Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. Thus, a systematic understanding of cellular systems requires methods that allow the activation of more than one specific signaling molecule or pathway within a cell. However, the construction of tools suitable for such multiplexed signal control remains challenging. In this work, we aimed to develop a platform for chemically manipulating multiple signaling molecules/pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). SLIPT is an emerging chemogenetic tool that controls protein localization and cell signaling using synthetic self-localizing ligands (SLs). Focusing on the inner leaflet of the plasma membrane (PM), where there is a hub of intracellular signaling networks, here we present the design and engineering of two new PM-specific SLIPT systems based on an orthogonal eDHFR and SNAP-tag pair. These systems rapidly induce translocation of eDHFR- and SNAP-tag-fusion proteins from the cytoplasm to the PM specifically in a time scale of minutes upon addition of the corresponding SL. We then show that the combined use of the two systems enables chemically inducible, individual translocation of two distinct proteins in the same cell. Finally, by integrating the orthogonal SLIPT systems with fluorescent reporters, we demonstrate simultaneous multiplexed activation and fluorescence imaging of endogenous ERK and Akt activities in a single cell. Collectively, orthogonal PM-specific SLIPT systems provide a powerful new platform for multiplexed chemical signal control in living single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.

中文翻译：

---

工程正交，等离子膜专用SLIPT系统，用于活体单细胞信号通路的多路化学控制。

大多数细胞行为是处理细胞刺激后产生的多个信号信息的结果。因此，对细胞系统的系统性理解需要允许激活细胞内一个以上特定信号分子或途径的方法。但是，构造适合于这种多路信号控制的工具仍然具有挑战性。在这项工作中，我们旨在开发一个平台，用于基于自定位配体诱导的蛋白质易位（SLIPT）化学处理活的哺乳动物细胞中的多种信号分子/途径。SLIPT是新兴的化学生成工具，可使用合成的自定位配体（SLs）控制蛋白质定位和细胞信号传导。着眼于细胞膜内信号网络枢纽的质膜（PM）内叶，在这里，我们介绍了基于正交eDHFR和SNAP标签对的两个新的PM专用SLIPT系统的设计和工程。这些系统迅速地诱导eDHFR和SNAP标签融合蛋白从细胞质到PM的易位，特别是在添加相应SL的几分钟内。然后，我们表明两种系统的组合使用可以在同一细胞中化学诱导诱导两种不同蛋白质的个体易位。最后，通过将正交SLIPT系统与荧光报告基因整合，我们证明了单个细胞中内源性ERK和Akt活性的同时多重激活和荧光成像。总而言之，正交于PM的SLIPT专用系统为活单细胞中的化学信号多路控制提供了强大的新平台，