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: 10709472
• 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/10709472
• 关键词: Address; Adopted; Adult; Animals; Awareness; Biochemical; Biological Models; Biosensor; Cell Differentiation process; Cell Surface Receptors; Cells; Chemicals; 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; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Measures; Membrane; Microscopy; Modality; Monitor; Mouse Cell Line; Mouse Strains; Mutation; NF-kappa B; 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; cell fixing; cell type; combinatorial; design; detection platform; detector; dynamic system; extracellular; human disease; improved; in vivo; invention; live cell microscopy; network architecture; new technology; prevent; promoter; prototype; ras Proteins; recombinase; response; success; 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.

项目概要 每个细胞都存在于复杂多变的环境中。为了应对复杂的环境，细胞 已经进化出不同的系统来感知外部线索（例如营养物质、压力或沟通） 来自邻近细胞的分子）并将这些信息存储在内部表示中。然而这件事的细节 内部表征仍然是神秘的。细胞用什么模式来代表蛋白质活性 有关其环境的信息？这些模式是如何产生的，它们控制着什么命运？ 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 以及各种下游基因。生长因子信号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（它控制着 伤口愈合）和癌症（生长因子信号基因的突变导致不受控制的生长和 肿瘤发生）。由于其重要性，生长因子信号传导受到越来越多的深入研究 解决。生物传感器现在可用于实时监测活细胞中的 Erk 活动，从而使 实验者追踪组织中从一个细胞到另一个细胞的生长因子信号的波动 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以前未被认识到的复杂性 活动。 Erk 不是在刺激时简单地从关闭变为打开，而是可以在细胞中快速打开和关闭，或者 甚至表现出在整个组织中传播的活动行波。然而该领域还没有 了解 Erk 脉冲是否导致细胞采取不同的功能状态，以及脉冲本身如何 由细胞内部或细胞之间的生化网络产生。这种情况并非 Erk 所独有：豆类 在许多其他信号通路中也被广泛观察到，但人们普遍对其知之甚少。 当前的提案旨在提供用于研究信号脉冲的新工具，以帮助他们在培养中进行研究 细胞和活体动物中。我们发明了一项新技术——充当 Erk 的原型基因电路 “脉冲探测器”——这将使研究人员无需实时成像即可研究 Erk 脉冲。这项技术 满足了一个重要的需求：目前，脉冲只能通过活体的高分辨率显微镜来检测 细胞，限制了研究它们的环境。在这里，我们建议开发我们的免成像生物传感器 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 显微镜很难或不可能进行。这项工作的成功完成将产生一个新的班级 生物传感器揭示对人类疾病具有潜在影响的复杂信号传导。