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

在本文中，我们打算进一步阐明PATJ （Pals1-associated Tight Junction protein）在果蝇发育中的作用，并将我们的研究范围从果蝇扩展到哺乳动物。特别是，PATJ如何调节顶端连接肌球蛋白动力学，从而支持粘附体与其他蛋白质冗余连接的稳定性是关键问题之一。此外，我们将研究PATJ如何参与苍蝇和哺乳动物中控制细胞大小和增殖的hippo信号通路。对于哺乳动物系统，除了培养细胞外，我们还将使用INADL（小鼠中编码PATJ的基因）的条件敲除系统。在这个模型中，我们将分析几种上皮和器官的缺陷，包括细胞极性和肌动蛋白-肌球蛋白细胞骨架的（错误）调节，以及细胞大小/增殖和组织/器官生长的变化。在第二部分，我们将一方面研究支架蛋白Bazooka及其哺乳动物同源物PAR-3在果蝇神经干细胞顶端连接和顶端皮层的冗余机制。另一方面，我们将描述Bazooka/PAR-3和aPKC之间的结构和功能关系。由于我们在Bazooka/PAR-3中检测到几个（迄今为止未知的）aPKC磷酸化位点，这些磷酸化事件的生理相关性将在苍蝇和培养的哺乳动物上皮细胞中进行测试。此外，还应利用晶体学建立巴祖卡- apkc复合物的三维模型。通过这些实验，我们希望更好地了解Bazooka/PAR-3在aPKC方面的功能。

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.