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Low-frequency SERS spectrum reveals that spectral changes in the fingerprint vibrations of pATP on a metal surface are not due to plasmon-mediated transformation to trans-azo compounds but due to photo-thermal variation of Au–S bond configuration.

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

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.

Intravital imaging of BrafV600E melanoma cells expressing GCaMP6s with or without treatment of a VEGFR inhibitor, motesanib. Images represent the fold increase in fluorescence intensity shown in IMD mode as in Figure 4C. Images were acquired every 6 seconds for 40 minutes. Motesanib was intravenously administered at 10 minutes. At only about 20 minutes after administration, the Ca2+ transients were significantly suppressed. Scale bar = 60 µm. Times are shown in minutes (m) and seconds (s).

Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF–TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies.Significance:This study identifies the VEGF–TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.

Intravital imaging of BrafV600E melanoma cells expressing GCaMP6s. Images represent the fold increase in fluorescence intensity shown in IMD mode as in Figure 1E. Images were acquired every 3 seconds for 15 minutes. From left to right: parental melanoma, Gnaq-/- melanoma, and Gnaq-/- melanoma cells introduced with Gnaq (Gnaq-/- + Gnaq). Genetic ablation of Gnaq abolished active Ca2+ transients observed in parental cells, which was restored by the introduction of Gnaq in Gnaq-/- melanoma. Scale bar = 60 µm. Times are shown in minutes (m) and seconds (s).

Supplementary Method, Reference, Figures 1-8, Tables 1-2

Live imaging of BrafV600E melanoma cells expressing GCaMP6s in vivo and in vitro. Pseudo colors represent the fluorescence intensity as in Figure 1C. Images were acquired every 6 seconds for 20 minutes. A small population of tumor cells continuously shows active Ca2+ transients repeatedly in vivo, but not in vitro. Scale bar = 60 µm. Times are shown in minutes (m) and seconds (s).

Live imaging of MDCK cells expressing the PKA biosensor. BrafV600E melanoma cells expressing the DREADD hM3 were stimulated by 1 μM CNO treatment at 60 seconds. PKA activation in surrounding MDCK cells was observed 30 seconds after stimulation. Scale bar = 100 µm. Times are shown in minutes (m) and seconds (s).

Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF–TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. Significance: This study identifies the VEGF–TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.

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 EC50 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.

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

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, mDcTMP. This mDcTMP 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. mDcTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.

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 (HoeAc2Fl) 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 (K3-DKDKC12 and DKCK12-K3) showed lower cytotoxicity (IC50 = 241 and 198 μM) among those having oligo-Lys, (Lys)n (n = 1, 3, 5; IC50 = 88-197 μM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys)3 at both N- and C-terminal sides (K3-DKDKC12-K3) (IC50 = 225 μM). In contrast, the CP-PG having (Arg)3 at the N-terminal side (R3-DKDKC12) showed higher cytotoxicity (IC50 = 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 K3-DKDKC12, DKCK12-K3, K3-DKDKC12-K3, DKCK12-K5, and R3-DKDKC12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC50) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 μM, respectively). Especially, the carrier abilities of DKCK12-K3 and DKCK12-K5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC50 = 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.

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.

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.

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., 細胞の集まりからリーダーが生まれる仕組み --出る杭はより出る--. 京都大学プレスリリース. 2022-09-29.

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.

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

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 Escherichiacoli dihydrofolate reductase (eDHFR)-fusion proteins toendomembranes. 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.

We report here efficient synthesis of artificial transmembrane molecules (ATMs), via click chemistry of a transmembrane part and membrane-penetrating parts. Synthesized ATMs penetrated the cell mem...

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 (λIII) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λIII of collagen gels was examined, and the slope (k1) and SHG light intensity at 0% type III collagen (k2) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k3) of the relationship was determined. The λIII in a collagen fiber bundle was estimated from these constants (k1-3) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained λIII = 84.7% ± 13.8% and λIII = 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.

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.

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-d-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.

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.

Prostaglandin E

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. We first present chemogenetic protein-recruiting and -releasing condensates, which rapidly inhibited and activated signaling proteins, respectively. An optogenetic condensate system was successfully constructed that enables reversible release and sequestration of protein activity using light. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step towards tailor-made engineering of synthetic protein condensates with various functionalities.

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 SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, 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.

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.

The ability to artificially attach lipids to specific intracellular protein targets would be a valuable approach for controlling protein localization and function in cells. We recently devised a chemogenetic method in which a SNAP-tag fusion protein can be translocated from the cytoplasm to the plasma membrane by post-translationally and covalently conjugating a synthetic lipopeptide in cells. However, the first-generation system lacked general applicability. Herein, we present an improved synthetic lipidation system that enables efficient plasma membrane translocation of SNAP-tag fusion proteins in cells. This second-generation system is now applicable to the control of various cell-signaling molecules, offering a new and useful research tool in chemical biology and synthetic biology.

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.

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., Bacterial infection of plants causes severe crop losses. A bacterial virulence factor, coronatine (1), facilitates infection by hijacking a plant’s defense pathway. We found an ER-mediated noncanonical pathway of 1-mediated infection in Arabidopsis.

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.

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.

Tumor-derived prostaglandin E2 (PGE2) promotes tumor progression through evasion of anti-tumor immunity. However, little is known about how PGE2 secretion is regulated within tumor tissues. Here, we show that host cell-derived thromboxane A2 (TXA2) triggers tumor cells to secrete PGE2, resulting in the evasion of anti-tumor immunity. An induction of Ca2+ transients in BrafV600E mouse melanoma cells is sufficient to trigger PGE2 secretion. The frequency of Ca2+ transients is markedly higher in melanoma cells implanted into mice than those in vitro. We show that t he melanoma cells deficient in TXA2 receptor or Gq subunit α do not exhibit Ca2+ transients in vivo nor evade anti-tumor immunity, as do not melanoma cells deficient in cyclooxygenases (COX). Motesanib, a VEGF receptor antagonist, suppresses Ca2+ transients, suggesting the involvement of tumor endothelial cells in the TXA2 production within tumor tissue. These resultsresults identifyresults identifyidentify TXA2 as a critical promotertargetof PGE2-dependent tumorigenesis. Citation Format: Kenta Terai, Yoshinobu Konishi, Hiroshi Ichise, Tetsuya Watabe, Yukari Sando, Takefumi Kondo, Choji Oki, Shinya Tsukiji, Yoko Hamazaki, Yasuhiro Murakawa, Akifumi Takaori-Kondo, Michiyuki Matsuda. Host cell-derived TXA2-mediated Gq signaling in tumor cells promotes tumor immune evasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-057.

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 (F520/F460) 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.