Engineering Orthogonal, Plasma Membrane-Specific SLIPT Systems for Multiplexed Chemical Control of Signaling Pathways in Living Single Cells.

Autor: Nakamura A; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan., Oki C; Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan., Kato K; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan., Fujinuma S; Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan., Maryu G; Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.; National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan., Kuwata K; Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan., Yoshii T; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.; PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan., Matsuda M; Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.; Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan., Aoki K; National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.; Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.; Department of Basic Biology, Faculty of Life Science, SOKENDAI, The Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan., Tsukiji S; Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.; Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.; Frontier Research Institute for Materials Science (FRIMS), Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
Jazyk: angličtina
Zdroj: ACS chemical biology [ACS Chem Biol] 2020 Apr 17; Vol. 15 (4), pp. 1004-1015. Date of Electronic Publication: 2020 Mar 20.
DOI: 10.1021/acschembio.0c00024
Abstrakt: 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.
Databáze: MEDLINE
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