Neurotransmitter receptor clustering at the synapse is critical for efficient. neurotransmission. We have identified a novel secreted protein, LEV-9, which is a. component of an extracellular scaffold required for levamisole-sensitive acetylcholine. receptor (L-AChR) clustering at C. elegans neuromuscular junctions (NMJs).. To unravel L-AChR clustering mechanisms, we undertook a genetic strategy using. levamisole, a nematode-specific cholinergic agonist that activates L-AChRs present at. NMJs. We previously demonstrated that partial resistance to levamisole could be the. consequence of a clustering defect.
lev-9 mutants were initially isolated by Jim Lewis as. weakly resistant to levamisole but remained uncharacterized. We cloned
lev-9 by SNP-. mapping and by characterizing a mutant generated by Mos1 mutagenesis.
lev-9 encodes a. predicted secreted protein, which contains an N-terminal protease modulatory domain and 8. protein-protein interaction domains. Mosaic analysis and tissue-specific rescue. demonstrated that
lev-9 is required in muscle.. To characterize the subcellular distribution of LEV-9, we engineered the
lev-9 locus by. homologous recombination using the MosTIC technique to introduce a short T7 tag at the. N-terminus of the protein. Immunostaining using anti-T7 antibodies indicates that LEV-9 is. a synaptic protein which co-localizes with L-AChRs at NMJs.. In
lev-9 mutants, L-AChRs are no longer detected by immunostaining, whereas GABA. receptors and nicotinic AChRs (ACR-16) are still present and clustered. Yet, western blot. analysis demonstrated that L-AChRs are still expressed at a wild-type level.. Electrophysiological recordings of body-wall muscle cells in
lev-9 mutants confirm that. L-AChRs are present at the muscle cell surface at levels similar to wild type but their. concentration is strongly reduced at the synapse. Hence, LEV-9 is necessary to localize L-. AChRs at the synapse.. We previously demonstrated that clustering of L-AChRs at NMJs requires the ectodomain. of LEV-10, a type I transmembrane protein expressed in muscle and clustered at synapses.. Interestingly, LEV-10 clusters are no longer detected in
lev-9 mutants, yet the protein is. still expressed. Reciprocally, we demonstrated that in mutants that do not express LEV-10,. LEV-9 is no longer detected at the synapse. Together, these results suggest that LEV-9 is. required to localize both L-AChRs and LEV-10 at the synapse.. To test potential interactions between LEV-9 and LEV-10, we expressed both proteins in a. heterologous system and demonstrated a direct interaction between LEV-9 and LEV-10.. Together, these results strongly suggest that LEV-9 and LEV-10 are part of a previously. unknown extracellular scaffold required for L-AChR clustering at chemical synapses.