The role of atypical protein kinase C in the control of cell polarity of neuroblasts and epithelia in Drosophila

极性的建立和维持对于上皮细胞和神经元细胞的功能至关重要。在果蝇中，多PDZ结构域蛋白Bazooka（Baz）是在上皮细胞和成神经细胞（中枢神经系统的干细胞）中建立顶-基底极性所必需的。我们最近发现，巴兹直接结合到果蝇蛋白激酶C（DaPKC）的非典型亚型，这两种蛋白质是相互依赖的正确的顶端定位。DaPKC的功能丧失突变体表现出顶端-基底极性的丧失、上皮细胞的多层化、细胞命运决定子的错误定位和成神经细胞中纺锤体的异常取向。总之，这些数据提供了强有力的证据，存在一个进化上保守的机制，控制上皮细胞和神经干细胞的顶端-基底极性。在拟议的项目中，我们希望研究DaPKC如何控制神经母细胞和上皮细胞极性的建立，以及DaPKC蛋白的激酶活性是否在功能上很重要。我们将在体内使用转基因果蝇和组织培养中检测通过体外诱变产生的几种DaPKC突变体的功能。此外，我们希望通过免疫亲和层析，然后通过质谱分析来确定DaPKC的相互作用伙伴和磷酸化靶点。

The establishment and maintenance of polarity is of fundamental importance for the function of epithelial and neuronal cells. In Drosophila, the multi-PDZ domain protein Bazooka (Baz) is required for establishment of apical-basal polarity in epithelia and in neuroblasts, the stem cells of the central nervous system. We have recently shown that Baz directly binds to the Drosophila atypical isoform of protein kinase C (DaPKC) and that both proteins are mutually dependent on each other for correct apical localization. Loss-of-function mutants of DaPKC show loss of apical-basal polarity, multilayering of epithelia, mislocalization of cell fate determinants and abnormal spindle orientation in neuroblasts. Together, these data provide strong evidence for the existence of an evolutionarily conserved mechanism that controls apical-basal polarity in epithelia and neuronal stem cells. In the proposed project we want to investigate how DaPKC controls the establishment of polarity in neuroblasts and epithelia and whether the kinase activity of the DaPKC protein is functionally important. We will assay the function of several mutant versions of DaPKC generated by in vitro mutagenesis both in vivo using transgenic flies and in tissue culture. In addition, we want to identify interaction partners and phosphorylation targets of DaPKC by immunoaffinity chromatography, followed by mass spectrometry analysis.