How does atypical protein kinase C signalling regulate actomyosin-mediated force generation in polarised cells?

上皮细胞是极化的，极性驱动的PAR复合体的定位与肌动球蛋白的定位显著重叠。肌动球蛋白在收缩和细胞结构中的作用驱动着发育过程中的形态发生。PAR复合体效应非典型蛋白激酶C(APKC)与肌动球蛋白的调控有关，但其调控机制的全面性尚不清楚。APKC还与包括神经退行性变和癌症进展在内的疾病有关。因此，我提出了一种无偏筛选法来鉴定新的aPKC底物。使用对类似物敏感的aPKC，底物将被硫代磷酸化，纯化，通过质谱学鉴定，并在体外验证与aPKC的相互作用。将研究HITS，用荧光显微镜表征它们的正常定位和在急性aPKC抑制下的定位。遗传学方法也将被用来阐明驱动肌动球蛋白由蛋白激酶C调控的机制。果蝇模型系统将允许分析不同的细胞类型，从而能够识别上下文相关的aPKC活性。

Epithelial cells are polarised, and the localisation of the polarity-driving PAR complex overlaps with that of actomyosin significantly. Actomyosin's roles in contractility and cell structure drive morphogenesis during development. The PAR complex effector atypical protein kinase C (aPKC) is linked to actomyosin regulation, yet the full scope of regulatory mechanisms is unclear. aPKC has also been implicated in disease contexts including neurodegeneration and cancer progression. I therefore propose an unbiased screen to identify novel aPKC substrates. Using an analogue-sensitive version of aPKC, substrates will be thiophosphorylated, purified, identified through mass spectrometry, and interactions with aPKC validatedin vitro. Hits will be investigated, with their normal localisation and localisation under acute aPKC inhibition characterised using fluorescence microscopy. Genetic approaches will also be used to elucidate mechanisms driving actomyosin regulation by aPKC. The Drosophila model system will allow analysis of different cell types, enabling the identification of context-dependent aPKC activity.