Development of a functional nervous system requires correct and precise targeting of individual axons and dendrites to their proper destinations. We have cloned and characterized the
unc-69 gene, which is previously known to be critical for axon outgrowth and fasciculation. UNC-69 is a 108 amino acid protein and has no recognizable motif except a coiled-coil domain in its C-terminus. Surprisingly, we also isolated an
unc-69 allele in a forward genetic screen for mislocalization of a synaptic vesicle marker SNB-1::GFP. The synaptic GFP puncta in
unc-69 mutant are bigger and irregular in position compared to wild type. The effect of UNC-69 is specific to axons based on following observations: (1) SNB-1::GFP is not significantly mislocalized to dendrites in bipolar ASI sensory neurons; (2) odorant receptor ODR-10 is not prevented from targeting to the AWB cilia. Similarly, dendritic growth of sensory neurons in
unc-69 mutants is not affected, as evidenced from dye filling and GFP reporter assay. In addition, we constantly observe ectopic branches emanating from soma or axonal shafts, but seldomly from dendrites, in
unc-69 mutants. Thus UNC-69 appears to control protein trafficking and membrane extension of the nervous system in a polarized fashion. In a yeast-two-hybrid screen using UNC-69 as a bait, we found that the previously characterized axonal guidance molecule UNC-76 associates physically with UNC-69. UNC-76 is a 385 amino acid protein ubiquitously expressed in neurons of C.elegans , and is similar to human FEZ1. In vitro pull-down studies demonstrated that the interaction between UNC-69 and UNC-76 is specific. Furthermore, amino acids 281-299 of UNC-76 are both necessary and sufficient for interaction with UNC-69, and a point mutation of either L281P or L287P totally disrupts the interaction. We show that
unc-69 and
unc-76 act together to control size and position of the synaptic GFP puncta, as well as axonal outgrowth. A tempting hypothesis is that UNC-69 and UNC-76 function to prevent uncontrolled vesicular fusion, and to ensure precise targeting of (synaptic) vesicles and fusion with the plasma membrane, leading to proper axonal outgrowth and fasciculation.