143-57.. 8.. Cipollo, J.F., A.M. Awad, C.E. Costello, and C.B. Hirschberg, J Biol Chem, 2005. 280(28): p. 26063-72.