Synapse Construction In C Elegans

Specification and assembly of synapses is a highly coordinated and regulated process. Knowledge of the position and connectivity of all C. elegans neurons makes it a highly useful organism for studying the underlying mechanisms that control synapse formation. Using cell-specific promoters and fluorescently-labeled synaptic vesicle proteins, we are able to monitor synapse formation in subsets of C. elegans neurons. Close observation of synapse formation in a single posterior motorneuron (DA9) led to the identification of a mutation in the alternative splicing regulator mbl-1 that changes the synaptic pattern. The cholinergic motorneuron DA9 is required for backwards locomotion and forms ~25 synapses onto both inhibitory neurons and body wall muscles in the dorsal nerve cord (DNC) of the worm. We found that the 10 most distal synapses of DA9 fail to form in mbl-1 mutants, visualized with the synaptic vesicle-associated protein RAB-3 and the active zone proteins SYD-2/liprin-α and UNC-10/Rim. In addition, some RAB-3 mis-localizes to the dendrite of DA9 and animals have a backwards locomotion defect consistent with a loss of synapses onto dorsal body wall muscles. mbl-1 is a member of the conserved MBNL (Muscleblind like) family of CCCH zinc-finger RNA binding proteins that regulate alternative splicing of target genes by directly binding to target mRNA. In the human disease myotonic dystrophy type 1 (DM1), a progressive muscular dystrophy, sequestration of MBNL proteins in nuclear foci leads to altered splicing of downstream genes. Mis-splicing of several genes is responsible for the muscular and cardiac symptoms present in individuals with DM1. Most work on the MBNL proteins has focused on their role in muscle morphogenesis and maintenance. However, C. elegans mbl-1 is expressed in a subset of motorneurons including DA9 and is required cell autonomously in these neurons to regulate proper synapse formation. Post-synaptic and muscle markers were unaffected in mbl-1 mutant animals. Thus, our work demonstrates that mbl-1 also functions in neurons to regulate synapse formation. In a separate set of experiments, we identified a new mutation in the coding region of the touch cell-specific beta-tubulin, mec-7(wy116) that causes a defect in synapse formation in the mechanosensory neuron PLM. Previous studies have shown that mec-7 is expressed exclusively in the six touch neurons of C. elegans and is required for sensing light touch. Our mec-7 mutation leads to a loss of synaptic vesicle accumulation at PLM synaptic sites in the ventral nerve cord and synaptic vesicles are visible at ectopic locations along the lateral axon of PLM. Localization of the synaptic proteins VAMP and GIT-1 is also defective in our mutant, but neuronal morphology is wild-type. mec-7(wy116) is mildly Mec, but other alleles of mec-7 (e1506, e1527) do not phenocopy the synaptic vesicle localization defect. mec-7(wy116) is a missense mutation that alters a highly conserved Thr at position 409 to Ile. Crystal structures of tubulin indicate that this residue is on the face of tubulin that interacts with kinesin motor. Because we see synaptic vesicles along the lateral axon of PLM, we believe that kinesin-mediated vesicle transport is less efficient in mec-7(wy116) mutants.