Thomas Boulin et al. (2008) C.elegans Neuronal Development Meeting "Reconstituting the levamisole-sensitive AChR of Caenorhabditis elegans in Xenopus laevis oocytes"

Pierre Paoletti, Marc Gielen, Janet Richmond, Thomas Boulin, Jean-Louis BESSEREAU

[C.elegans Neuronal Development Meeting, 2008]

Acetylcholine receptors sensitive to the nematode-specific cholinergic agonist levamisole (L-AChRs), are hetero-pentameric ligand-gated ion channels that mediate fast excitatory neurotransmission at C. elegans neuromuscular junctions (NMJs). The cell biological mechanisms and genes involved in the assembly, trafficking and synaptic clustering of levamisole-sensitive AChR start to be well understood. However, its basic pharmacological properties remain largely unknown due to the lack of a reliable heterologous expression system. We have succeeded in getting robust expression of the L-AChR in Xenopus oocytes by using information derived from C. elegans genetics. In vivo, at least eight genes are necessary to produce L-AChR: five genes (lev-1, lev-8, unc-29, unc-38, unc-63) encode AChR subunits and three genes encode proteins involved in intracellular trafficking and protein maturation (ric-3, unc-50, unc-74). We have co-injected the corresponding cRNAs into Xenopus oocytes and observed robust expression of the L-AChR 2 to 3 days after injection. Strikingly, each of the eight genes is required for efficient L-AChR expression in Xenopus oocytes. Acetylcholine elicits currents in the microA range, with fast activation kinetics and almost no desensitization. Similarly, robust responses are obtained for levamisole, which behaves as a partial agonist. d-Tubocurarine, MLA and hexamethonium, classical antagonists of AChR, inhibit the currents. Acetylcholine and levamisole ECs calculated in dose-response experiments are 25 microM and 10 microM, respectively. Surprisingly, nicotine, the prototypic agonist of ionotropic acetylcholine receptor, does not activate the L-AChR, but rather acts as a strong antagonist. In the future, we will use this new expression system to examine the molecular basis for the unusual pharmacological properties of L-AChR. In addition, the system will be useful to study the function of the novel gene molo-1 which acts as a positive allosteric modulator of L-AChR in vivo.

Virginie Hachet et al. (2006) European Worm Meeting "Centrosomes Dictate Timing of Mitotic Entry in C. elegans Embryos"

Pierre Gonczy, Virginie Hachet

[European Worm Meeting, 2006]

Virginie Hachet and Pierre Gonczy Mitosis is a tightly regulated process that allows equal repartition of the genetic material to daughter cells. Activation of the Cyclin B1/Cdk1 complex is key for initiating entry into mitosis. Intriguingly, in human cells, the active form of Cyclin B1/Cdk1 is detected first at centrosomes, the major microtubule organizing centre (MTOC) of animal cells. However, it is not known in any systems whether centrosomes are required for promoting mitotic entry. We set out to investigate the mechanisms underlying timing of mitotic entry, in the early C. elegans embryo. To this end, we developed a novel assay to monitor timing of mitotic entry with high spatial and temporal resolution. Using this assay, we showed that neither anterior-posterior (A-P) polarity, nor microtubules, dictate timing of mitotic entry. By contrast, we established that centrosome integrity is essential. Moreover, we found that the centrosomal Aurora-A kinase AIR-1 is essential for proper mitotic timing. Finally, by using appropriate mutant embryos, we could demonstrate that centrosomes are not only necessary but also sufficient to promote entry into mitosis. Our findings lead us to propose that centrosomes serve as integrative centres for mitotic regulators and thus dictate the timing of mitotic entry.