unc-73 mutants have a variety of defects in axon guidance and cell migration. The axons of many neurons fail to reach their targets and often travel along abnormal pathways. The strongest loss of function allele of
unc-73 exhibits maternally rescued embryonic and larval lethality. The largest UNC-73 isoform of 2,463 a.a. contains, in order, eight spectrin-like repeats, two dbl homologous (DH)/ pleckstrin homologous (PH) tandem domain elements that flank an SH3 domain, an Ig domain and a FnIII domain. Smaller UNC-73 isoforms also exist. Published experiments suggest that UNC-73 function involves signaling through the Rac pathway to regulate the actin cytoskeleton in migrating cells and growth cones. The
unc-73(
rh40) mutation (S1216F) is located in a highly conserved region of the DH-1 domain. This mutation eliminates the RacGEF and actin polymerization activities of the DH-1 domain.
unc-73(
rh40) animals were screened for suppression of the Unc phenotype following mutagenesis with EMS. A single nonUnc
rh40 suppressor was identified in a screen of 200,000 F1 animals and 120,000 haploid genomes in the F2 generation. This suppressor is dominant and tightly linked to
unc-73. Sequencing of the
unc-73 gene in the suppressor strain revealed that the suppressor mutation (L1269F) is located close to the
rh40 mutation within the DH-1 domain of
unc-73. Examination of the 3D structure of the DH domain indicates that the suppressor mutation may structurally compensate for the defect caused by the
rh40 mutation. The effect of the suppressor mutation on RacGEF and actin polymerization activity was tested in vitro. The introduction of the (L1269F) suppressor mutation into a DH-1 domain containing the (S1216F)
rh40 mutation restored the actin polymerization activity of the DH-1 domain as tested in Rat2 cells. The in vitro RacGEF activity of the S1216F/L1269F DH-1 domain, however, was not restored. Additional genetic screens are underway in the hope that genes that interact with
unc-73 will be identified. Studies of the human UNC-73 homologue, Trio, have revealed a potential interaction between Trio and the PTPase LAR. Genetic evidence of an interaction between
unc-73 and a C. elegans LAR-like gene,
ptp-1 (M. Hentgartner, p.c.), will be presented.