Jean-Louis Bessereau et al. (2002) European Worm Meeting "Characterization of Mos1-mediated mutagenesis."

Thomas Boulin, Jean-Louis BESSEREAU

[European Worm Meeting, 2002]

Mos1-mediated mutagenesis relies on the mobilization of the Drosophila transposon Mos1 in the C. elegans germline (Nature, 2001, 413, 70-74). Since Mos1 insertions are easily localized in the genome using inverse PCR, the transposon can be used as a molecular tag to rapidly identify a gene that has been mutated by a Mos1 insertion. The mobilization of Mos1 requires two extrachromosomal arrays. oxEx166[hsp::MosTransposase; unc-122::GFP] is the "enzyme array" which drives the expression of the transposase under the control of a heat shock promoter. oxEx229[Mos1; myo- 2::GFP] is the "substrate array" which contains multiple copies of the transposon. The two transgenes are maintained in independent strains because transposition efficiency decreases when double transgenic animals are propagated for many generations. Moreover, we observed that the substrate array can be "silenced" over time: a substrate array which had been propagated in the laboratory for about a year had a low transposition rate (10 6 % of F1s with at least one insert, n = 3 experiments). When the original version of oxEx229 was thawed and retested, we recovered high transposition rates (49 15 %, n=13 experiments). To measure the mutagenicity of Mos1, we performed three screens for mutants resistant to levamisole using the Mos1 transposon. Of 13,940 Mos1-mutagenised F1s, we identified 6 levamisole resistant mutants. Under similar screening conditions, we recovered 8 mutants from 1,710 EMS-mutagenised F1s. There are 6 loci which can be mutated to confer strong levamisole resistance, and the observed rate of EMS-induced mutations is consistent with the known mutagenicity of EMS and the target size for levamisole resistance. Therefore, Mos1 mediated mutagenesis is about 10 times less efficient than chemical mutagenesis. Mobilization of transposons can generate mutants that do not contain a copy of a transposon (hit and run' events). However, these events are rare with Mos1: among the mutants that were identified in different Mos1screens conducted in our laboratory, a Mos1 insertion was present in 14 of 15 mutated genes. In conclusion, Mos1 is a less efficient mutagen than EMS but the identification of a mutated gene is extremely fast. Before using Mos1 for mutagenesis, one must evaluate the trade-off between time spent screening for mutants versus the time spent mapping and rescuing a mutation.

Robert, Valerie J P et al. (2009) International Worm Meeting "MosLIB: A PCR-screenable Mos1 insertion library."

Robert, Valerie J P, Bessereau, Jean-Louis

[International Worm Meeting, 2009]

MosTIC (for Mos1 excision transgene-instructed gene conversion)1 provides a means to engineer the Caenorhabditis elegans genome. In MosTIC, transgene-instructed gene conversion occurs during the repair of a DNA double-strand break generated by the excision of a pre-existing Mos1 insertion located in the genomic region to engineer. Mos1 is a DNA transposon isolated in Drosophila mauritiana. In C. elegans, expression of the Mos transposase is sufficient to induce the mobilization of Mos1 from an extrachromosomal array into the genome2. Such genomic insertions are indispensable reagents to initiate MosTIC. Here, we present the development and the characterization of MosLIB, a Mos1 insertion library consisting of 40,000 independent clonal strains containing Mos1 insertions3. MosLIB is made of 719 pools of 48 to 72 clonal strains. Each pool is represented by a duplicated frozen nematode stock and the corresponding genomic DNA. We estimated that at least 80,000 Mos1 insertions are present in MosLIB, 65 percent of them being close enough to coding regions to be valuable reagents to engineer genes by MosTIC. To screen MosLIB, we first perform a single PCR on the DNA stock with one primer in Mos1 and one specific primer designed in the genomic region of interest. Once a positive signal is obtained and its identity confirmed by sequencing, we thaw one tube of the corresponding nematode stock and we make 20 to 40 pools of 20 animals which survived freezing. These pools are screened by PCR for the presence of animals carrying the insertion of interest and rounds of sib-selection are made until a single homozygous animal carrying the insertion of interest is isolated. To optimize the screening of MosLIB, we now aim at using high-throughtput sequencing technologies to map the Mos1 insertions present in MosLIB. We would like to thank Y. Duverger, J. Ewbank and members of the Bessereau''s lab for their help in the making of the library. 1.Robert, V. and Bessereau, J. L. Embo J 26, 170-83 (2007). 2.Bessereau, J. L., Wright, A., Williams, D. C., Schuske, K., Davis, M. W. and Jorgensen, E. M. Nature 413, 70-4 (2001). 3.Duverger, Y., Belougne, J., Scaglione, S., Brandli, D., Beclin, C. and Ewbank, J. J. Nucleic Acids Res 35, e11 (2007).

Bonneau, Benjamin et al. (2019) International Worm Meeting "Characterization of the mechanisms of adaptation to levamisole."

Bessereau, Jean-Louis, Bonneau, Benjamin, Jospin, Maelle

[International Worm Meeting, 2019]

At the neuromuscular junction, acetylcholine receptors (AChRs) mediate fast excitatory neurotransmission. In C. elegans, a class of AChRs is sensitive to the agonist levamisole. Prolonged exposure to levamisole induces irreversible worm paralysis. However, worms with mutations in genes involved in AChR biosynthesis or clustering are able to adapt to levamisole and recover their motility. Detailed analysis demonstrated that all worms first become hypercontracted and paralyzed. Wild type animals then relaxed and remained paralyzed while adapting worms remained hypercontracted and started to move after few hours. Accordingly, calcium measurement revealed that in adaptating mutants calcium concentrations remain high in body wall muscles all along levamisole exposure. Our research aims to decipher the mechanisms underlying this adaptation process and, in particular, to understand how muscle cells can remain functional when facing prolonged hypercontraction. Our results indicate that elevated Ca2+ concentration in the muscle could activate the Ca2+-dependent phosphatase tax-6/Calcineurin, which then play a key role in the adaptation process. Muscle-specific degradation of TAX-6 before levamisole exposure prevents the adaption of mutants that normally adapt. TAX-6 could in turn activate different pathways including a CREB-dependent transcriptional response as mutation of crh-1, a CREB1 homolog, impairs adaptation. TAX-6 also seems to control the levels of AChRs at the membrane, which is reduced during adaptation. Finally, we observed that mitochondria become fragmented in the muscle of non-adapting worms while they remain fused in adapting worms. Altogether these results suggest that muscle cells can be reprogramed at different levels to overcome prolonged activation and contraction

Rapti, Georgia et al. (2009) International Worm Meeting "A single immunoglobulin domain protein required for the localization of acetylcholine receptors at the C.elegans neuromuscular junction."

Rapti, Georgia, Richmond, Janet, Bessereau, Jean-Louis

[International Worm Meeting, 2009]

Acetylcholine receptors sensitive to the nematode-specific cholinergic agonist levamisole (L-AChRs) form clusters at the C. elegans neuromuscular junctions (NMJs) and their proper localization is critical for efficient excitatory neurotransmission. Here we demonstrate that oig-4 (one Ig domain-4) is a new gene required for L-AChR synaptic clustering. We have previously shown that L-AChR clustering requires two proteins, LEV-9 and LEV-10, which form an extracellular synaptic scaffold. LEV-9, a CCP-domain rich secreted protein, physically interacts with the ectodomain of LEV 10, a CUB-domain rich type 1 transmembrane protein. Interestingly, none of the proteins of the L-AChR/LEV-9/LEV-10 complex forms clusters at the synapse by itself, indicating that additional components are necessary for synaptic localization of this complex. lev-10 and lev-9 mutants show partial resistance to levamisole: they sense levamisole and hypercontract but do not die on drug concentrations lethal for wild-type animals. To identify new components of the L-AChR clustering machinery we performed a screen for mutants with partial resistance to levamisole after EMS mutagenesis and we isolated a new mutant allele, kr39. Using classical genetic mapping we identified a mis-sense point mutation in the predicted gene oig-4. We isolated a second oig-4 mutant allele in a non-complementation screen. In both mutants, levamisole-resistance is rescued by providing an oig-4 genomic fragment. Immunostaining of L-AChR subunits and in vivo imaging of knock-in strains with fluorescent L-AChR subunits demonstrate that L-AChRs do not localize properly at the NMJs of oig-4 mutants. Moreover, the LEV-10 protein is no longer detected at synapses of these mutants. However, presynaptic boutons are properly formed. OIG-4 is specifically required for the L-AChR clustering as the UNC-49 GABA receptor and the ACR-16 N-AChRs are clustered normally at NMJs of oig-4 mutants. oig-4 is predicted to encode a secreted protein with a single immunoglobulin (Ig) domain. We demonstrated that OIG-4 is expressed predominantly in the body wall muscle. Its muscle expression is sufficient to rescue the partial levamisole-resistance phenotype. A functional OIG-4-GFP protein is secreted when expressed by the muscle and it forms clusters at NMJs. Altogether, these results suggest that we have identified a new extracellular synaptic protein that might be part of the L-AChR/LEV-9/LEV-10 scaffold and may link this complex to local synaptic determinants.

Pierron, Marie et al. (2013) International Worm Meeting "Genetic analysis of cholinergic synaptogenesis in Caenorhabditis elegans."

Pierron, Marie, Bessereau, Jean-Louis, Pinan-Lucarre, Berangere

[International Worm Meeting, 2013]

At the Caenorhabditis elegans neuromuscular junction, acetylcholine receptors (AChRs) form post-synaptic clusters precisely facing acetylcholine release sites and mediate excitatory transmission. The number and the localization of these receptors are important to regulate the strength of synaptic transmission. To identify novel mechanisms involved in the formation and the maintenance of cholinergic synapses, we performed a novel genetic screen based on the direct visualization of fluorescently-tagged AChRs. Mutations causing abnormal AChR localization were identified using whole genome re-sequencing. Out of 29 mutants isolated from the screen 8 mutants had abnormal AChR distribution patterns that had not been described previously. For each mutant candidate genes were identified using whole genome re-sequencing. Causative mutations were confirmed by complementation test and rescue experiments. In some mutants, presynaptic defects (axonal outgrowth and branching) were most likely responsible for AChR distribution defects (mislocalized clusters or missing clusters). In other mutants, AChR cluster distribution was altered independently of presynaptic defects. Mutants from this group are currently being characterized.

Pinan-Lucarre, Berangere et al. (2011) International Worm Meeting "An ADAMTS-Like protein is required for acetylcholine receptor localization."

Bessereau, Jean-Louis, Robert, Valerie, Pinan-Lucarre, Berangere

[International Worm Meeting, 2011]

The C. elegans neuromuscular junction (NMJ) is a genetically tractable model to decipher the molecular mechanisms underlying synaptic formation. At the NMJ both heteromeric and homomeric ionotropic acetylcholine receptors (AChRs) mediate fast synaptic neurotransmission. The anthelmintic drug levamisole activates heteromeric AChRs (the levamisole-sensitive AChRs or L-AChRs) and causes muscle hypercontraction and death at high concentration. Screens for resistance to levamisole have been conducted for decades and identified genes required for biosynthesis, assembly, trafficking and localization of L-AChRs at the synapse. Specifically, screening for mutants that adapt after overnight exposure to levamisole identified 3 proteins, LEV-9, LEV-10 and OIG-4, which build an extracellular scaffold for clustering L-AChRs at cholinergic NMJs. However, how this scaffold localizes at synapses is not well understood. To directly identify novel genes required for proper localization of L-AChRs, we recently conducted a visual screen for abnormal localization of fluorescently-tagged L-AChRs. Specifically, the TagRFP sequence was introduced in the L-AChR subunit locus unc-29 using the MosTIC technique. Animals were mutagenized with EMS and the F2 progeny was screened for abnormal fluorescence pattern. We screened 3,000 haploid genomes so far and isolated 29 mutants. Half were resistant to levamisole and represented alleles of genes encoding L-AChR subunits or their scaffolding proteins. In addition, the screen identified novel targets that are not resistant to levamisole and that have no obvious locomotory defects. Among these novel genes, we characterized in more details a mutant displaying redistribution of L-AChRs at extrasynaptic areas of muscle cell membrane. By rough SNP mapping and whole genome sequencing, we identified a mutation in a gene coding for a non characterized ADAMTS-Like protein (A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif-Like protein). These proteins are predicted to be secreted and to interact with basal membrane components. An overview of mutants retrieved during the screen together with the ongoing analysis of the ADAMTS-Like mutant will be presented at the meeting. This work was supported in part by the Fondation pour la Recherche Medicale.

Richard, Magali et al. (2011) International Worm Meeting "Control of acetylcholine receptor maturation and ER homeostasis by a new transmembrane complex."

Boulin, Thomas, Richmond, Janet, Richard, Magali, Bessereau, Jean-Louis

[International Worm Meeting, 2011]

Nicotinic acetylcholine receptors (AChRs) are ligand-gated ion channels that have been widely conserved throughout evolution and are implicated in numerous physiological and pathological processes in humans. In C. elegans, levamisole activates AChRs present at the neuromuscular junction causing death at high concentrations. Using Mos1-mediated insertional mutagenesis, we performed a large screen for mutants partially resistant to levamisole. Among 69 resistant strains, we identified one mutant allele of the previously uncharacterized gene, which we tentatively named emc-6. emc-6(kr150) mutants rapidly paralyze on levamisole but survive and adapt to prolonged exposure to the drug. This decreased levamisole sensitivity can be rescued by providing an emc-6 genomic fragment. emc-6 encodes a 111 amino-acid protein containing two predicted transmembrane domains. It is widely conserved from plant to human, but remains uncharacterized in any species. Based on immunostaining, in vivo imaging, western-blot analysis and electrophysiological recording, we demonstrated that AChR expression was reduced by 80 percent in emc-6 mutants, yet the remaining functional AChRs were still clustered at neuromuscular junctions. Based on transcriptional reporter constructs, EMC-6 appears to be expressed ubiquitously. Using tissue-specific rescue experiments, we demonstrated that it acts cell autonomously in body-wall muscle to restore wild-type AChR function. emc-6(kr150) is likely a hypomorphic allele because kr150 over deficiency is lethal and emc-6 RNAi causes embryonic and larval lethality. Recently, the yeast ortholog of EMC-6 was identified as a member of the ER Membrane protein Complex (EMC). In Saccharomyces cerevisiae, loss of EMC leads to accumulation of misfolded proteins and activation of the Unfolded Protein Response (UPR). This complex is composed of six proteins highly conserved through evolution. Preliminary results of immunocytochemistry and colocalization experiments with known intracellular compartments markers indicate that C. elegans emc-6 might localize in ER. RNAi targeting of additional C. elegans EMC members causes developmental defects and activation of stress reporters. These results suggest that EMC is required for ER homeostasis in metazoans, and especially for proper folding or assembly of AChR subunits. Current progress on emc-6 function will be presented at the meeting. Specifically, AChR biosynthesis is being characterized as well as EMC function in UPR response.

Laine, Viviane et al. (2015) International Worm Meeting "Adaptation to cholinergic agonist levamisole, a new insight of AChR regulation at neuromuscular junctions."

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

[International Worm Meeting, 2015]

Myasthenia gravis is the most prevalent form of neuromuscular transmission disorders. This pathology, characterized by a fluctuating muscle weakness and an excessive fatigability, is often due to a downregulation of the acetylcholine receptors (AChR) expressed at the surface of muscle cells. Current treatments use acetylcholinesterase inhibitors to restore efficient neuromuscular transmission. Unfortunately these inhibitors can sometimes lead to a cholinergic overstimulation, called cholinergic crisis: a respiratory failure can be observed as muscles stop responding to acetylcholine. To prevent cholinergic crises, alternative therapeutic strategies relying on the modulation of AChR metabolism or function are considered. So far, however, only few cellular factors are known to regulate AChRs. As these receptors are conserved in many organisms, we use the model organism Caenorhabditis elegans to better understand the regulation mechanisms of AChRs at excitatory neuromuscular junctions.As levamisole-sensitive AChRs (L-AChRs) do not desensitize to the cholinergic agonist levamisole, prolonged muscle activation with this drug induces fast worm paralysis and death in a few hours. Yet some mutants are able to recover locomotion while they are exposed to levamisole. This phenotype, referred as levamisole-mediated adaptation, is observed in mutants of L-AChR regulators. Thus the levamisole-mediated adaptation provides a suitable experimental approach to investigate pathways involved in AChR regulation.As AChRs are permeant to calcium we monitored the Ca2+ time course over adaptation using a GCaMP indicator expressed in muscle cells. We showed that the long-term adaptation is determined by early variations of Ca2+ levels in muscle cells. We elaborated a model for levamisole-mediated adaptation, in which the AChR cluster signaling is decoupled from the downstream mechanisms leading to intracellular Ca2+ variations. We suggest that a negative modulator being part of the clusters inhibits AChR signaling pathway. Finally, using a large-scale genetic screen, we isolated several mutants displaying a levamisole-adaptation phenotype. Among them, a shorter product of lev-10 gene, described in the clustering of L-AChRs at synapses, discloses a role of LEV-10 at the interface between scaffolding components and Ca2+ signaling pathways.

Richard, Magali et al. (2009) International Worm Meeting "A visual screen for abnormal localization of cholinergic receptors tagged using homologous recombination."

Robert, Valerie, Duboin, Aurelien, Bessereau, Jean-Louis, Richard, Magali, Studer, Vincent

[International Worm Meeting, 2009]

Ionotropic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that have been conserved throughout evolution. At the C. elegans neuromuscular junctions (NMJ) two types of AChRs mediate excitatory neurotransmission: N-AChRs, likely homomeric channels composed of the ACR-16 subunits, which are sensitive to nicotine, and L-AChRs, heteropentameric receptors activated by the drug levamisole. Several components involved in L-AChR formation and function have been identifed in the past using forward screens for mutants with decreased sensitivity to levamisole. However, resistance to levamisole is quite an indirect index of the numerous molecular mechanisms controling AChR composition, assembly, trafficking and localization. To study the biology of L-AChRs, we propose a novel approach based on the in vivo vizualization of tagged AChRs. Using the MosTIC technique recently developped in our laboratory, we have introduced, by homologous recombination, different fluorescent tags into the endogenous locus unc-29, which encodes one L-AChR subunit. We then used two different approaches to inactivate gene function (i) a chemical mutagenesis using EMS (ethylmethane sulfonate), (ii) RNA interference (RNAi) targeting 1300 genes selected for putative role in synaptic function. First, we will present mutants isolated in the EMS screen for AChRs clusters disappearance at the nerve ring. Second, we will describe a RNAi screen, for abnormal localization of AChRs at the NMJ, and the characterization of some putative candidates in which AChRs clusters seem to be altered. Third, we will demonstrate how microfluidic devices can improve the efficiency of visual screens.

Zhan, Hong et al. (2013) International Worm Meeting "Towards correlated localization of synaptic proteins at light and electron microscopy using a new generation of quantum dots."

Nasilowski, Michel, Bessereau, Jean-Louis, Stigloher, Christian, Dubertret, Benoit, Zhan, Hong

[International Worm Meeting, 2013]

At the active zone (AZ) of chemical synapses depolarization of presynaptic terminals triggers the opening of Voltage-Dependent Calcium Channels (VDCCs). The rise of calcium concentration that can trigger the fusion of synaptic vesicles (SVs) is restricted to nanodomains within less than a 100 nm from the VDCCs. Therefore, both the number and the distribution of VDCCs are crucial parameters that control synaptic efficiency. Using electron tomography we previously demonstrated that SVs docked at the plasma membrane are retained in close contact with the dense projection of C. elegans neuromuscular junctions. However, the relative distribution of docked SVs and VDCCs at these synapses is still enigmatic. To localize VDCCs in 3D with a nanometer resolution we are implementing a novel approach combining in vivo labeling of VDCCs via genetically-encoded extracellular epitope tags and electron tomography. In a first step we genetically introduced a GFP tag at the extracellular N-terminus of UNC-36, the a-2-d subunit of VDCCs, without impairing channel function. We then use an improved generation of multishell quantum dot (QD) as both fluorescent and electron dense probe. QDs are coupled to anti-GFP antibodies and injected in the pseudoceolomic cavity of C. elegans to label the VDCCs present at NMJs. In order to have access to near-to-native ultrastructure, worms are instantly immobilized by high pressure freezing and subsequently freeze substituted. Worms are imaged with light microscopy just before freezing, which provides a means to later image region of interest by transmission electron microscopy and visualize QD distribution. Using electron tomography we should eventually get access to the tridimensional distribution of VDCCs at the active zone with a few nanometer resolution. Current progress will be presented at the meeting.