Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD5 1EH, UK.
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD5 1EH, UK.
Development. 2019 Jan 29;146(2):dev170589. doi: 10.1242/dev.170589.
Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the time point of phenotypic analysis. This is particularly true when studying kinases that have pleiotropic functions and multiple substrates. neuroblasts (NBs) are rapidly dividing stem cells and an important model system for the study of cell polarity. Mutations in multiple kinases cause NB polarity defects, but their precise functions at particular time points in the cell cycle are unknown. Here, we use chemical genetics and report the generation of an analogue-sensitive allele of atypical Protein Kinase C (aPKC). We demonstrate that the resulting mutant aPKC kinase can be specifically inhibited and Acute inhibition of aPKC during NB polarity establishment abolishes asymmetric localization of Miranda, whereas its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC helps to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.
在循环细胞中使用遗传学研究蛋白质的功能很复杂，因为基因失活和表型分析之间常常存在时间延迟。当研究具有多种功能和多个底物的激酶时，情况尤其如此。神经母细胞 (NBs) 是快速分裂的干细胞，是研究细胞极性的重要模型系统。多种激酶的突变会导致 NB 极性缺陷，但它们在细胞周期的特定时间点的确切功能尚不清楚。在这里，我们使用化学遗传学并报告了非典型蛋白激酶 C (aPKC) 的类似物敏感等位基因的产生。我们证明，由此产生的突变 aPKC 激酶可以被特异性抑制，并且在 NB 极性建立过程中急性抑制 aPKC 会导致 Miranda 的不对称定位被消除，而在正常有丝分裂的时间范围内抑制其不会消除。然而，aPKC 通过在核膜破裂后使 Miranda 保持在顶端和侧皮质之外，有助于使 Miranda 的模式更加清晰。
