Multiplexed Chemical Control of Signaling Pathways by Orthogonal, Plasma Membrane-Specific SLIPT Systems

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. A systematic understanding of cellular systems requires methods that activate multiple signaling molecules or pathways in single cells. However, the construction of tools for such multiplexed signal control is challenging. Here we present orthogonal chemogenetic systems that allow control of multiple signaling pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). Two orthogonal SLIPT systems were constructed to enable chemically inducible, individual translocation of two proteins from the cytoplasm to the inner-leaflet of the plasma membrane in the same cell. The SLIPT systems combined with fluorescent reporters achieved simultaneous multiplexed activation and monitoring of endogenous Ras/ERK and PI3K/Akt pathways in single cells. Thus, orthogonal SLIPT systems provide a powerful platform for multiplexed chemical signal control in single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.