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

摘要 蛋白酶和激酶控制广泛的细胞过程。蛋白酶和激酶调节异常 活动导致包括癌症在内的许多疾病。此外，激酶信号传导的时间动力学可以 引起不同的细胞反应。为了在活体动物中对蛋白酶和激酶活性的动态进行成像， 编码的荧光报道分子是理想的，因为它们不需要外源分子并且是非侵入性的。 虽然基于绿色荧光蛋白（GFP）的Förster共振能量转移（FRET）报告基因可以被用于 用于成像培养细胞中激酶信号传导的动力学，在那里它们可以实现亚细胞分辨率， 由于区室化的激酶信号传导，基于FRET的激酶（和蛋白酶）报告基因的体内使用是困难的 因为荧光团的荧光变化幅度小。我们寻求设计，展示 并应用包括基于GFP的报道基因的新类型的荧光报道基因，在激酶激活后， 相分离并通过多价蛋白质-蛋白质相互作用形成高度浓缩的液滴。我们 最近的工作表明，多价相互作用驱动蛋白质相分离， 形成蛋白质液滴，使蛋白质浓缩约10倍。基于相分离的激酶报告基因具有 因为荧光团集中在离散的点中，所以具有大的动态范围和高的亮度。这 点状信号模式是独特的，并且在整个动物中容易检测，并且对于体内检测 激酶活性报告基因还在秒到分钟的时间尺度上实现快速动力学，并且是可逆的， 在转基因动物中没有明显的毒性。为了进一步展示新的设计原则，我们的目标是 设计并应用基于相分离的激酶报告子，以可视化几种关键激酶在 动物的发展和疾病。我们还将开发荧光蛋白酶报告， 在活体动物中的凋亡信号传导。此外，我们将联合收割机将绿色荧光激酶报告基因与 红外荧光半胱天冬酶报告基因，以同时可视化激酶信号传导和细胞凋亡， 研究它们如何与胚胎发生和组织形成过程中的形态发生过程相关 体内平衡我们还将应用这些报告子研究脑内动态细胞信号网络 肿瘤发生

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.