Synaptic cell-adhesion molecules (sCAMs) are thought to mediate the formation of neuronal circuits by recognizing appropriate trans-synaptic partners and initiating the assembly of pre- and post-synaptic specializations. Of the sCAMs, neurexins are among the most extensively studied, and all neurexin family members have been identified as risk factors for autism and other neurodevelopmental disorders. The human genome contains three neurexin genes which, through alternative splicing, encode ~4000 long (alpha), medium (beta) and short (gamma) isoforms that differ in their extracellular domain but retain an identical intracellular domain. The C. elegans genome contains a single neurexin gene
nrx-1, which is expressed only as several long (alpha) and short (gamma) isoforms that also share a common intracellular domain. To evaluate whether
nrx-1 isoforms are differentially expressed across the C. elegans nervous system, we generated promoter reporters for the short (gamma) and long (alpha) isoforms, and found differential expression patterns. We are now using the NeuroPal tool to identify the specific neurons in which these isoforms are expressed. In addition, we have generated long and short isoform-specific knockouts of endogenously-tagged
nrx-1 to reveal the contribution of each isoform to overall synaptic expression. We have previously shown that a short (gamma) isoform of NRX-1 that doesn't include any canonical extracellular binding domains nonetheless localizes to presynaptic active zones and regulates presynaptic maturation and stability. We now show that expression of the intracellular domain alone, targeted to the membrane with a myristoylation sequence, can rescue the
nrx-1 null phenotype (although a cytosolic version does not), indicating that the membrane-bound NRX-1 intracellular domain is sufficient for mediating its presynaptic assembly functions. To identify mediators of NRX-1 localization at synapses, we performed a candidate screen of potential intracellular interactors using endogenously-tagged NRX-1 and identified the synaptic vesicle kinesin UNC-104, as well as active zone scaffold molecules SYD-1 and SYD-2. A deletion of the intrinsically disordered domain of SYD-2, shown previously to mediate its ability to undergo phase separation and thereby recruit additional active zone molecules, affected NRX-1 localization to the same degree as
syd-2 null mutants, implicating phase separation as a potential mechanism for NRX-1 recruitment to synapses.