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Atsuta-Tsunoda K

Bio: Atsuta-Tsunoda K is an academic researcher from Nagoya Institute of Technology. The author has contributed to research in topics: Cell signaling & Protein tag. The author has an hindex of 1, co-authored 1 publications receiving 2 citations.

Papers
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Posted ContentDOI
16 Mar 2021-bioRxiv
TL;DR: In this article, a bioorthogonal iK6DHFR/mDcTMP-based self-localizing ligand-induced protein translocation (SLIPT) approach was proposed.
Abstract: Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

5 citations


Cited by
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Journal ArticleDOI
TL;DR: In this paper , the authors show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration, and they show that lamellipodial extension after the release from mechanical confinement causes sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner.

4 citations

Journal ArticleDOI
TL;DR: In this paper, a photoactivatable self-localizing ligand (paSL) is used to recruit tag-fused proteins of interest from the cytoplasm to the plasma membrane within seconds upon light illumination.
Abstract: Manipulating subcellular protein localization using light is a powerful approach for controlling signaling processes with high spatiotemporal precision. The most widely used strategy for this is based on light-induced protein heterodimerization. The use of small synthetic molecules that can control the localization of target proteins in response to light without the need for a second protein has several advantages. However, such methods have not been well established. Herein, we present a chemo-optogenetic approach for controlling protein localization using a photoactivatable self-localizing ligand (paSL). We developed a paSL that can recruit tag-fused proteins of interest from the cytoplasm to the plasma membrane within seconds upon light illumination. This paSL-induced protein translocation (paSLIPT) is reversible and enables the spatiotemporal control of signaling processes in living cells, even in a local region. paSLIPT can also be used to implement simultaneous optical stimulation and multiplexed imaging of molecular processes in a single cell, offering an attractive and novel chemo-optogenetic platform for interrogating and engineering dynamic cellular functions.

3 citations

Journal ArticleDOI
TL;DR: In this article , the authors showed that the lipid acyl chain of the myristoyl-DCys motif can be as short as 10-carbons and still retain the palmitoylation-dependent Golgi localization property in cells.

1 citations

Journal ArticleDOI
TL;DR: A review of the most prominent bacterial enzymes used for site-specific modification of proteins, in vivo protein labeling, proximity labeling, interactome mapping, signaling pathway manipulation, and therapeutic discovery can be found in this paper .
Book ChapterDOI
TL;DR: In this article, a self-localizing ligand-induced protein translocation (SLIPT) system was proposed to control protein localization in living mammalian cells using synthetic SLs.
Abstract: Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.