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上皮细胞和神经细胞的功能至关重要。在果蝇中，多pdz结构域蛋白Bazooka （Baz）是在上皮细胞和神经母细胞（中枢神经系统的干细胞）中建立顶基极性所必需的。我们最近的研究表明，Baz直接与果蝇蛋白激酶C （DaPKC）的非典型亚型结合，并且这两种蛋白相互依赖以实现正确的顶端定位。DaPKC功能缺失突变体在神经母细胞中表现为尖基极性缺失、上皮多层、细胞命运决定因素定位错误和纺锤体取向异常。总之，这些数据为控制上皮细胞和神经干细胞的顶基极性的进化保守机制的存在提供了强有力的证据。在该项目中，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具有重要的功能。我们将在体内用转基因果蝇和组织培养中检测几种通过体外诱变产生的DaPKC突变体的功能。此外，我们希望通过免疫亲和层析鉴定DaPKC的相互作用伙伴和磷酸化靶点，然后进行质谱分析。

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.