This is consistent with the absence of β-neurexins from the invertebrate genomes of Drosophila and C. elegans ( Tabuchi and Südhof, 2002). We next determined the subcellular localization of endogenous ApNRX by immunocytochemical analysis by using an affinity-purified polyclonal antibody generated against the cytoplasmic tail region of ApNRX (Figure S1).

In isolated sensory neurons, ApNRX immunostaining was distributed along neurites in a punctate pattern (Figure 2C). The punctate appearance of endogenous ApNRX cluster is similar to the distribution reported for endogenous neurexin in mammals (Dean et al., 2003). Interestingly, clusters of ApNRX also appear to be distributed along the neurites of the motor neuron in sensory-to-motor cocultures (Figure 2D). When GFP was expressed in sensory neurons as a whole-cell marker, it became

3-Methyladenine manufacturer clear that some ApNRX clusters do not colocalize with the neurites of the sensory neuron outlined by GFP fluorescence; thus they presumably are located on the motor neuron. These ApNRX clusters in the motor neuron may represent a Selleckchem SAHA HDAC pool of ApNRX in postsynaptic compartments since there is a significant amount of neurexin in postsynaptic compartments in mammals (Taniguchi et al., 2007). Moreover, it became clear that some ApNRX clusters overlap with some presynaptic sensory neuron varicosities in contact with the major receptive surface of the postsynaptic motor neuron where the majority of functionally competent synaptic connections are found in culture (Figure 2D, Kim et al., 2003). These ApNRX clusters overlapping with presynaptic varicosities may represent enrichment of ApNRX in presynaptic sensory neuron varicosities, clustering

of ApNRX in postsynaptic motor neuron in apposition to presynaptic sensory neuron varicosities, or perhaps both. Since many known functions of neurexin and neuroligin require their binding to each other across the synaptic cleft, we set out to find whether ApNRX and ApNLG also bind to each other. To address this point, we generated Ig fusion constructs that contain the extracellular domain of either HA-tagged found ApNLG or VSV-G-tagged ApNRX with the Ig Fc at their C-termini (ApNLG-Fc and ApNRX-Fc). We then incubated soluble ApNRX-Fc with cell lysates prepared from GFP-ApNLG transfected HEK293 cells and soluble ApNLG-Fc with cell lysates prepared from GFP-ApNRX transfected HEK293 cells, respectively. We found that ApNRX-Fc specifically binds to GFP-ApNLG, but not to GFP control (Figure 3A), and conversely, ApNLG-Fc binds to GFP-ApNRX, but not to GFP control (Figure 3B). These results provide evidence that recombinant ApNRX and ApNLG bind to each other, as is the case for their vertebrate counterparts. Next, we wondered whether endogenous ApNRX and ApNLG colocalize at the synapse. Thus, we immunostained sensory-to-motor neuron cocultures with both ApNRX and ApNLG antibodies.