Zheng Lirong, Yan Yixiao, Zhou Bingxin, Lim Jormay, Shi Dongqing, An Bobae, Ko BumJin, Klyder Emily, Pitchiaya Sethuramasundaram, Cai Denise J, Boyden Edward S, Wei Donglai, Liò Pietro, Linghu Changyang
Department of Cell and Developmental Biology, Medical School, University of Michigan, Ann Arbor, MI, USA.
Michigan Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA.
bioRxiv. 2025 May 10:2025.05.10.653182. doi: 10.1101/2025.05.10.653182.
Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape-a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-, , , and activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.
基因表达不断受到基因调控网络的调节，该网络由多个调控组件组成，以介导细胞功能。一种用于分析基因调控过程的理想工具能够同时测量基因调控网络中许多组件的动态变化，但现有的方法难以长时间同时追踪这些组件的动态变化。在此，我们展示了CytoTape——一种基因编码的、模块化且可扩展的模拟记录器，用于在单细胞分辨率下连续、多路复用记录多天及数周内的基因调控动态变化。CytoTape由通过人工智能引导的合理设计构建的灵活、丝状、可伸长的细胞内蛋白质自组装体组成。基因调控动态变化以及用于重建连续时间轴的时间戳，通过沿着活细胞中单个CytoTape组装体分布的不同分子标签直接编码，固定后通过标准免疫荧光成像进行大规模读出。CytoTape记录器采用模块化设计，用于记录由多种活性依赖型启动子驱动的基因表达。我们在哺乳动物胚胎肾细胞、癌细胞、神经胶质细胞和神经元中展示了CytoTape的实用性，以时空可扩展的方式在单个细胞内同时记录了五种与细胞可塑性相关的转录因子活性和即刻早期基因表达水平，即CREB、c-、 、 和 的活性。CytoTape揭示了这些细胞活性之间复杂的波形和非线性时间耦合，从而能够研究基因调控历史和内在信号状态如何塑造转录逻辑。我们设想CytoTape在基础细胞生物学研究和疾病相关细胞生物学研究中都有广泛应用。
