Polarization of the C. elegans zygote along the anteriorposterior axis depends on cortically enriched (PAR) and cytoplasmic (MEX-5/6) proteins, which function together to localize determinants (e.g. PIE-1) in response to a polarizing cue associated with the sperm asters. Using timelapse microscopy and GFP fusions, we have analyzed the localization dynamics of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 in wild-type and mutant embryos. These studies reveal that polarization involves two genetically and temporally distinct phases.

Caenorhabditis elegans embryonic polarity requires the asymmetrically distributed proteins PAR-3, PAR-6 and PKC-3. The rho family GTPase CDC-42 regulates the activities of these proteins in mammals, flies and worms. To clarify its mode of action in C. elegans we disrupted the interaction between PAR-6 and CDC-42 in vivo, and also determined the distribution of GFP-tagged CDC-42 in the early embryo. Mutant PAR-6 proteins unable to interact with CDC-42 accumulated asymmetrically, at a reduced level, but this asymmetry was not maintained during the first division. We also determined that constitutively active GFP::CDC-42 becomes enriched in the anterior during the first cell cycle in a domain that overlaps with PAR-6. The asymmetry is dependent on PAR-2, PAR-5 and PAR-6. Furthermore, we found that overexpression of constitutively active GFP::CDC-42 increased the size of the anterior domain. We conclude that the CDC-42 interaction with PAR-6 is not required for the initial establishment of asymmetry but is required for maximal cortical accumulation of PAR-6 and to maintain its asymmetry.

PAR-6 is a conserved protein important for establishment and maintenance of cell polarity in a variety of metazoans. PAR-6 proteins function together with PAR-3, aPKC and CDC-42. Mechanistic details of their interactions, however, are not fully understood. We studied the biochemical interactions between C. elegans PAR-6 and its binding partners and tested the requirements of these interactions in living worms. We show that PB1 domain-mediated binding of PAR-6 to PKC-3 is necessary for polarity establishment and PAR-6 cortical localization in C. elegans embryos. We also show that binding of PAR-6 and PAR-3 is mediated in vitro by a novel type of PDZ-PDZ interaction; the βC strand of PAR-6 PDZ binds the βD strand of PAR-3 PDZ1. However, this interaction is dispensable in vivo for PAR-6 function throughout the life of C. elegans. Mutations that specifically abolish conventional ligand binding to the PAR-6 PDZ domain also failed to affect PAR-6 function in vivo. We conclude that PAR-6 binding to PKC-3, but not to PAR-3 nor to a conventional PDZ ligand, is required for PAR-6 cortical localization and function in C. elegans.