Koopman, Mandy et al. (2021) International Worm Meeting "Optogenetic manipulation of individual or whole population Caenorhabditis elegans worms with an under hundred-dollar tool: the OptoArm"

Koopman, Mandy, Janssen, Leen, Nollen, Ellen

[International Worm Meeting, 2021]

Optogenetic tools have revolutionized the study of neuronal circuits in Caenorhabditis elegans. The expression of light-sensitive ion channels or pumps under specific promotors allows researchers to modify the behavior of excitable cells. Several optogenetic systems have been developed to spatially and temporally photoactivate light-sensitive actuators in C. elegans. Nevertheless, their high costs and low flexibility have limited access to optogenetics for a broad public. Here, we developed an inexpensive, easy-to-build and highly adjustable optogenetics device for use with different microscopes and wormtrackers, called the OptoArm. The OptoArm allows for single-and multiple-worm illumination during imaging and is adaptable in terms of light intensity, lighting profiles and light-color. We demonstrate the system's versatility by performing multi-parameter behavioral analysis upon cholinergic and GABAergic stimulation. Furthermore, we leveraged the OptoArm's power in a population-based study to dissect the contributions of motor circuit cells to age-related motility decline. We discovered that the functional decline of cholinergic neurons corresponds with motor decline, while GABAergic neurons and muscle cells appear relatively age-resilient. This would suggest a rate-limiting, cell type-specific vulnerability to ageing, which may underlie neuronal circuit aging.

Steven J. Husson et al. (2006) European Worm Meeting "Impaired Processing of FLP and NLP Precursors in C. elegans Lacking Active Prohormone Convertase 2 (EGL-3) and Carboxypeptidase E (EGL-21): Mutant Analysis by Mass Spectrometry"

Geert Baggerman, Steven J. Husson, Tom Janssen, Elke Clynen, Liliane Schoofs

[European Worm Meeting, 2006]

Impaired Processing of FLP and NLP Precursors in C. elegans Lacking Active Prohormone Convertase 2 (EGL-3) and Carboxypeptidase E (EGL-21): Mutant Analysis by Mass Spectrometry. Steven J. Husson, Elke Clynen, Geert Baggerman, Tom Janssen and Liliane Schoofs. Biologically active (neuro)peptides are synthesized as larger proprotein precursors which are further processed by the concerted action of a cascade of enzymes. Among these, proprotein convertase 2 (PC2), which cleave the precursors after dibasic residues, and carboxypeptidase E (CPE), which remove these basic amino acids from the carboxyterminals, play a pivotal role. We have examined the role of the C. elegans orthologues PC2/EGL-3 and CPE/EGL-21 in the processing of FMRFamide-like peptide (FLP) precursors and neuropeptide-like protein (NLP) precursors. Recently, we performed the peptidomic analysis of C. elegans by two dimensional nanoscale liquid chromatography - Quadrupole Time-Of-Flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). This setup yielded the identification of sixty endogenously present (neuro)peptides. We now expand this list of identified peptides by using an off-line approach in which HPLC fractions are analyzed by a Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometer (MALDI-TOF MS). This way we compared the peptide profile of different C. elegans strains, among which PC2/EGL-3 and CPE/EGL-21 mutants. This differential peptidomics approach now unambiguously proves the role of these enzymes in the processing of FLP and NLP precursors.

Buechner M et al. (2000) Japanese Worm Meeting "A faciogenital dysplasia gene homologue regulates the formation of organ structure in C. elegans"

Hisamoto N, Nishiwaki K, Matsumoto K, Suzuki N, Buechner M, Hall DH

[Japanese Worm Meeting, 2000]

The molecular controls governing the formation of organ structure in the development are less understood. The Caenorhabditis elegans excretory system, like the renal organs of other animals, is believed to regulate osmoregulation and water elimination1. The excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in the excretory canal. Here we report that exc-5 encodes a protein homologous to guanine nucleotide exchange factors (GEFs). EXC-5 contains, in order, a Dbl/Plecstrin homology (DH/PH) domain, a cysteine-rich FYVE domain and a second PH element. This motif architecture is similar to that of FGD1 (ref. 2), which is responsible for faciogenital dysplasia or Aarskog-Scott syndrome. Ultrastructural results suggest that exc-5 is required for the proper placement of cytoskeleton at the apical epithelial surface. Furthermore, EXC-5 associates with KEL-2, a protein similar to the actin-associated protein Drosophila Kelch3,4. We speculate that EXC-5 controls structural organization of the excretory canal by linking between the actin cytoskeleton and a Rho family GTPase. References: 1. Nelson, F. K., Albert, T. S. & Riddle, D. L. Fine structure of the Caenorhabditis elegans secretory-excretory system. J. Ultrastruct. Res. 82, 156-171 (1983) 2. Broeks, A., Janssen, H. W., Calafat, J. & Plasterk, R. K. A P-glycoprotein protects Caenor ha bditis elegans against natural toxins. EMBO J. 14, 1858-1866 (1995) 3. Pasteris, N. G. et al. Isolation and characterization of the faciogenital dysplasia (Aarskog-scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor. Cell 79, 669-678 (1994) 4. Chitwood, M. B. & Chitwood, B. G. The excretory system. In Introduction to Nematology:(eds. Chitwood, B. G. & Chitwood, M. B.; University Park Press, Baltimore) 126-135 (1974)

Meelkop, Ellen et al. (2013) International Worm Meeting "Neuropeptides in neuronal development, maintenance and regeneration."

Hilliard, Massimo A., Meelkop, Ellen

[International Worm Meeting, 2013]

Neuropeptides consist of short sequences of amino acids and function through G protein-coupled receptors and second messengers such as calcium and cAMP. Their involvement in processes such as learning, memory, and behaviour (e.g. locomotion and social behaviour) has widely been established1. However, their role in neuronal development, maintenance and regeneration has only started to be explored. Recent work has shown that the neuropeptide pigment dispersing factor (PDF) is involved in neuronal regeneration in C. elegans2. Mutant animals lacking PDF-1 peptides showed a 25% decrease of regrowth, 24 hours after axotomy in the mechanosensory PLM neuron. So far, it remains unknown where, when and how these neuropeptides stimulate regeneration. In C. elegans, neuronal regeneration after a laser-induced axotomy occurs through regrowth of the proximal fragment (attached to the cell soma), which is capable of reconnecting and eventually fusing with the distal fragment (separated from the cell soma). Whenever reconnection and fusion fail to occur, the distal fragment degenerates in stereotypical pattern. In order to elucidate the involvement of the PDF system in these events in C. elegans, we are characterizing the mechanosensory ALM and PLM neurons during development and regeneration in pdf-1, pdf-2 and pdfr-1 mutant backgrounds. While ALM and PLM both develop normally, axonal regeneration and reconnection to the distal fragment is particularly compromised in the PLM neurons of pdf-1 and pdfr-1 mutant animals. Simultaneously, the distal fragments degenerate at a slightly faster rate. By expressing receptor fusion proteins under the pdfr-1 promoter, we were able to confirm their expression in the mechanosensory neurons3,4 and postulate that the observed effects are controlled cell-autonomously. By using C. elegans as a model for neuronal development, maintenance and regeneration, we are now able to genetically dissect the pathways through which specific neuropeptides modulate these crucial biological processes.1 L. Frooninckx et al., Front. Endocrin. 3 (2012). 2 L. Chen et al., Neuron 71, 1043 (2011). 3 A. Barrios et al., Nat. Neurosci. 15, 1675 (2012). 4 T. Janssen et al., JBC 283, 15241 (2008).

Tom Janssen et al. (2007) International Worm Meeting "The clock ticks on with the discovery of PDF and its receptors in Caenorhabditis elegans."

Inge Mertens, Steven J. Husson, Johan Geysen, Gert Jansen, Liliane Schoofs, Tom Janssen, Suzanne Rademakers, Kris Ver Donck, Karen Verstraelen, Marleen Lindemans

[International Worm Meeting, 2007]

Circadian clocks control the daily rhythms in physiology and behavior of organisms ranging from bacteria to mammals, as an adaptation to the earths 24-hour rotation. The basic mechanism underlying these circadian rhythms follows a similar general plan across the phylogenetic spectrum. In the fruit fly Drosophila melanogaster, a neuropeptide called pigment dispersing factor (PDF) is the key outgoing signal of the circadian system. Caenorhabditis elegans also exhibits a convincing circadian rhythm in locomotor behavior and in resistance to hyperosmotic stress (1-2), but the underlying mechanism still remains elusive. Here, we report the identification and characterization of a neuropeptide signaling system, similar to the key output signals of the circadian clock in Drosophila melanogaster. We identified three pigment dispersing factors (Ce-PDF-1a, b and Ce-PDF-2) and their G protein-coupled receptors in C. elegans, and demonstrate their involvement in the regulation of locomotor behavior (3). The three C. elegans PDFs were isolated from a peptide-enriched extract and identified by LC-MS. GFP fusions show that they are primarily expressed in specific head and tail neurons. The three identified GPCRs all display a specific affinity for each of the 3 Ce-PDFs. Receptor expression was observed in particular neurons and all somatic muscle cells. Many of the PDF and/or PDF-receptor expressing cells play a role in the integration of environmental stimuli and the control of locomotion. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit antagonistic effects on locomotion. These findings indicate that the Ce-PDFs may be a key outgoing signal in the C. elegans circadian clock, changing the locomotor behavior by acting through these (muscle) receptors. Our findings suggest that PDF signaling might be well conserved during evolution, at least in invertebrates (3). 1. Kippert,F. et al. Caenorhabditis elegans has a circadian clock. Curr. Biol. 12, R47-R49 (2002). 2. Saigusa,T. et al. Circadian behavioural rhythm in Caenorhabditis elegans. Curr. Biol. 12, R46-R47 (2002). 3. Janssen et al. The clock ticks on with the discovery of PDF and its receptors in the nematode Caenorhabditis elegans. Neuron submitted.

Janssen, T. et al. (2011) International Worm Meeting "GPCR signaling in free-living and parasitic nematode models: the cholecystokinin story."

Beets, I., Suetens, N., Janssen, T., Peeters, L., Temmerman, L., Meelkop, E., Schoofs, L., Grant, W.

[International Worm Meeting, 2011]

Members of the cholecystokinin/gastrin family of peptides, including the arthropod sulfakinins, and their cognate receptors, play an important role in the regulation of feeding behavior and energy homeostasis. The C. elegans genome contains one gene (two splice-isoforms) with strong homology to the mammalian cholecystokinin receptors and their invertebrate counterparts, the sulfakinin receptors. By using these potential C. elegans CCK receptors as bait, we have isolated and identified two CCK-like neuropeptides encoded by a peptide precursor protein called NLP-12, as the endogenous ligands of these receptors (Janssen et al. 2008, 2009, 2010). The NLP-12 peptides have a very limited neuronal expression pattern and seem to be very well conserved within four clades of nematodes. Both receptors and ligands share a high degree of structural similarity with their vertebrate and arthropod counterparts and also display similar biological activities with respect to digestive enzyme secretion and fat storage. Our recent microarray studies revealed that C. elegans CK signaling directly or indirectly affects the expression of several hundred genes, many of which are involved in sugar and fat metabolism and transport, reproduction and the response to starvation. These effects seem to be highly dependent on the animal's feeding status. The expression of the nematode CK system (receptor/ligand) itself is also reversibly upregulated under starvation, which is the opposite of the situation in mammals where feeding stimulates CCK expression and secretion. In addition, food-consumption tests revealed an inhibitory effect of nematode CK signaling on satiety, which is the complete opposite of their function in vertebrates and arthropods. It seems that some of the CCK functions have been conserved during evolution (i.e. related to fat storage, digestive enzyme secretion) whereas others have changed (i.e. opposite effects on satiety). Also L1 diapause and arrested dauer animals show an astonishing increase in CK receptor and ligand transcript levels. Many similarities exist between the arrested dauer larvae of free-living nematodes and the infective L3 stage of parasitic nematodes. This has led to suggestions that they are analogous lifecycle stages and has triggered us to investigate whether the nlp-12/ckr-2 system is present and differentially expressed in infectious (iL3) or parasitic stage animals of the parasitic nematode Parastrongyloides trichosuri.

Hung, Wesley et al. (2011) International Worm Meeting "An F-box protein FSN-1 Regulates Retrograde Insulin Signalling."

Hwang, Christine, Chitturi, Jyothsna, Li, Hang, Wang, Ying, Hung, Wesley, Zhen, Mei, Liao, Edward

[International Worm Meeting, 2011]

The formation of a functional synapse requires the co-ordinated growth of both pre-synaptic and post-synaptic termini. Communication between the pre- and post-synaptic termini involves many proteins and factors. The pre-synaptic F-box protein FSN-1 is an evolutionarily conserved protein that plays an important role in axon growth and synapse development. FSN-1 is a part of an SCF-like E3 ubiquitin ligase complex that comprises of the RING finger domain protein RPM-1, Cullin and Skp (1). FSN-1/RPM-1 complex negatively regulates a MAPK kinase cascade comprised of DLK-1/MKK-4/PMK-3 during synapse formation (2). We report here that FSN-1 also regulates synapse development, in part, through the regulation of a retrograde insulin/IGF-signalling pathway in the post-synaptic terminal. Mutation in fsn-1 causes over-development of some synapses along the dorsal nerve cord while other areas of the dorsal cord have little or no synapses. In fsn-1 mutants there is an increased activity of the insulin/IGF signalling pathway in the post-synaptic muscle cells. Accordingly, loss-of-function mutations in the insulin/IGF pathway components suppressed the synaptic morphology defects of fsn-1 mutants. We identified several neuronal insulin-like ligands that regulate the postsynaptic insulin/IGF signal during synapse development; all require a proprotein convertase, EGL-3 (3, 4), for maturation. We further demonstrate that, in vivo, EGL-3::GFP's level is increased in fsn-1 mutants. in vitro, FSN-1 interacts with, and ubiquitinates EGL-3. We propose that FSN-1 modulates the activity of the post-synaptic insulin/IGF pathway via its regulation of EGL-3. 1. Liao, E.H., W. Hung, et al. (2004). An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430: 345-350. 2. Nakata K, B. Abrams , et al. (2005). Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120:407- 420. 3. Kass, J., T.C. Jacob, et al. (2001).The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans. J Neurosci 21: 9265-9272. 4. Husson, S. J., T. Janssen, et al. (2007). Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analyzed by mass spectrometry. J Neurochem 102: 246-60.

Laine, Viviane et al. (2017) International Worm Meeting "Fifty years of levamisole: do we really know how it works?"

Laine, Viviane, Bonneau, Benjamin, Jospin, Maelle, Bessereau, Jean-Louis, Briseno-Roa, Luis

[International Worm Meeting, 2017]

Levamisole was identified in 1966 by Paul Janssen and collaborators as a potent broad spectrum anthelminthic. It activates acetylcholine receptors (AChR) at the neuromuscular junction, which causes worm hypercontraction and irreversible paralysis at high concentrations. Genetic screens conducted over 4 decades for mutants either insensitive to the drug (resistance) or able to recover movement after initial paralysis (adaptative-resistance) have been instrumental to identify subunits and regulators of the levamisole-sensitive AChR (L-AChR). Yet, the precise physiological mechanisms of the levamisole response in the wild type (WT) and in adaptative-resistant mutants remain elusive. Using a combination of electrophysiological, Ca2+ imaging and genetic tools we have identified a complex sequence of events that underlie the behavioral features observed at the whole-organism level. First, when worms are dropped onto plates containing 1 mM levamisole, their muscles hypercontract within a few minutes. This initial response is similar in WT and adaptative-resistant mutants, and abolished in mutants lacking L-AChRs. We show that this early contraction is not due to the entry of Ca2+ only through L-AChR, but requires the activation of the voltage-gated Ca2+ channels (VGCC) EGL-19. Second, after 10 minutes on levamisole, WT start to relax, whereas adaptative-resistant mutants such as lev-10 remain hypercontracted. This long-lasting contraction is correlated with the maintenance of a high intracellular Ca2+ concentration in muscle cells. The difference of behavior between WT and lev-10 worms is puzzling: in lev-10 mutants L-AChRs are spread over the muscle surface instead of being clustered at post-synaptic sites, but the total number of L-AChRs is unchanged (Gally 2004). How can levamisole have an effect dependent on L-AChR subcellular localization? We show that the difference between WT and mutants is explained by VGCC properties. In WT, L-AChRs remain opened on levamisole and the muscle depolarization causes a complete inactivation of VGCC. In lev-10 worms, muscle cells are less depolarized on levamisole and a fraction of VGCC is still open, explaining why lev-10 mutants remain hypercontracted when WT worms relax over time. Third, after 8 hours of levamisole exposure, WT worms are still paralyzed whereas lev-10 mutants partially recover locomotion. In these mutants, we show that the membrane resting potential is back to normal, suggesting that L-AChRs are not functional anymore. Our results demonstrate that slightly reducing the magnitude of muscle depolarization during the acute phase of levamisole exposure is the key to explain adaptative-resistance. A new large-scale screen conducted in our laboratory has demonstrated that levamisole-resistance screens are not saturated and could still reveal new molecular players involved in the implementation of L-AChR activation.