Bertolin Giulia, Le Marchand Gilles, Tramier Marc
Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France;
Univ Rennes, CNRS, IGDR (Genetics and Development Institute of Rennes), UMR 6290, F-35000 Rennes, France.
J Vis Exp. 2020 Jul 30(161). doi: 10.3791/61611.
Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.
上皮癌通常以丝氨酸/苏氨酸激酶极光激酶A/AURKA的过表达为特征。AURKA是一种多功能蛋白，在苏氨酸288位点发生自磷酸化后被激活。AURKA的丰度在有丝分裂时达到峰值，在有丝分裂过程中它控制着有丝分裂纺锤体的稳定性和保真度以及有丝分裂的整体效率。尽管在结构层面已有充分研究，但缺乏对整个细胞周期中AURKA激活情况的持续监测。一种可能的解决方案是使用基因编码的荧光共振能量转移（FRET）生物传感器，以足够的时空分辨率深入了解AURKA的自磷酸化情况。在此，我们描述了一种构建检测苏氨酸288自磷酸化的FRET生物传感器的方案，以及如何在有丝分裂过程中追踪这种修饰。首先，我们概述了可能的供体/受体FRET对，并展示了AURKA FRET生物传感器在哺乳动物细胞中的可能克隆和插入方法。然后，我们提供了在定制装置上通过荧光寿命成像显微镜（FLIM）进行快速FRET测量的分步分析。不过，该方案也适用于其他可用的商业解决方案。我们通过考虑基于AURKA的生物传感器最合适的FRET对照，并强调未来可能的改进以进一步提高该工具的灵敏度来得出结论。
