Munneke, Allyson et al. (2015) International Worm Meeting "The G protein-coupled receptor FSHR-1 regulates the balance of excitatory to inhibitory transmission at the C. elegans neuromuscular junction."

Wasilk, Amy, Kolnik, Julie, Munneke, Allyson, Damler, Erica, Kowalski, Jennifer, Olofsson, Sarah

[International Worm Meeting, 2015]

Regulation of excitatory and inhibitory synaptic transmission is critical for nervous system function; misregulation of this signaling occurs in neurological disorders1. G protein-coupled receptors (GPCRs) are mediators of neuronal signaling and may directly or indirectly control synaptic transmission2. Here, we report a novel role for the conserved GPCR FSHR-1 as a key regulator of synaptic signaling at the C. elegans neuromuscular junction (NMJ) where a balance of excitatory cholinergic and inhibitory GABA signaling from presynaptic motor neurons controls muscle activity. In addition to controlling C. elegans innate immunity and germline differentiation3,4, the glycopeptide hormone receptor FSHR-1 was previously shown to regulate cholinergic signaling for muscle excitation and hypothesized to act as neuropeptide receptor5. The specific mechanism and site of FSHR-1 activity at the NMJ, however, are unknown. We recently identified FSHR-1 as a candidate substrate of the Anaphase-promoting complex (APC), an ubiquitin ligase that promotes GABA signaling at the NMJ6. FSHR-1 contains an APC recognition motif and the reduced muscle excitation seen in fshr-1(ok778) mutants suppresses the enhanced muscle excitation of APC loss of function mutants. Rescue of fshr-1 expression only in GABA neurons (where the APC functions) restores near wild type muscle contraction. Consistent with a role for FSHR-1 in both cholinergic and GABA signaling, fshr-1 mutants not only exhibit synaptic vesicle accumulation at cholinergic presynapses5, indicative of decreased acetylcholine release, but also show decreased synaptic vesicle accumulation at GABA presynapses, consistent with increased GABA release. These effects may involve canonical Galphas and adenylyl cyclase signaling4, as gain of function (gf) mutations in gsa-1(ce81) and acy-1(md1756), which cause increased muscle excitation, suppress the reduced muscle excitation of fshr-1 mutants, suggesting these genes act downstream of fshr-1. Neuronal activation of this pathway is important, as neuronal overexpression of an ACY-1 gf construct suppresses the fshr-1 loss of function phenotype. Current work involves further elucidating the role of FSHR-1 signaling in each motor neuron class, the APC-FSHR-1 relationship, and potential FSHR-1 interactions with neuropeptides. 1) Casillas-Espinosa PM et al. 2012. Epilepsia, 53:41-58; 2) Betke KM et al. 2012. Prog Neuro, 96: 304-21; 3) Powell JR et al. 2009. PNAS, 106:2782-87; 4) Cho S et a.l 2007. Curr Biol, 17:203-12; 5) Sieburth D et al. 2005. Nature, 436:510-17; 6) Kowalski JR et al 2014. Mol Cell Neuro, 58:62-75.

Olofsson, Sarah et al. (2017) International Worm Meeting "The G protein-coupled receptor FSHR-1 is a candidate APC substrate that controls excitatory to inhibitory signaling balance at the C. elegans neuromuscular junction."

Kowalski, Jennifer, Munneke, Allyson, Damler, Erica, Ryskamp, David, Kolnik, Julie, Godfrey, Amy, Olofsson, Sarah

[International Worm Meeting, 2017]

Regulation of excitatory to inhibitory synaptic signaling (E:I balance) is critical for nervous system function, and E:I imbalances occur in neurological disorders. G protein-coupled receptors (GPCRs) are common mediators of neuronal signaling, and many GPCRs modulate synaptic transmission. We are investigating a role for the conserved GPCR and glycopeptide hormone receptor FSHR-1 as a regulator of the C. elegans neuromuscular junction (NMJ), where a balance of excitatory cholinergic and inhibitory GABA signaling from motor neurons controls muscle activity. FSHR-1 controls several physiological processes in C. elegans, including innate immunity, germline differentiation, and stress responses. FSHR-1 also affects cholinergic signaling for muscle excitation and may be a neuropeptide receptor; yet, the mechanism and sites of FSHR-1 activity at the NMJ are unknown. We identified FSHR-1 as a candidate substrate of the anaphase-promoting complex (APC), an ubiquitin ligase that promotes GABA signaling at the NMJ. FSHR-1 has an APC recognition motif and fshr-1(ok778) loss of function (lf) mutations, which cause decreased muscle activity, suppress enhanced muscle excitation in APC lf animals. Re-expression of fshr-1 in all neurons or only GABA neurons (where APC acts) restores near wild type muscle activity to fshr-1 mutants, as does fshr-1 expression in cholinergic neurons. FSHR-1 signaling likely involves Gas and adenylyl cyclase, as gain of function (gf) mutations in gsa-1(ce81) and acy-1(md1756), which promote muscle excitation, suppress the reduced muscle excitation of fshr-1 mutants. Neuronal activation of this pathway is important, as overexpression of an acy-1 gf construct only in neurons suppresses fshr-1 lf phenotypes, while acy-1 gf only in GABA neurons gives partial suppression. Fluorescently tagged synaptic vesicles accumulate at cholinergic presynapses, indicative of decreased acetylcholine release, and at GABA presynapses of fshr-1 mutants, which may result from decreased cholinergic drive. GABA neuron-specific restoration of fshr-1 fails to rescue the synaptic vesicle accumulation of fshr-1 mutants, suggesting rescue of muscle contraction by GABA neuron-specific fshr-1 may be due to FSHR-1 indirectly influencing muscle activity. Current work will determine FSHR-1 function in each motor neuron class, including using CRISPR to identify sites of endogenous fshr-1 expression, and evaluate the APC-FSHR-1 relationship and FSHR-1-neuropeptide interactions.

Buckley, Morgan et al. (2021) International Worm Meeting "FSHR-1 regulates cholinergic synaptic vesicle and active zone protein localization to control neuromuscular signaling balance in C. elegans"

Kowalski, Jennifer, Wei, Barry, Kolnik, Julie, Olofsson, Sarah, Godfrey, Amy, Buckley, Morgan, Cherry, Kyle, Munneke, Allyson, Ritter, Alyssa, Padgett, Lauryn, Emch, David

[International Worm Meeting, 2021]

Excitatory to inhibitory signaling balance is essential to nervous system function. The G protein-coupled receptor FSHR-1 is the Caenorhabditis elegans ortholog of mammalian glycopeptide hormone receptors and modulates signaling at the C. elegans neuromuscular junction, where excitatory cholinergic and inhibitory GABAergic inputs control muscle excitation. Inhibition of fshr-1 expression was previously shown to cause reduced muscle contraction and accumulation of fluorescently labeled synaptic vesicles in cholinergic motor neurons. Here, we used a combination of aldicarb-induced paralysis and swimming assays to confirm the neuromuscular defects of fshr-1(ok778) loss-of-function mutants, which we found are exacerbated in aged animals, as well as in animals exposed to chronic oxidative stress. Expression of fshr-1 in all neurons, as well as in cholinergic or GABAergic neurons alone, could restore neuromuscular activity to these animals under normal conditions. Surprisingly, however, this same fshr-1 re-expression in cholinergic or GABAergic neurons failed to restore wild type synaptic vesicle localization, and endogenous fshr-1 expression was detected in what appear to be pharyngeal neurons and arcade cells but not in body wall motor neurons, suggesting a complex site of action. fshr-1(ok778) mutants also exhibited build-up of the synaptic vesicle priming factor, UNC-10/RIM, and its interaction partner, SYD-2/Liprinalpha, primarily in cholinergic motor neurons. No such effects were observed for the active zone protein, CLA-1/Clarinet, or the INS-22 dense core vesicle marker. Finally, gain-of-function mutations in gsa-1/GaS or acy-1/adenylyl cyclase suppressed the reduced muscle excitation of fshr-1 mutants. Together, these data support a model in which FSHR-1 may act cell non-autonomously, as well as in body wall motor neurons, to promote muscle excitation upstream of GSA-1 and ACY-1, ultimately controlling UNC-10 localization and cholinergic vesicle release. We are currently further exploring FSHR-1 site(s) of action, working to identify potential ligands that may control FSHR-1's neuromuscular effects, as well as investigating potential connections of FSHR-1 to lipid signaling and other stress-related pathways as they relate to neuromuscular and other neuronal signaling events in physiological and stress conditions.

Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Yohann Duverger et al. (2007) International Worm Meeting "A French functional genomics platform."

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Sarah Scaglione, Daniel Wong

[International Worm Meeting, 2007]

For the last four years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates up to 1000 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented. This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community. This French functional genomics platform has been made possible through funding from the National Genopole&#174; network, Marseille-Nice genopole&#174;, INSERM, the CNRS and support from Union Biometrica.

May Tran et al. (2005) International Worm Meeting "RNAi knockout of the mitochondrial carrier protein ucp-4: effects on metabolism"

May Tran, Craig LaMunyon, Aidyl Gonzalez-Serricchio

[International Worm Meeting, 2005]

We are investigating the phenotype of ucp-4 RNAi knockout worms. Mitochondrial uncoupling protein (UCP) function in humans has been linked to obesity. UCPs are mitochondrial carrier proteins, and human UCP-1, -2, and -3 are protonophores, transporting protons from the intermembrane space to the matrix, bypassing ATP synthase, and thereby raising resting metabolic rate in response to certain cues. The function of human UCP-4 is not clear, but it may be a mitochondrial fatty-acid transporter, and it is a homolog to the C. elegans ucp-4 (K07B1.3). Initial RNAi experiments by Dr. Julie Ahringers lab deemed knockout worms to be wild-type. However, we wondered if the RNAi phenotype might be a subtle alteration of metabolic activity. We obtained Dr. Ahringers RNAi feeding clone from the UK HGMP Resource Centre and measured egg laying and defecation rates. Compared to control worms fed HT115 bacteria containing the empty feeding vector, ucp-4 RNAi fed young adults that were exposed beginning as L1 larvae laid eggs at a significantly slower rate, although preliminary data on defecation rate suggests no difference between these treatments. If the trends in these data hold, it would suggest that ucp-4 RNAi knockout worms are processing food at the same rate, but converting that energy into offspring at a reduced rate.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

P Askjaer et al. (2004) European Worm Meeting "IDENTIFICATION OF GENES REGULATING NUCLEAR ENVELOPE ASSEMBLY BY GENOME-WIDE RNAI"

V Galy, T Zimmermann, I Mattaj, M Gorjanacz, P Askjaer

[European Worm Meeting, 2004]

The nuclear envelope (NE) is essential for many cellular processes including regulation of gene expression, nuclear positioning and chromatin segregation during mitosis. In metazoans the NE disassembles when the cell enters mitosis and reassembles during anaphase or telophase to reestablish compartmentalization of the cell. Despite the importance of this process remarkably few genes have been identified, which are required for formation of a closed NE. One likely explanation is that overlapping and redundant pathways at least partially control NE formation: We and others (Liu et al., PNAS, 100:4598-4603) have found that while each of the two NE proteins emerin and MAN1 are dispensable in the worm co-depletion of the two proteins causes 100% embryonic lethality. To identify genes important for NE formation we have characterized an emerin deletion strain and use this strain in parallel with a control strain in a genome-wide RNAi screen to identify genes interacting with emerin. L1 larvae are incubated with RNAi-inducing bacteria from Julie Ahringer's RNAi library in 96-well plates enabling rapid screening of high numbers of samples. The individual wells are scored in three categories: wild type, adults without progeny (emb and ste), and larvae only (lva and lvl). In addition to visual analysis we have developed tools for automated scoring thereby standardizing data acquisition and providing documentation for each RNAi experiment. Data from our control strain allows us to compare our results with published data. Using a stringent threshold where only phenotypes with >80% penetrance are scored 13% of the RNAi constructs induce non-viable phenotypes, which is similar to other investigations. Furthermore, for previously identified essential genes we observe lethality or sterility in two thirds of the cases. We are currently retesting candidate genes in a secondary screen and will provide detailed information at the meeting.

Sylvia E J Fischer et al. (2002) European Worm Meeting "Characterization and identification of genes involved in transposon silencing."

Pawel Pasierbek, Nadine L Vastenhouw, Ronald H A Plasterk, Sylvia E J Fischer

[European Worm Meeting, 2002]

The transposon Tc1 is active in somatic cells of all C. elegans isolates. However, Tc1 activity in the germ line is restricted to certain strains: Tc1 is active in the germ line of Bergerac BO, but silenced in Bristol N2. Mori et al. showed that multiple genetic loci are involved in regulation of germ-line transposition (Mori et al., 1988). In an EMS screen for activation of Tc1 in the germ line of Bristol N2, 43 mutants were isolated that fall into two classes: mutants that are resistant to pos-1 RNAi, such as mut-7(pk204)(Ketting et al. 1999) and mut-14(pk738)(Tijsterman et al. 2002), and mutants that are wild type for pos-1 RNAi. The latter class consists of 20 mutants. These mutants are sensitive to RNAi directed against germline-expressed genes (pos-1, rba-2), as well as somatically-expressed genes (unc- 22). Furthermore, one of the mutants, mut-11 (pk724), was tested for gld-1 cosuppression, and found to be sensitive. Similarly, two previously isolated Tc1 mutators, mut-4(st700) and mut- 6(st702) are completely sensitive to both RNAi (Tabara et al., 1999; our data) and cosuppression (Ketting and Plasterk, 2000). RNAi-sensitive mutators do share some additional phenotypes with mut-7(pk204). Both RNAi- resistant and RNAi-sensitive mutators are thermo-sensitive sterile. DAPI-staining of the gonads several mutants shows a reduction of gonad size and the presence of univalents in diakinesis. Like mut-7, most RNAi-sensitive mutators show a high incidence of males. Classical genetic mapping of the Tc1-excision phenotype has placed the mut-13(pk766) mutation in the center of chromosome I. As an alternative approach to rapidly identify genes involved in transposon silencing in C. elegans, we are performing genome-wide screens using the RNAi feeding library to inactivate each C. elegans gene (in collaboration with Julie Ahringer, Cambridge, UK). We identified three putative mutator genes on chromosome I by scoring for Tc1 excision after feeding of unc- 22::Tc1 worms.