The gene
unc-69 is critical for axonal outgrowth, targeting and fasciculation in C. elegans. Loss of
unc-69 function caused pleiotropic effects on axon outgrowth and fasciculation. A rescuing UNC-69::GFP protein is expressed throughout development in nearly all of the neurons and a subset of migratory cells including muscles, DTCs and excretory canals. UNC-69 is a 108 amino acid protein with no recognizable protein motif except a coiled-coil domain in its C-terminus. Both of the canonical alleles
unc-69(
e587) and
unc-69(
e602) are nonsense mutations inside the coiled-coil domain, and are possibly genetic nulls. We found
unc-69 homologues in C. briggsae, flies, Zebrafish, mice and humans based on sequence homology, and the coiled-coil domain is highly evolutionarily conserved with 74% identity across all species. In a previous yeast-two-hybrid screen using UNC-69 as a bait, we found that the previously characterized axon guidance molecule UNC-76 associates physically with UNC-69. UNC-76 is a 385 amino acid protein and is ubiquitously expressed in neurons of C. elegans (Bloom and Horvitz 1997). UNC-76 protein is similar to human FEZ1 (fasciculation and elongation proteins; zygin/zeta-1). in vitro GST pull-down studies demonstrated that the interaction between UNC-69 and UNC-76 is specific. We have furthered our investigation and found that amino acids 135-369 of UNC-76 are necessary for its interaction with UNC-69, whereas a fragment containing amino acids 135-243 is incapable of binding. Previous data has shown that amino acids 1-197 of UNC-76 direct exogenous proteins to axons of C. elegans (Bloom and Horvitz 1997). We propose that UNC-69 acts by integrating and transducing guidance cues to downstream effectors via its association with UNC-76, possibly through their respective coiled-coil domains. While the C-terminal portion of UNC-76 (amino acids 244-369) is responsible for its interaction with UNC-69, its N-terminus (amino acids 1-197) is required for targeting into the axons. We are performing a forward genetics screen for suppressors of the Unc-69 locomotion defect. We have screened over 1,000,000 haploid genomes and found at least 10 strong suppressors, many of which are dominant and are currently being mapped.