Cellular FM-radios: seeing, probing, and perturbing single-cell protein activity dynamics in biological systems with frequency-barcoded spatiotemporal signaling circuits

项目总结/摘要 生物学是由蛋白质活动在空间和时间上的自我组织提供动力的， 像运动和信息处理这样的生物化学反应的涌现行为。在多细胞 系统，这些单细胞行为的正确执行对于发育，免疫， 监控和伤口愈合因此，许多人类疾病都起源于免疫调节失调。 蛋白质活性动态，包括癌症和自身免疫性疾病。因此，迫切需要 了解细胞的动态蛋白质硬件的正常运作以及它在疾病中是如何失败的。 contexts.拟议的研究描述了一种创新的战略，安装“蜂窝无线电”电路到细胞 其产生基于蛋白质的频率可调制（FM）条形码信号。该信号可以用于 可视化，探测和干扰任何复杂生物环境中单细胞的蛋白质活性动力学。 我们的方法是通过我们成功地实现了一个遗传编码的正交图案电路 （MinDE）在人类细胞中可以产生前所未有的细胞规模时空蛋白质 动态和模式。MinDE电路的快速振荡产生独特的单细胞FM条形码， 荧光信号可以锁定到其他重叠细胞上，并使用频率- 域图像处理工具，我们已经开发了基于傅立叶，小波和希尔伯特变换。使用 蛋白质工程和合成生物学，我们将开发一个通用平台，用于设计MinDE电路， 与细胞中任何动态蛋白质活动有关。这将允许MinDE电路可以读出 并在细胞独特的FM条形码信号上同时广播多种蛋白质活动，使我们能够 明确跟踪单个细胞的内部状态如何随着多细胞过程的演变而变化; MinDE电路可以作为遗传编码的控制信号，干扰任何靶蛋白的动力学 感兴趣的是，通过显微镜和高通量技术实现细胞行为关键节点的动态分析， 基于测序的测定。我们将设计和应用特定的MinDE电路来研究致癌信号 癌细胞和肿瘤类器官中的动力学和异常信息处理， 上游致癌驱动突变通过ERK、mTOR和PKA破坏下游信号动力学 激酶。与此同时，我们将产生MinDE电路，刺激ERK，mTOR或PKA信号在不同的水平。 时间尺度来定义信号传输的时间约束如何在不同的致癌基因中被破坏， 背景及其对肿瘤类器官发育的影响。虽然本提案中的应用程序侧重于 致癌信号传导，我们的平台很容易应用于任何感兴趣的动态蛋白质活性。因此，我们的工作将 为理解和工程化生物系统中的动态蛋白质活性建立了新的范例， 为基础和转化生物学提供了新的见解，具有很高的治疗应用潜力。

Project Summary/Abstract Biology is powered by the self-organization of protein activities in space and time, allowing cells to build emergent behaviors like motility and information-processing out of biochemical reactions. In multicellular systems, proper execution of these single-cell behaviors is critical for processes like development, immune surveillance, and wound healing. As a result, many human diseases have their origin in the dysregulation of protein activity dynamics, including cancer and autoimmune disorders. There is thus a pressing need to understand both the normal operation of the cell’s dynamic protein-based hardware and how it fails in diseased contexts. The proposed research describes an innovative strategy for installing “cellular radio” circuits into cells that generate a protein-based frequency-modulatable (FM) barcoded signal. This signal can then be used for visualizing, probing, and perturbing the protein activity dynamics of single cells in any complex biological setting. Our approach is enabled by our successful implementation of a genetically-encoded orthogonal patterning circuit (MinDE) in human cells that can produce an unprecedented breadth of cell-scale spatiotemporal protein dynamics and patterns. The fast oscillations of MinDE circuits generate a unique single-cell FM-barcoded fluorescent signal that can be locked on to and spectrally separated from other overlapping cells using frequency- domain image processing tools we have developed based on Fourier, Wavelet and Hilbert Transforms. Using protein-engineering and synthetic biology, we will develop a general platform for designing MinDE circuits that can be connected to any dynamic protein activity in the cell. This will allow for MinDE circuits that can read out and broadcast multiple protein activities simultaneously on a cell’s unique FM-barcoded signal, enabling us to unambiguously track how the internal state of individual cells changes as multicellular processes evolve; and MinDE circuits that can act as genetically-encoded control signals that perturb the dynamics of any target protein of interest, enabling dynamic profiling of key nodes of cell behavior by microscopy and high-throughput sequencing based assays. We will design and apply specific MinDE circuits to investigate oncogenic signaling dynamics and aberrant information processing in cancer cells and tumor organoids, asking how different upstream oncogenic driver mutations corrupt downstream signaling dynamics through ERK, mTOR, and PKA kinases. In parallel, we will generate MinDE circuits that stimulate ERK, mTOR, or PKA signaling at different timescales to define how temporal constraints on signal transmission are corrupted in different oncogenic backgrounds and their impact on tumor organoid development. While the applications in this proposal focus on oncogenic signaling, our platform is easily applied to any dynamic protein activity of interest. Our work will thus establish a new paradigm for understanding and engineering dynamic protein activities in biological systems, providing new insights into basic and translational biology with high potential for therapeutic applications.