Marie Gendrel, Dan Williams1, Bill Schafer2, Erik Jorgensen1, Janet Richmond3, Jean-Louis Bessereau Clustering neurotransmitter receptors at the synapse is critical for efficient neurotransmission. Most of the proteins involved in receptor clustering function through cytoplasmic interactions with a sub-synaptic scaffold. However, we previously demonstrated that clustering acetylcholine receptors (AChRs) at C.elegans neuromuscular junctions (NMJs) requires LEV-10, a type I transmembrane protein that contains protein-protein interaction domains in its extracellular region. Expression of the LEV10 ectodomain in muscle cells rescues the AChR clustering defects of lev10 mutants. In the wild type, LEV10 is localized in post-synaptic membranes at NMJs. In mutants that do not express AChRs, LEV10 is expressed but no longer clustered at the synapse. These results suggest that AChRs and LEV10 are part of a complex that interacts with additional synaptic components to be recruited or stabilized at the synapse.. lev-10 mutants are weakly resistant to levamisole, a nematode specific nicotinic agonist. To identify additional components of the AChR clustering machinery, we immuno-stained AChRs in weak levamisole-resistant mutants. We observed that AChR clusters are no longer detected in lev-9 mutants. Electrophysiological recording of body-wall muscle cells in lev9 mutants indicated that the overall response to levamisole was the same as in wild type. These data demonstrate the presence of functional receptors at the muscle cell surface. Interestingly, LEV10 clusters are no longer detected in lev9 mutants, suggesting that this gene might be required to localize both AChR and LEV10 at the synapse.. SNP mapping experiments localized lev9 in a region that contained a Mos1 insertion previously isolated in a screen for resistance to the acetylcholinesterase inhibitor aldicarb. We demonstrated by genetic complementation that ox177::Mos1 is a lev9 allele. In this strain, Mos1 is inserted in the first exon of a gene that encodes a putative secreted protein with multiple protein-protein interaction domains. lev9 identity was confirmed by single ORF rescue and identification of mutations in three independent lev9 alleles. Mosaic experiments demonstrated that lev-9 was required in muscle. Experiments are in progress to localize LEV9 at the subcellular level and to test possible physical interactions between LEV-9, LEV-10 and AChRs. LEV9 might be part of a putative extracellular scaffold involved in AChR clustering at the synapse.