The N-Terminus of UNC-53 Interacts with Regulators of the Actin Cytoskeleton

The unc-53 gene is required for the guidance and extension of a subset of cells along the anterior-posterior axis of Caenorhabditis elegans, including several neurons, the sex muscles and the excretory canals. Immunofluorescence of adult hermaphrodites with antisera raised against the calponin homology (CH) domain in the amino terminus of UNC-53 stains the excretory canals, vulva and coelomocytes, suggesting that the full length UNC-53 protein is required in these cells after migration and outgrowth. Staining in n152 and n166 mutants indicates that these animals express truncated UNC-53 products. In order to identify proteins that interact with UNC-53, a yeast two-hybrid screen was conducted. Using the N-terminal CH domain as 'bait' and a C. elegans cDNA library as 'prey', 18 strong candidate interactors were identified. Seven of these correspond to the C. elegans genomic locus B0336.6, which encodes a SH3 containing protein suggesting a role in signal transduction. The SH3 domain of the Drosophila homologue, Abi, binds to the polyproline motif of Abelson tyrosine kinase and the yeast homologue, Ysc84p, is thought to couple the actin cytoskeleton to the endocytic machinery. Four candidate cDNAs encode C. elegans REF(2) P like protein that contains a zinc finger motif of the ZZ type found in the cytoskeletal protein dystrophin, and a ubiquitin associated domain. The yeast homologue, verprolin, is an actin binding protein that presumably modulates polarization of the actin cytoskeleton. The yeast protein interaction database identified yeast verprolid/C. elegans T12G3.1 and yeast Ysc84p/Drosophila Abi/C. elegans B0336.6 as interactors with yeast Sla1p, a multifunctional protein that couples the yeast endocytic machinery to proteins regulating actin dynamics. These data suggest that UNC-53, together with verprolin/T12G3.1, Ysc84p/Abi/B0336.6 and Slalp may form a complex at the cytoskeleton that stabilizes or promotes actin polymerization. The remaining 7 candidates define molecules with diverse cellular activities. Interestingly, none of the candidate UNC-53 interactors have been discovered in previous genetic screens aimed at identifying genes involved in cell migration and outgrowth.