Characterization on PAR-3 in early Xenopus laevis development

Abstract

Polarized cell movements are essential to the cell rearrangements that occur during morphogenesis. In Xenopus, cell polarity is reflected in the directional cell intercalations that drive the morphogenetic movements characterizing gastrulation. While these cell behaviours are well described, the molecular mechanism underlying this cell polarity is unknown. PAR-3 is a multi-domain scaffolding protein and a key regulator of cell polarity. I have isolated a cDNA encoding Xenopus PAR-3 and generated several mutant constructs, each lacking a conserved domain. Initial characterization of GFP-tagged PAR-3 in A6 cells demonstrates localization to points of cell-cell contact in epithelial sheets, as well as at the leading edge of migrating cells. PAR-3 constructs lacking the CR1 or PDZ1 domain fail to compartmentalize properly and are found in the cytoplasm. Eliminating the PDZ3 domain resulted in a loss of contact inhibition. Mutation of the aPKC phosphorylation site created a membrane hyper-accumulation phenotype. Together these data suggest that the CR1 and PDZ1 domains mediate membrane compartmentalization that is modulated through aPKC phosphorylation, while the PDZ3 domain is required for contact inhibition. In embryos, PAR-3 is expressed throughout gastrulation and over-expression of PAR-3 inhibits blastopore closure indicating a requirement during gastrulation. Inhibition is relieved when the construct lacking the CR1 domain is over-expressed. PAR-3 was localized to the cell periphery in axial mesoderm. Localization was abolished with deletion of the CR1 domain indicating that membrane targeting of PAR-3 is required for gastrulation and this targeting is dependent on oligomerization of PAR-3. This investigation also suggests PAR-3 functions independent of the PAR complex in Xenopus embryos indicating involvement of a different PAR-3 signaling pathway.