Ravindran Pavithran T, McFann Sarah, Thornton Richard H, Toettcher Jared E
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA.
Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
Cell Syst. 2022 Feb 16;13(2):131-142.e13. doi: 10.1016/j.cels.2021.10.002. Epub 2021 Nov 4.
Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution. Here, we describe an approach to infer pulsatile signaling dynamics from a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuits with the capability to selectively detect signaling pulses, revealing an incoherent feedforward topology that robustly performs this computation. We implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our "recorder of Erk activity dynamics" (READer) responds sensitively to spontaneous and stimulus-driven Erk pulses. READer circuits open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.
细胞利用细胞内信号通路来感知并响应其外部环境的变化。近年来，活细胞生物传感器揭示了许多信号通路中复杂的脉动动力学，但这些信号动力学的研究受到高时空分辨率活细胞成像必要性的限制。在此，我们描述了一种使用脉冲检测基因电路从固定细胞的单次测量中推断脉动信号动力学的方法。我们通过计算筛选出具有选择性检测信号脉冲能力的电路，揭示了一种能稳健执行此计算的非相干前馈拓扑结构。我们使用单一工程化转录因子和荧光蛋白报告基因，通过实验为细胞外调节蛋白激酶（Erk）信号通路实现了该基序。我们的“Erk活性动力学记录器”（READer）对自发和刺激驱动的Erk脉冲敏感响应。READer电路为永久标记瞬态、动态细胞群体打开了大门，以阐明信号动力学的机制基础和生物学后果。
