Many neurons undergo rapid elongation of the selected neurite, a process known as axonal outgrowth. We found that human CRMP-2 (Collapsin response mediator protein-2) plays pivotal roles for axonal outgrowth. CRMP-2 is highly conserved from worms to mammals. In Caenorhabditis elegans ,
unc-33 encodes a homologue of CRMP-2. UNC-33 has three splicing forms with extension at N-terminus (UNC-33L, UNC-33M, and UNC-33S). All splicing forms are expressed in nervous tissue.To investigate the functions of UNC-33 during neuronal morphogenesis, we examined the neuronal morphology in several
unc-33 mutants. We found that one
unc-33 mutant,
e204 , showed lower penetrance of axonal outgrowth defects in D-type motoneurons, compared to other
unc-33 null mutants. We confirm expression of UNC-33 in
e204 by western blotting and immunostaining. In wild type N2, UNC-33 was localized at axonal process. Interestingly,
unc-33 (
e204 ) mutant protein is mislocalized from axonal process. These results suggested that
unc-33 (
e204 ) is a hypomorphic allele of the
unc-33 gene. To characterize the molecular lesions of UNC-33 caused by the
e204 mutation, we determined mutation site and found that the mutation in
unc-33 (
e204 ) mutant allele was a missense mutation with amino acid substitution of Asp (D) to Asn (N). The substituted residue is highly conserved in UNC-33/CRMP protein family. Furthermore, we constructed the UNC-33 mutant form corresponding to the mutant protein expressed in
unc-33 (
e204 ) mutant, namely UNC-33L D389N . When we introduced UNC-33L or UNC-33L D389N into
unc-33 null mutant, the expression of UNC-33L recovered abnormal behavior and axonal outgrowth defects in the
unc-33 mutant. UNC-33L also was localized at axonal processes. However, UNC-33L D389N could not rescue and was mislocalized as same as the original mutant.These results suggested that the localization of UNC-33 at axonal processes is important for neuronal morphogenesis and functions in C. elegans . At present, to examine the molecular mechanism of UNC-33 localization at axonal processes, we search mutants in which UNC-33 is mislocalized from axonal process.