A photoactivatable self-localizing ligand with improved photosensitivity for chemo-optogenetic control of protein localization in living cells.

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 Ca2+ oscillations using a GFP-based Ca2+ biosensor in living cells.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

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.

Chemo-optogenetic Protein Translocation System Using a Photoactivatable Self-Localizing Ligand.

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.

Intravital Imaging Identifies the VEGF-TXA2 Axis as a Critical Promoter of PGE2 Secretion from Tumor Cells and Immune Evasion.

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.

Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells.

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.

Designer Palmitoylation Motif-Based Self-Localizing Ligand for Sustained Control of Protein Localization in Living Cells and Caenorhabditis elegans.

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.

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

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.

Stabilization of Charge Carriers in Picket-Fence Polythiophenes Using Dielectric Side Chains.

Insulated molecular wires (IMWs) are π-conjugated polymers that are molecularly sheathed with an insulating layer and are structurally analogous to electric power cords at the nanoscale. Such unique architectures are expected in molecular electronics and organic devices. Herein, we propose a new molecular design concept of IMWs, in which the sheaths can be customized, thereby enabling the modulation of the electronic properties of the interior π-conjugated systems. To this end, we focused our attention on the dielectric constant of the sheaths, as it governs the electrostatic interaction between charges. Upon doping, charge carriers, such as polaron and bipolaron, were generated regardless of the dielectric properties of the sheaths. Flash-photolysis time-resolved microwave conductivity measurements revealed that intrawire charge carrier mobility was independent of the sheaths. However, we found that the charge carriers could be stabilized by the sheaths with a high dielectric constant owing to the charge screening effect. We expect that IMWs designed in this way will be useful in a variety of applications, where the nature of charge carriers plays an important role, and particularly when redox switching is required (e.g., electrochromic, magnetic, and memory applications).

Synthetic self-localizing ligands that control the spatial location of proteins in living cells.

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.