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

Abstract

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.<br>

Chemogenetic Control of Protein Anchoring to Endomembranes in Living Cells with Lipid-Tethered Small Molecules

“…16 However, despite its significant potential, the organelles we can target for protein translocation are currently limited to the plasma membrane (PM), nucleus, and microtubules. 15,16 Therefore, to extend the versatility of the SLIPT approach, expanding the repertoire of targetable sites (i.e., localization motifs) is of fundamental importance. Here, we report a new SLIPT system that targets the surface of the endoplasmic reticulum (ER) and Golgi membranes, referred to as endomembranes (Figure 1a).…”

“…The development of a strategy for manipulating the localization of a target protein using only a single small molecule (without relying on protein heterodimerization) is an important challenge in chemical biology. To this end, we recently devised a new chemogenetic platform, called Self-localizing Ligand-Induced Protein Translocation (SLIPT), which enables controlling protein localization in a “single protein-single ligand” manner (Figure a). , This technique uses a new class of synthetic ligands, termed self-localizing ligands (SLs), which consists of three parts: (i) a ligand for a target protein, (ii) a small molecule localization motif that binds to the intended organelle or subcellular site, and (iii) a linker connecting them. Because of this design, after entering into cells, the SL itself can bind to its target protein and relocate it to the target site.…”

Chemogenetic Control of Protein Anchoring to Endomembranes in Living Cells with Lipid-Tethered Small Molecules

“…To this end, we recently devised a new chemogenetic platform, called Self-localizing Ligand-Induced Protein Translocation (SLIPT), which enables controlling protein localization in a "single protein-single ligand" manner (Figure 1a). 15,16 This technique uses a new class of synthetic ligands, termed selflocalizing ligands (SLs), which consists of three parts: (i) a ligand for a target protein, (ii) a small molecule localization motif that binds to the intended organelle or subcellular site, and (iii) a linker connecting them. Because of this design, after entering into cells, the SL itself can bind to its target protein and relocate it to the target site.…”

“…In addition, because of the "single protein-single ligand" nature of this system, the development of orthogonal SLIPT systems is straightforward using various protein-specific ligands, making the SLIPT approach suitable as a platform for multiplexing. 16 However, despite its significant potential, the organelles we can target for protein translocation are currently limited to the plasma membrane (PM), nucleus and microtubules. 15,16 Therefore, to extend the versatility of the SLIPT approach, expanding the repertoire of targetable sites (i.e., localization motifs) is of fundamental importance.…”

“…16 However, despite its significant potential, the organelles we can target for protein translocation are currently limited to the plasma membrane (PM), nucleus and microtubules. 15,16 Therefore, to extend the versatility of the SLIPT approach, expanding the repertoire of targetable sites (i.e., localization motifs) is of fundamental importance.…”