Quantitative control of subcellular protein localization with a photochromic dimerizer.

Artificial control of intracellular protein dynamics with high precision provides deep insight into complicated biomolecular networks. Optogenetics and caged compound-based chemically induced dimerization (CID) systems are emerging as tools for spatiotemporally regulating intracellular protein dynamics. However, both technologies face several challenges for accurate control such as the duration of activation, deactivation rate and repetition cycles. Herein, we report a photochromic CID system that uses the photoisomerization of a ligand so that both association and dissociation are controlled by light, enabling quick, repetitive and quantitative regulation of the target protein localization upon illumination with violet and green light. We also demonstrate the usability of the photochromic CID system as a potential tool to finely manipulate intracellular protein dynamics during multicolor fluorescence imaging to study diverse cellular processes. We use this system to manipulate PTEN-induced kinase 1 (PINK1)-Parkin-mediated mitophagy, showing that PINK1 recruitment to the mitochondria can promote Parkin recruitment to proceed with mitophagy.

Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System.

The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein-protein interactions. However, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein-labeling technology by applying a caged ligand to the BL-tag system, a covalent protein labeling system that uses mutant ß-lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL-tag and HaloTag ligands, and achieved light-induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo-CID system enabled quick protein translocation to a laser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Light-Wavelength-Based Quantitative Control of Dihydrofolate Reductase Activity by Using a Photochromic Isostere of an Inhibitor.

Photopharmacology has attracted research attention as a new tool for achieving optical control of biomolecules, following the methods of caged compounds and optogenetics. We have developed an efficient photopharmacological inhibitor-azoMTX-for Escherichia coli dihydrofolate reductase (eDHFR) by replacing some atoms of the original ligand, methotrexate, to achieve photoisomerization properties. This fine molecular design enabled quick structural conversion between the active "bent" Z isomer of azoMTX and the inactive "extended" E isomer, and this property afforded quantitative control over the enzyme activity, depending on the wavelength of irradiating light applied. Real-time photoreversible control over enzyme activity was also achieved.

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Perfluorocarbon-Based 19 F†MRI Nanoprobes for In†Vivo Multicolor Imaging.

In vivo multicolor imaging is important for monitoring multiple biomolecular or cellular processes in biology. 19 F magnetic resonance imaging (MRI) is an emerging in vivo imaging technique because it can non-invasively visualize 19 F nuclei without endogenous background signals. Therefore, 19 F MRI probes capable of multicolor imaging are in high demand. Herein, we report five types of perfluorocarbon-encapsulated silica nanoparticles that show 19 F NMR peaks with different chemical shifts. Three of the nanoprobes, which show spectrally distinct 19 F NMR peaks with sufficient sensitivity, were selected for in vivo multicolor 19 F MRI. The nanoprobes exhibited 19 F MRI signals with three colors in a living mouse. Our in vivo multicolor system could be utilized for evaluating the effect of surface functional groups on the hepatic uptake in a mouse. This novel multicolor imaging technology will be a practical tool for elucidating in vivo biomolecular networks by 19 F MRI.

Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular Dynamics.

Single-molecule imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein-protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant ß-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, single-molecule imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein-protein interaction in response to ligand stimulation.

Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.

Transcellular Mg(2+) transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg(2+) extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg(2+) extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg(2+) by exchanging intracellular Mg(2+) with extracellular Na(+). Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg(2+) extrusion activity. These results demonstrate the crucial importance of Mg(2+) extrusion by CNNM4 in organismal and topical regulation of magnesium.

Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of ß-lactamase, which forms specific covalent bonds with ß-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic ß-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

Activatable 19F MRI nanoparticle probes for the detection of reducing environments.

(19)F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the (19)F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction-responsive PFC-encapsulated nanoparticle probe, FLAME-SS-Gd(3+) (FSG). This is the first example of an activatable PFC-encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd(3+) complexes per nanoparticle was more than approximately 5.0×10(2), resulting in the high signal augmentation.

Multifunctional core­shell silica nanoparticles for highly sensitive (19)F magnetic resonance imaging.

19F magnetic resonance imaging (19F MRI) is useful for monitoring particular signals from biological samples, cells, and target tissues, because background signals are missing in animal bodies. Therefore, highly sensitive 19F MRI contrast agents are in great demand for their practical applications. However, we have faced the following challenges: 1) increasing the number of fluorine atoms decreases the solubility of the molecular probes, and 2) the restriction of the molecular mobility attenuates the 19F MRI signals. Herein, we developed novel multifunctional core­shell nanoparticles to solve these issues. They are composed of a core micelle filled with liquid perfluorocarbon and a robust silica shell. These core­shell nanoparticles have superior properties such as high sensitivity, modifiability of the surface, biocompatibility, and sufficient in vivo stability. By the adequate surface modifications, gene expression in living cells and tumor tissue in living mice were successfully detected by 19F MRI.

Development of luminescent coelenterazine derivatives activatable by ß-galactosidase for monitoring dual gene expression.

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect ß-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing ß-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain ß-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for ß-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect ß-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by ß-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.

pH induced dual "OFF-ON-OFF" switch: influence of a suitably placed carboxylic acid.

The design and synthesis of molecular probes competent for pH signaling within or beyond a certain range is a complicated matter. Herein a new mechanism for ''OFF-ON-OFF'' absorbance and fluorescence intensities vs. pH behaviour is described. The probe design is based on the connection of carboxylic acid derivatized benzoxazole and 7-hydroxycoumarin/iminocoumarin parts. The protonation/deprotonation of the carboxylic acid (-COOH), N atom of benzoxazole ring and hydroxy part of the coumarin ring have been used for this mechanistic study. We have designed the molecule in such a fashion that deprotonation of the hydroxy part takes place at a lower pK(a) compared to deprotonation of the -COOH. The dual ''OFF-ON-OFF'' properties of our probes depend on the C-C bond between the two different heterocyclic parts. Quantum mechanical calculations showed that the particular 'C-C' bond has an additional π-character. The twisting around this bond in different forms is responsible for such an ''OFF-ON-OFF'' property. This mechanism is new in fluorescence alteration processes. The delocalization of charge from one heterocyclic part to the other heterocyclic part in the mono- and dianionic forms controls the ''OFF-ON-OFF'' properties. The role of the carboxylic acid group was examined using an acetyl substituted derivative. One of our probes was successfully applied in live cell imaging studies in media at different pH.

Cellsketch: Simplified Cell Representation for Label-free Cell and Nuclei Segmentation.

This paper presents a novel technique for cell segmentation, named "Cellsketch," which generates an RGB mask containing simplified representations of cells (including nuclei, whole-cell, and cell boundaries) from microscopic images, and applies the watershed algorithm to produce segmentation masks of cells and nuclei. The RGB mask is generated using a generator model trained with a combination of L1 loss and adversarial loss. The method achieved accurate cell and nuclei segmentation from differential interference contrast (DIC) images using only automatically annotated training data and shows potential for a generalizable algorithm for cell segmentation. The code is freely available at: https://github.com/iranovianti/cellsketchClinical Relevance- This method simultaneously detects individual cells and nuclei from DIC images.

Long-term imaging of intranuclear Mg2+ dynamics during mitosis using a localized fluorescent probe.

A novel fluorescent Mg2+ probe was developed based on a small molecule-protein hybrid. This probe enables subcellular targeting, long-term imaging, and high selectivity for Mg2+ over Ca2+. Using ratiometric fluorescence microscopy with a co-localized standard fluorophore, the variations in intranuclear Mg2+ concentrations during mitosis could be visualized.

Arylazopyrazole-Based Photoswitchable Inhibitors Selective for Escherichia coli Dihydrofolate Reductase.

Selective inhibitors of Escherichia coli dihydrofolate reductase (eDHFR) are crucial chemical biology tools that have widespread clinical applications. We developed a set of eDHFR-selective photoswitchable inhibitors by derivatizing the structure of our previously reported methotrexate (MTX) azolog, azoMTX. Substitution of the skeletal p-phenylene group of azoMTX with bulky bis-alkylated arylazopyrazole moieties significantly increased its selectivity toward eDHFR over human DHFR. Owing to the physical properties of arylazopyrazoles, the new ligands exhibited nearly complete Z-to-E photoconversion and high thermostability of Z-isomers. In addition, real-time photoreversible control of eDHFR activity was achieved by alternatively switching the illumination light wavelengths.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis.

Small molecule labeling techniques for cellular proteins under physiological conditions are very promising for revealing new biological functions. We developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant ß-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegradation were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase analysis, the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addition, membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins.

Development of a fluorogenic probe with a transesterification switch for detection of histone deacetylase activity.

Histone deacetylases (HDACs) are key enzymatic regulators of many cellular processes such as gene expression, cell cycle, and tumorigenesis. These enzymes are attractive targets for drug development. However, very few simple methods for monitoring HDAC activity have been reported. Here, we have developed a fluorogenic probe, K4(Ac)-CCB, which consists of the histone H3 peptide containing acetyl-Lys and a coumarin fluorophore with a carbonate ester. By the simple addition of the probe to a HDAC solution, enzyme activity was clearly detected through spontaneous intramolecular transesterification, which renders the probe fluorescent. In addition, K4(Ac)-CCB can be applied to the evaluation of HDAC inhibitor activity. This is the first report to demonstrate the monitoring of HDAC activity by using a one-step procedure. Thus, our novel fluorogenic probe will provide a powerful tool for epigenetic research and the discovery of HDAC-targeted drugs.

Simple and real-time colorimetric assay for glycosidases activity using functionalized gold nanoparticles and its application for inhibitor screening.

The development of real-time assays for enzymes has been receiving a great deal of attention in biomedical research recently. Self-immolative elimination is the spontaneous and irreversible disassembly of a multicomponent construct into its constituent fragments through a cascade of elimination processes, in response to external stimuli. Here, we report a simple and real-time colorimetric assay for glycosidases (ß-galactosidase and ß-glucosidase). Self-immolative elimination was utilized to release amines to give rise to aggregation and color change by electrostatic attraction after cleavage of the trigger by enzymes displayed on functionalized gold nanoparticles (Gal-Lip-AuNPs and Glc-Lip-AuNPs, where AuNPs denotes gold nanoparticles). The detection limits for ß-galactosidase and ß-glucosidase were as low as 9.2 and 22.3 nM at 20 min, and they improved slightly over time. Thus, glycosidase activity was detected successfully in real time, and this technique could be used for glycosidase inhibitor screening, based on real-time colorimetric variation.