Abstract 899: A Structure/Function Analysis of Zebrafish Nagie oko/Mpp5, a Regulator of Heart Morphogenesis
The establishment and maintenance of cell polarity is essential for cardiac morphogenesis in zebrafish. However, a systematic approach to dissect molecules implicated in apical junction formation has not yet been attempted in vertebrates. We performed a structure/function analysis of nagie oko (nok)/MAGUK p55 subfamily member 5 (mpp5), a main cell polarity regulator, to uncover whether a functional apical tight junction (TJ) - and associated with it Crumbs-PATJ and Par6-PRKCi-Par3 protein complexes - is essential for heart morphogenesis. The deletion constructs that we generated include forms that encode different Myc-tagged deletion mutants: NokΔECR1 (abolishes binding to Par6-PRKCi-Par3), NokΔL27 (abolishes binding to Lin-7 and PATJ), NokΔL27N (abolishes binding to Patj), NokΔPDZ (abolishes binding to the transmembrane receptor Crumbs), and the NokΔC-terminus [including the Guanylate kinase domain (NokΔC)-direct binding partners to this protein-protein interaction domain are currently unknown]. Based on the fact that nok/mpp5 mutants can be completely rescued by injection of wt nok/mpp5 mRNA, the function of the deletion proteins was assessed by mRNA injection and the subcellular localization of each deletion protein was characterized. We found that the PDZ domain (association with the Crumbs intracellular tail) is essential for correct function and apical localization of Nok/Mpp5 to the tight junction. Similarly, the C-terminal GUK domain is essential for correct function, however, is not required for apical membrane association. In contrast, NokΔL27 and NokΔECR1 deletion proteins are functional which suggests the binding of Nok/Mpp5 to Patj/Lin-7 and Par6-aPKC is not essential in the context of early vertebrate development. This observation changes our understanding of the relevance of in vitro protein-protein interaction data and suggests that Nok/Mpp5 mainly functions to stabilize the apical localization of Crumbs and that the Par6-aPKC complex is tethered to the Crumbs complex via alternative binding sites. Together, our study may potentially indicate the relevance of some genes for human congenital heart disease.