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

无需活细胞显微镜即可检测信号动态的新型生物传感器

• 批准号: 10337472
• 负责人: Jared E Toettcher
• 金额: $ 31.86万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10337472
• 关键词: Address; Adopted; Adult; Animals; Awareness; Biochemical; Biological Models; Biosensor; Cell Differentiation process; Cell Surface Receptors; Cells; Communication; Complex; Coupled; Cues; Cultured Cells; Defect; Detection; Development; Discrimination; Disease; Embryonic Development; Environment; Exhibits; Genes; Genetic Screening; Goals; Growth; Growth Factor; Homeostasis; Human Cell Line; Image; Individual; Label; Lead; Ligands; Light; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Membrane; Microscopy; Modality; Monitor; Mouse Strains; Mus; Mutation; Nutrient; Organ; Organogenesis; Output; Pathway interactions; Pattern; Phenotype; Phosphotransferases; Physiologic pulse; Physiology; Play; Proteins; Research Personnel; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Protein; Stress; Syndrome; System; TP53 gene; Technology; Testing; Time; Tissues; Transcriptional Activation; Transgenic Animals; Translational Repression; Travel; Variant; Work; base; cell type; chemical genetics; combinatorial; design; detector; extracellular; human disease; in vivo; live cell microscopy; network architecture; new technology; prevent; promoter; prototype; ras Proteins; recombinase; response; tissue regeneration; tool; tumor; tumorigenesis; wound healing

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.

项目摘要 每个单元都存在于复杂而不断变化的环境中。为了应对他们复杂的环境，牢房 已经发展了多种系统以感知外部线索（例如营养，压力或交流 来自相邻细胞的分子）并将这些信息存储在内部表示中。但是这个细节 内部表示仍然是神秘的。细胞使用哪些蛋白活性模式来表示 有关他们的环境的信息？这些模式是如何产生的，它们控制了什么命运？ 生长因子信号传导是理解细胞信号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的激活，激酶ERK 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），成人组织再生（它控制各个方面 伤口愈合）和癌症（生长因子信号基因中的突变驱动了不受控制的生长和 肿瘤发生）。由于其重要性，生长因子信号传导完好无损 解决。生物传感器现在可以实时和活细胞中监测ERK活动，使得能够 实验学家在生长因子信号传导中从一个细胞到另一个细胞的痕迹波动，跨组织和 不同的蜂窝环境。 使用ERK生物传感器的研究表明，先前未贴上的生长因子信号的复杂性 活动。 ERK不简单地从刺激上打开刺激，而是可以在细胞中快速脉动和脉动，或 即使是裸露的活动浪潮，可以在整个组织中传播。但是该领域还没有 了解ERK脉冲是否导致细胞采用不同的功能状态，或者脉冲本身是如何采用的 由细胞内部或细胞之间的生化网络生成。这种状况不是ERK独有的：脉冲 在许多其他信号通路中也被广泛观察到，并且通常对知识较少。 当前的建议旨在提供新的工具来研究信号脉冲以帮助其研究 细胞和活动物。我们发明了一种新技术 - 一种原型基因电路，充当ERK “脉冲检测器” - 这将使研究人员能够在无效成像的情况下研究ERK脉冲。这项技术 解决了一个重要的需求：目前，只能通过高分辨率显微镜检测到脉冲 细胞，限制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 在鼠标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，以用于研究 显微镜很难或不可能执行。成功完成这项工作将产生新的类别 生物传感器可以阐明复杂信号传导，并可能影响人类疾病。