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Canonical epidermal growth factor (EGF) receptor (EGFR) activation involves the binding of seven EGFR ligands (EGFRLs); however, their extracellular dynamics remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding, we show that epiregulin (EREG), a low-affinity EGFRL, rapidly and efficiently activates EGFR in Madin-Darby canine kidney (MDCK) epithelial cells and mouse epidermis. During collective cell migration, EGFR and extracellular signal-regulated kinase (ERK) activation waves propagate in an a disintegrin and metalloprotease 17 (ADAM17) sheddase- and EGFRL-dependent manner. Upon induced EGFRL shedding, low-affinity ligands EREG and amphiregulin (AREG) mediate faster and broader ERK waves than high-affinity ligands. Tight/adherens junction integrity is essential for ERK activation propagation, suggesting that tight intercellular spaces prefer the low-affinity EGFRLs for efficient signal transmission. In EREG-deficient mice, ERK wave propagation and cell migration were impaired during skin wound repair. We additionally show that heparin-binding EGF-like growth factor (HBEGF) primarily promotes surrounding cell motility. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo , we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER-PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Cells sense extracellular stimuli through membrane receptors and process information through an intracellular signaling network. Protein translocation triggers intracellular signaling, and techniques such as chemically induced dimerization (CID) have been used to manipulate signaling pathways by altering the subcellular localization of signaling molecules. However, in the fission yeast Schizosaccharomyces pombe , the commonly used FKBP-FRB system has technical limitations, and therefore, perturbation tools with low cytotoxicity and high temporal resolution are needed. We here applied our recently developed self-localizing ligand-induced protein translocation (SLIPT) system to S. pombe and successfully perturbed several cell cycle-related proteins. The SLIPT system utilizes self-localizing ligands to recruit binding partners to specific subcellular compartments such as the plasma membrane or nucleus. We optimized the self-localizing ligands to maintain the long-term recruitment of target molecules to the plasma membrane. By knocking in genes encoding the binding partners for self-localizing ligands, we observed changes in the localization of several endogenous molecules and found perturbations in the cell cycle and associated phenotypes. This study demonstrates the effectiveness of the SLIPT system as a chemogenetic tool for rapid perturbation of endogenous molecules in S. pombe , providing a valuable approach for studying intracellular signaling and cell cycle regulation with an improved temporal resolution.

Fingerprint Raman features of para -aminothiophenol (pATP) in surface-enhanced Raman scattering (SERS) spectra have been widely used to measure plasmon-driven catalytic activities because the appearance of characteristic spectral features is purported to be due to plasmon-induced chemical transformation of pATP to trans-p , p '-dimercaptoazobenzene ( trans -DMAB). Here, we present a thorough comparison of SERS spectra for pATP and trans -DMAB in the extended range of frequencies covering group vibrations, skeletal vibrations, and external vibrations under various conditions. Although the fingerprint vibration modes of pATP could be almost mistaken with those of trans -DMAB, the low-frequency vibrations revealed distinct differences between pATP and DMAB. Photo-induced spectral changes of pATP in the fingerprint region were explained well by photo-thermal variation of the Au-S bond configuration, which affects the degree of the metal-to-molecule charge transfer resonance. This finding indicates that a large number of reports in the field of plasmon-mediated photochemistry must be reconsidered.

Membrane contact sites (MCSs) are morphologically defined intracellular structures where cellular membranes are closely apposed. Recent progress has significantly advanced our understanding of MCSs with the use of new tools and techniques. Visualization of MCSs in living cells by split fluorescence proteins or FRET-based techniques tells us the dynamic property of MCSs. Manipulation of MCSs by chemically-induced dimerization (CID) or light-induced dimerization (LID) greatly contributes to our understanding of their functional aspects including inter-organelle lipid transport mediated by lipid transfer proteins (LTPs). Here we highlight recent advances in these tools and techniques as applied to MCSs, and we discuss their advantages and limitations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Conjugating small-molecule ligands to synthetic motifs that can localize to specific organelles or membranes in living cells is a practical approach to develop compounds as chimeric tools or drugs that can manipulate biological processes in a subcellular site-specific manner. However, the number of available organelle-targeted synthetic motifs for small-molecule localization is limited. We have recently developed a synthetic myristoyl- D Cys motif for small-molecule localization that undergoes S -palmitoylation via the cellular palmitoylation machinery and localizes to the Golgi surface. Herein, we show that the lipid acyl chain of the myristoyl (C14)- D Cys motif can be as short as 10-carbons and still retain the palmitoylation-dependent Golgi localization property in cells. This discovery led to the identification of four new derivatives for small-molecule localization: tridecanoyl (C13)-, dodecanoyl (C12)-, undecanoyl (C11)-, and decanoyl (C10)- D Cys motifs. We demonstrated that even the short decanoyl- D Cys palmitoylation motif could be used to generate small-molecule ligand conjugates that functioned as chemical tools for controlling protein localization and cell signaling. The miniaturized synthetic palmitoylation motifs identified in this work may find applications in creating various Golgi-localizable chimeric molecules for use in chemical biology and drug development.

Cage-like supramolecular assemblies called molecular cages, which possess attractive functions, have been prepared. Although biomolecule-based nanocages are required for biological/medical applications such as drug delivery systems, the majority of nanocages are constructed using aromatic compounds with lower biocompatibility and biodegradability. In this study, the construction of a peptide nanocage consisting of an oligoproline conjugate is demonstrated. The conjugate was easy to prepare and had high biocompatibility. The oligoproline moiety of the conjugate had a rigid, rod-like structure suitable for the backbone of the supramolecular nanocage. The conjugates self-assembled to form peptide nanocages with a huge inner cavity.

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (m D cTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for m D cTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and m D cTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations., Competing Interests: Declarations of interest S. Suzuki, A.N., T.Y., and S.T. are co-inventors of a patent application related to this work. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Light-mediated control of protein localization in living cells is a powerful approach for manipulating and probing complex biological systems. By incorporating a classical 6-nitroveratryloxycarbonyl (NVOC) caging group into the inner plasma membrane (PM)-localizing trimethoprim ligand, we recently developed a photoactivatable self-localizing ligand (paSL) that can rapidly recruit engineered Escherichia coli dihydrofolate reductase-fusion proteins from the cytoplasm to the PM upon violet (ca. 400 nm)-light illumination. However, because the photosensitivity of the NVOC-caged paSL is low to moderate, photouncaging experiments require high light intensity, which may not be ideal for many cell applications. Herein, we present a new 7-diethylaminocoumarin (DEAC)-caged paSL with improved photosensitivity. DEAC-caged paSL induced efficient protein recruitment upon violet-light irradiation, even at the low intensity under which NVOC-caged paSL does not respond. DEAC-caged paSL was insensitive to excitation light used to image green fluorescent proteins (GFPs), and it was applicable for simultaneous optical stimulation of Gαq signaling and fluorescence imaging of subsequent Ca 2+ oscillations using a GFP-based Ca 2+ biosensor in living cells., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Here, we describe a protocol for the translational regulation of transfected messenger RNAs (mRNAs) using light in mammalian cells. We detail the steps for photocaged ligand synthesis, template DNA preparation, and mRNA synthesis. We describe steps for mRNA transfection, treatment of cells with a photocaged ligand followed by light irradiation, and analysis of the transgene expression. The protocol enables spatiotemporally regulated transgene expression without the risk of insertional mutagenesis. For complete details on the use and execution of this protocol, please refer to Nakanishi et al. (2021)., Competing Interests: Kyoto University holds a patent regarding the translational regulators (JP2020015891). H.N. and H.S. are the inventors of record listed on the patents. H.S. is an outside director of aceRNA Technologies, Co., Ltd., (© 2022 The Author(s).)

Here, we describe the design and synthesis of a new reduction-cleavable spacer (RCS) based on a nitrobenzene scaffold for constructing reduction-responsive oligonucleotides according to standard phosphoramidite chemistry. In addition, we demonstrate that the introduction of the RCS in the middle of an oligonucleotide (30 nt) enables the construction of a self-assembled microsphere capable of exhibiting a reduction-responsive disassembly., (© 2022 Wiley-VCH GmbH.)

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung., Competing Interests: HI, ST, TH, YK, CO, ST, SI, AM, KS, KT, MM No competing interests declared, (© 2022, Ichise et al.)

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC 50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

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.

Prostaglandin E 2 (PGE 2 ) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE 2 , little is known whether PGE 2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A 2 (TXA 2 ) triggers Ca 2+ transients in tumor cells, culminating in PGE 2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca 2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca 2+ transients were monitored as the surrogate marker of PGE 2 secretion. Intravital imaging of Braf V600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca 2+ transients is markedly higher in vivo than in vitro . The TXA 2 receptor was indispensable for the Ca 2+ transients in vivo , high intratumoral PGE 2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca 2+ transients and reduced TXA 2 and PGE 2 concentrations in tumor tissues. These results identify the VEGF-TXA 2 axis as a critical promoter of PGE 2 -dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. SIGNIFICANCE: This study identifies the VEGF-TXA 2 axis as a potentially targetable regulator of PGE 2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies., (©2021 American Association for Cancer Research.)

The ratio of type III to type I collagen is important for properly maintaining functions of organs and cells. We propose a method to quantify the ratio of type III to total (type I + III) collagen (λ III ) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λ III of collagen gels was examined, and the slope (k 1 ) and SHG light intensity at 0% type III collagen (k 2 ) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k 3 ) of the relationship was determined. The λ III in a collagen fiber bundle was estimated from these constants (k 1-3 ) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained λ III  = 84.7% ± 13.8% and λ III  = 17.5% ± 15.2%, respectively. These values concurred with those obtained with a typical quantification method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings demonstrated that the method proposed is useful to quantify the ratio of type III to total collagen in a collagen fiber bundle.

The photo-regulation of transgene expression is one effective approach in mammalian synthetic biology due to its high spatial and temporal resolution. While DNAs are mainly used as vectors, modified RNAs (modRNAs) are also useful for medical applications of synthetic biology, because they can avoid insertional mutagenesis and immunogenicity. However, the optogenetic control of modRNA-delivered transgenes is much more difficult than that of DNA-delivered transgenes. Here, we develop two types of photo-controllable translational activation systems that are compatible with modRNAs. One is composed of a heterodimerization domain-fused split translational activator protein and a photocaged heterodimerizer. The other is composed of a destabilizing domain-fused translational activator protein and a photocaged stabilizer. The destabilized type can be used for not only translational activation but also translational repression of the modRNAs. These photo-controllable translation systems will expand the application of mammalian synthetic biology research., Competing Interests: Declaration of interests Kyoto University has filed a patent application regarding the translational regulators (JP2020015891). H.N. and H.S. are the inventors of record listed on the patents. H.S. is an outside director of aceRNA Technologies, Co., Ltd., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

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, DHFR iK6 , 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, m D cTMP. The m D cTMP-mediated PM recruitment of DHFR iK6 -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 m D cTMP/DHFR iK6 -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.

Organelle-localizable small-molecule ligands are valuable tools for spatiotemporally controlling protein localization and signaling processes in living cells. Here, we present synthetic ligands that specifically localize to the Golgi surface. The ligands can rapidly recruit their target proteins from the cytoplasm to the Golgi and be applied to manipulate signaling proteins and lipids on the Golgi membrane, offering a new useful chemical tool for the study and control of Golgi/cell functions.

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAP f protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAP Δ , is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl- D Cys lipopeptidomimetic, called m D cBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

The development of methods that allow detection of ligand-target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand-target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug-target engagement in cells.

Inducing protein translocation to the plasma membrane (PM) is an important approach for manipulating diverse signaling molecules/pathways in living cells. We previously devised a new chemogenetic system, in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation turned out to be transient and spontaneously reversed within 1 h, limiting its application. Here, we first demonstrated that the spontaneous reverse translocation was caused by cellular degradation of mgcTMP, presumably by proteases. To address this problem, we newly developed a proteolysis-resistant SL, m D cTMP. This m D cTMP now allows sustained PM localization of eDHFR-fusion proteins (over several hours to a day), and it was applicable to inducing prolonged signal activation and cell differentiation. m D cTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.

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.

In plants, stomata regulate water loss through transpiration for plant growth and survival in response to various environmental stressors; and simple methods to assess stomatal dynamics are needed for physiological studies. Herein, we report a fluorescence-imaging-based method using fluorescein diacetate tagged with Hoechst 33342, a nuclear staining chemical probe (HoeAc 2 Fl) for the qualitative assessment of stomatal dynamics. In our method, the stomatal movement is inferred by simple monitoring of the fluorescence intensity in the nucleus of the stomata.

Recently, development of techniques to deliver pharmacologically active biomacromolecules such as peptides and proteins to cytosol has gained much interest. Here, we applied the peptide gemini (PG)-surfactants to a novel platform to design cell penetration lipopeptides (CP-PGs), which can deliver exogenous peptides and proteins to cytosol. Among the number of candidate CP-PGs having different peptide sequences at the X-, Y-, and Z-positions, we focused on those having two C12 alkyl chains appended to the side chain of two Cys residues, the betaine sequence -Asp-Lys-Asp-Lys- between the alkylated Cys residues (i.e., at the X-position), and having different cationic peptide sequences of oligo-Lys or oligo-Arg at the Y- and/or Z-positions. With respect to cytotoxicity for mammalian cells such as NIH3T3 cells upon 1 h exposure, those having (Lys) 3 ( K 3 -DKDKC 12 and DKCK 12 -K 3 ) showed lower cytotoxicity (IC 50 = 241 and 198 μM) among those having oligo-Lys, (Lys) n ( n = 1, 3, 5; IC 50 = 88-197 μM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys) 3 at both N- and C-terminal sides ( K 3 -DKDKC 12 -K 3 ) (IC 50 = 225 μM). In contrast, the CP-PG having (Arg) 3 at the N-terminal side ( R 3 -DKDKC 12 ) showed higher cytotoxicity (IC 50 = 88 μM). Carrier abilities of the CP-PGs for exogenous peptides were evaluated using the proapoptotic domain (PAD) peptide, which induces apoptosis by disturbing mitochondrial membranes after delivery into cytosol. As a result, the CP-PGs of K 3 -DKDKC 12 , DKCK 12 -K 3 , K 3 -DKDKC 12 -K 3 , DKCK 12 -K 5 , and R 3 -DKDKC 12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC 50 ) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 μM, respectively). Especially, the carrier abilities of DKCK 12 -K 3 and DKCK 12 -K 5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC 50 = 6.8 μM). Together, our results indicate that the PG-surfactant molecular framework could be a potential new platform to design efficient cell penetration carrier materials.

The self-localizing ligand-induced protein translocation (SLIPT) system is an emerging platform that controls protein localization in living cells using synthetic self-localizing ligands (SLs). Here, we report a chemogenetic SLIPT system for inducing protein translocation from the cytoplasm to the surface of the endoplasmic reticulum (ER) and Golgi membranes, referred to as endomembranes. By screening a series of lipid-trimethoprim (TMP) conjugates, we found oleic acid-tethered TMP (oleTMP) to be the optimal SL that efficiently relocated and anchored Escherichia coli dihydrofolate reductase (eDHFR)-fusion proteins to endomembranes. We showed that oleTMP mediated protein anchoring to endomembranes within minutes and could be reversed by the addition of free TMP. We also applied the endomembrane SLIPT system to artificially activate endomembrane Ras and inhibit the active nuclear transport of extracellular signal-regulated kinase (ERK), demonstrating its applicability for manipulating biological processes in living cells. We envision that the present oleTMP-based SLIPT system, which affords rapid and reversible control of protein anchoring to endomembranes, will offer a new unique tool for the study and control of spatiotemporally regulated cell signaling processes.

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405nm, the hoeFL-DNA complex displayed two fluorescence bands around 460nm and 520nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F 520 /F 460 ) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Coronatine ( 1 ), a small-molecular virulence factor produced by plant-pathogenic bacteria, promotes bacterial infection by inducing the opening of stomatal pores, the major route of bacterial entry into the plant, via the jasmonate-mediated COI1-JAZ signaling pathway. However, this pathway is also important for multiple plant functions, including defense against wounding by herbivorous insects. Thus, suppression of the COI1-JAZ signaling pathway to block bacterial infection would concomitantly impair plant defense against herbivorous wounding. Here, we report additional, COI1-JAZ-independent, action of 1 in Arabidopsis thaliana guard cells. First, we found that a stereoisomer of 1 regulates the movement of Arabidopsis guard cells without affecting COI1-JAZ signaling. Second, we found using alkyne-tagged Raman imaging (ATRI) that 1 is localized to the endoplasmic reticulum (ER) of living guard cells of Arabidopsis . The use of arc6 mutant lacking chloroplast formation was pivotal to circumvent the issue of autofluorescence during ATRI. These findings indicate that 1 has an ER-related action on Arabidopsis stomata that bypasses the COI1-JAZ signaling module. It may be possible to suppress the action of 1 on stomata without impairing plant defense responses against herbivores.

Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases.

Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K(+) channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides.

Synthetic small-molecule ligands that control the intracellular location of proteins would be powerful tools for regulating cellular systems. However, the creation of such molecules has long remained unexplored because of the lack of a design methodology. Here, we introduce a new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target (exogenously expressed and endogenous) proteins and relocate them rapidly from the cytoplasm to their targeting sites. SLL-induced protein translocation is applicable to manipulate diverse synthetic/endogenous signaling pathways. These results validate the utility of SLLs in the spatial control of intracellular protein localization and signaling processes, opening a new direction in the design of small-molecule-based chemical tools or drugs for cell regulation.

Introducing nongenetically encoded, synthetic probes into specific proteins is now recognized as a key component in chemical biology. In particular, the ability to chemically modify specific "native" proteins in various contexts from in vitro to cellular systems is of fundamental importance to study biological systems. We developed a protein-labeling technique termed ligand-directed tosyl (LDT) chemistry for this purpose. This method is capable of labeling specific native proteins with diverse synthetic probes with high site specificity and target selectivity without compromising protein function. Here we describe the principle of the LDT chemistry and the protocol for selective chemical labeling of native carbonic anhydrase in vitro, in blood cells (ex vivo), and in living mice (in vivo).

The ability to introduce any chemical probe to any endogenous target protein in its native environment, that is in cells and in vivo, is anticipated to provide various new exciting tools for biological and biomedical research. Although still at the prototype stage, the ligand-directed tosyl (LDT) chemistry is a novel type of affinity labeling technique that we developed for such a dream. This chemistry allows for modifying native proteins by various chemical probes with high specificity in various biological settings ranging from in vitro (in test tubes) to in living cells and in vivo. Since the first report, the list of proteins that are successfully labeled by the LDT chemistry has been increasing. A growing number of studies have demonstrated its utility to create semisynthetic proteins directly in cellular contexts. The in situ generated semisynthetic proteins are applicable for various types of analysis and imaging of intracellular biological processes. In this review, we summarize the basic properties of the LDT chemistry and its applications toward in situ engineering and analysis of native proteins in living systems. Current limitations and future challenges of this area are also described., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

Small-molecule ligands that control the spatial location of proteins in living cells would be valuable tools for regulating biological systems. However, the creation of such molecules remains almost unexplored because of the lack of a design methodology. Here we introduce a conceptually new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target proteins and relocate (tether) them rapidly from the cytoplasm to their targeting sites, thus serving as synthetic protein translocators. SLL-induced protein translocation enables us to manipulate diverse synthetic/endogenous signaling pathways. The method is also applicable to reversible protein translocation and allows control of multiple proteins at different times and locations in the same cell. These results demonstrate the usefulness of SLLs in the spatial (and temporal) control of intracellular protein distribution and biological processes, opening a new direction in the design of small-molecule tools or drugs for cell regulation.

Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells., (© 2012 American Chemical Society)

Supramolecular hydrogels constructed through molecular self-assembly of small molecules have unique stimuli-responsive properties; however, they are mechanically weak in general, relative to conventional polymer gels. Very recently, we developed a zwitterionic amino acid tethered amphiphilic molecule 1, which gave rise to a remarkably stiff hydrogel comparable with polymer-based agarose gel, retaining reversible thermal-responsive properties. In this study, we describe that rational accumulation of multiple and orthogonal noncovalent interactions in the supramolecular nanofibers of 1 played crucial roles not only in the mechanical reinforcement but also in the multistimuli responsiveness. That is, the zwitterionic amino acid moiety and the C-C double bond unit of the hydrogelator 1 can function as a pH-responsive unit and a light-responsive unit, respectively. We also demonstrated that this stiff and multistimuli-responsive supramolecular hydrogel 1 is applied as a unique mold for 2D and 3D-patterning of various substances. More significantly, we succeeded in the fabrication of a collagen gel for spatial patterning, culturing, and differentiation of live cells by using hydrogel 1 molds equipped with 2D/3D microspace channels (100-200 μm in diameter)., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Catalysts hold promise as tools for chemical protein modification. However, the application of catalysts or catalyst-mediated reactions to proteins has only recently begun to be addressed, mainly in in vitro systems. By radically improving the affinity-guided DMAP (4-dimethylaminopyridine) (AGD) catalysts that we previously reported (Koshi, Y.; Nakata, E.; Miyagawa, M.; Tsukiji, S.; Ogawa, T.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 245.), here we have developed a new organocatalyst-based approach that allows specific chemical acylation of a receptor protein on the surface of live cells. The catalysts consist of a set of 'multivalent' DMAP groups (the acyl transfer catalyst) fused to a ligand specific to the target protein. It was clearly demonstrated by in vitro experiments that the catalyst multivalency enables remarkable enhancement of protein acylation efficiency in the labeling of three different proteins: congerin II, a Src homology 2 (SH2) domain, and FKBP12. Using a multivalent AGD catalyst and optimized acyl donors containing a chosen probe, we successfully achieved selective chemical labeling of bradykinin B(2) receptor (B(2)R), a G-protein coupled receptor, on the live cell-surface. Furthermore, the present tool allowed us to construct a membrane protein (B(2)R)-based fluorescent biosensor, the fluorescence of which is enhanced (tuned on) in response to the antagonist ligand binding. The biosensor should be applicable to rapid and quantitative screening and assay of potent drug candidates in the cellular context. The design concept of the affinity-guided, multivalent catalysts should facilitate further development of diverse catalyst-based protein modification tools, providing new opportunities for organic chemistry in biological research., (© 2011 American Chemical Society)

In this study, assisted by affinity-guided DMAP strategy, we developed a novel (19)F-modified lectin as a biosensor for specific detection and imaging of glycoproteins. Exploited the large chemical shift anisotropy property of (19)F nuclei, glycoproteins detected by our (19)F-biosensor are signatured by broadened peaks in (19)F NMR, hence enabled the distinction between glycoproteins and small molecule saccharides. Such signal on/off switching was also applied to glycoprotein imaging by (19)F MRI., (Copyright © 2011 Elsevier Ltd. All rights reserved.)