A new class of biosensors for detecting signaling dynamics without live-cell microscopy

项目摘要 每一个细胞都生存在一个复杂多变的环境中。为了应对复杂的环境，细胞 已经进化出多种系统来感知外部线索（如营养、压力或交流 来自相邻细胞的分子）并将该信息存储在内部表示中。然而， 内部表征仍然是个谜。细胞用什么样的蛋白质活动模式来代表 关于环境的信息？这些模式是如何产生的，它们控制着什么样的命运？ 生长因子信号传导是理解细胞信号传导原理的重要模型系统，其中 细胞表面受体的激活与膜定位蛋白Ras，激酶Erk的激活相关联 和各种下游基因。生长因子信号传导在胚胎发育中起着至关重要的作用（其中 生长因子触发细胞分化）、成体组织再生（其中它控制细胞的各个方面）。 伤口愈合）和癌症（其中生长因子信号传导基因的突变驱动不受控制的生长， 肿瘤发生）。由于其重要性，生长因子信号传导在越来越高的水平上被深入研究。 分辨率生物传感器现在可用于实时监测Erk活性和活细胞， 实验学家追踪生长因子信号从一个细胞到另一个细胞的波动， 不同的细胞环境。 使用Erk生物传感器的研究揭示了生长因子信号传导中前所未有的复杂性 活动Erk不是简单地在刺激时从关闭变为打开，而是可以在细胞中快速地脉冲打开和关闭，或者 甚至表现出在整个组织上传播的活动行波。然而，该领域还没有 理解Erk脉冲是否导致细胞采取不同的功能状态，也不知道脉冲本身是如何 由细胞内或细胞间的生化网络产生。这种情况并不是Erk独有的：脉冲 在许多其他信号通路中也被广泛观察到，但通常知之甚少。 目前的建议旨在为研究信号脉冲提供新的工具，以帮助他们在培养的细胞中的研究。 细胞和活的动物。我们发明了一种新技术-一种原型基因电路， “脉冲探测器”-这将使研究人员能够在没有实时成像的情况下研究Erk脉冲。这项技术 解决了一个重要的需求：目前，脉冲只能通过高分辨率的活体显微镜来检测， 细胞，限制了可以研究它们的环境。在这里，我们建议开发我们的无成像生物传感器， 在小鼠和人类细胞系中快速部署，将其设计扩展到其他途径和信号传导 动力学，并建立表达生物传感器的转基因动物，用于在许多组织中进行研究， 显微术很难或不可能进行。这项工作的成功完成将产生一个新的类 生物传感器揭示了对人类疾病有潜在影响的复杂信号传导。

PROJECT SUMMARY Every cell exists in a complex and changing environment. To deal with their complex surroundings, cells have evolved diverse systems to sense external cues (such as nutrients, stresses, or communication molecules from neighboring cells) and store this information in an internal representation. Yet the details of this internal representation are still mysterious. What patterns of protein activity do cells use to represent information about their environment? How are these patterns generated, and what fates do they control? Growth factor signaling is an important model system for understanding principles of cell signaling, where activation of cell surface receptors is coupled to activation of a membrane-localized protein Ras, the kinase Erk and various downstream genes. Growth factor signaling plays crucial roles in embryo development (where growth factors trigger cells to differentiate), adult tissue regeneration (where it controls various aspects of wound healing), and cancer (where mutations in growth factor signaling genes drive uncontrolled growth and tumorigenesis). Owing to its importance, growth factor signaling is intensely studied at increasingly high resolution. Biosensors are now available to monitor Erk activity in real-time and in living cells, enabling the experimentalist to trace fluctuations in growth factor signaling from one cell to another across a tissue and in different cellular contexts. Studies using Erk biosensors have revealed previously-unappreciated complexity in growth factor signaling activity. Instead of simply turning from off to on upon stimulation, Erk may pulse on and off rapidly in cells, or even exhibit traveling waves of activity that propagate across entire swaths of tissue. Yet the field does not yet understand whether Erk pulses lead cells to adopt distinct functional states, nor how the pulses themselves are generated by biochemical networks inside or between cells. This state of affairs is not unique to Erk: pulses have also been widely observed in many other signaling pathways and are generally poorly understood. The current proposal aims to provide new tools for studying signaling pulses to aid their study in cultured cells and in living animals. We have invented a new technology – a prototype gene circuit that acts as an Erk “pulse detector” – which will allow researchers to study Erk pulses without live imaging. This technology addresses an important need: currently, pulses can only be detected by high resolution microscopy of living cells, limiting contexts where they can be studied. Here, we propose to develop our imaging-free biosensor for rapid deployment in mouse and human cell lines, to expand its design to other pathways and signaling dynamics, and to establish transgenic animals expressing the biosensor for studies in many tissues where microscopy is difficult or impossible to perform. Successful completion of this work will produce a new class of biosensors to shed light on complex signaling with potential impact on human disease.