Designing a new class of fluorescent reporters for imaging dynamic cell signaling in live animals

摘要 蛋白水解酶和蛋白激酶控制着广泛的细胞过程。蛋白水解酶和蛋白激酶的失调 运动会导致许多疾病，包括癌症。此外，激酶信号的时间动力学可以 引发不同的细胞反应。对活体动物中的蛋白水解酶和激酶活性的动态成像，从基因上 编码的荧光报告是理想的，因为它们不需要外源分子，而且是非侵入性的。 尽管基于绿色荧光蛋白(GFP)的Förster共振能量转移(FRET)记者可以 用于成像培养细胞中的激酶信号动力学，在那里它们可以实现亚细胞分辨和 在体内使用基于FRET的激酶(和蛋白水解酶)报告是困难的 由于荧光团的荧光变化幅度很小。我们寻求设计、展示 并应用新类别的荧光记者，包括基于绿色荧光蛋白的记者，在激酶激活后， 通过多价蛋白质-蛋白质相互作用进行相分离并形成高度集中的液滴。我们的 该方法的灵感来自于最近的研究表明，多价相互作用推动蛋白质相分离 形成蛋白质液滴，使蛋白质浓缩10倍左右。基于相分离的激酶报告具有 大动态范围和高亮度都是因为荧光团集中在离散的斑点上。这 点状信号模式是独特的，在整个动物中很容易检测到，对于活体检测是可靠的 激活酶活性。记者还在秒到分钟的时间尺度上实现了快速动力学，并且是可逆的， 在转基因动物身上没有明显的毒性。为了进一步证明新的设计原则，我们的目标是 设计和应用基于相分离的激酶报告器来可视化过程中几个关键激酶的动态 动物发育和疾病。我们还将开发荧光蛋白水解酶记者来可视化动态 活体动物体内的细胞凋亡信号。此外，我们还将把绿色荧光激酶记者与 红外荧光半胱氨酸天冬氨酸氨基转移酶同时显示信号转导和细胞凋亡 研究它们与胚胎发育和组织的形态发生过程之间的关系 动态平衡。我们还将利用这些记者来研究大脑中动态的细胞信号网络 肿瘤发生学。

Abstract Proteases and kinases control a broad range of cellular processes. Dysregulation of protease and kinase activities causes many diseases including cancer. Furthermore, temporal dynamics of kinase signaling can elicit distinct cellular responses. To image dynamics of protease and kinase activity in live animals, genetically encoded fluorescent reporters are ideal because they require no exogenous molecule and are non-invasive. Although green fluorescent protein (GFP)-based Förster resonance energy transfer (FRET) reporters can be used to image dynamics of kinase signaling in cultured cells where they can achieve subcellular resolution and compartmentalized kinase signaling, in vivo use of the FRET-based kinase (and protease) reporters is difficult because of small magnitude of the fluorescence changes of the fluorophores. We seek to design, demonstrate and apply new classes of fluorescent reporters including the GFP-based reporters that, upon kinase activation, phase separate and form highly concentrated droplets via multivalent protein-protein interactions. Our approach is inspired by recent work showing that multivalent interactions drive protein phase separation to form protein droplets that concentrate the protein ~10 fold. The phase separation-based kinase reporter has both large dynamic range and high brightness because fluorophores concentrate in discrete punctae. This punctal signal pattern is distinctive and easily detectable in whole animals and robust for in vivo detection of kinase activity. The reporter also achieves fast kinetics at the second-to-minute timescale and is reversible, with no apparent toxicity in transgenic animals. To further demonstrate the new design principle, we aim to design and apply phase separation-based kinase reporters to visualize dynamics of several key kinases during animal development and disease. We will also develop fluorogenic protease reporters to visualize dynamic apoptosis signaling in live animals. Furthermore, we will combine the green fluorescent kinase reporters with the infrared fluorogenic caspase reporters to simultaneously visualize kinase signaling and apoptosis and investigate how they are correlated to morphogenetic processes during embryogenesis and tissue homeostasis. We will also apply these reporters to investigate dynamic cell signaling network during brain tumorigenesis.