Structural and functional characterization of new complexes of known polarity determinants in Drosophila and vertebrates

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In this proposal we intend to further elucidate the role of PATJ (Pals1-associated Tight Junction protein) in the development of Drosophila melanogaster and we aim to extent our studies from the fly to mammals. In particular, how PATJ regulates apical-junctional Myosin dynamics and thereby support adherens junction stability in redundancy with other proteins is one of the key questions. Furthermore, we will investigate how PATJ is involved in the Hippo-signaling pathway controlling cell size and -proliferation in the fly and in mammals. For the mammalian system we will use in addition to cultured cells a conditional knock-out system for INADL (the gene encoding PATJ in the mouse). In this model we will analyze several epithelia and organs for defects regarding cell polarity and (mis)regulation of the Actin-Myosin cytoskeleton as well as for changes in cell size/proliferation and tissue/organ growth.In the second part we are going to investigate on the one hand which redundant mechanisms target the scaffolding protein Bazooka and its mammalian homologue PAR-3 to the apical junctions and to the apical cortex of Drosophila neural stem cells. On the other hand we will describe the structural and functional relationship between Bazooka/PAR-3 and aPKC. As we detected several (up to now unknown) phosphorylation sites in Bazooka/PAR-3 for aPKC, the physiological relevance of these phosphorylation events will be tested in the fly and in cultured mammalian epithelial cells. In addition a three-dimensional model of the Bazooka-aPKC complex using crystallography should be developed. From these experiments we hope to obtain a better understanding in how Bazooka/PAR-3 functions with respect to aPKC.