An orange fluorescent glutamate sensor for multicolor single-synapse imaging.

High-resolution imaging of glutamate is essential for understanding synaptic transmission. While fluorescent sensors capable of detecting glutamate release with single-synapse resolution have been developed, these sensors are almost exclusively green-emitting. This spectral constraint hinders multicolor imaging experiments designed to investigate the interplay between glutamate dynamics and other cellular activities.

Recent advances in cancer detection using dynamic, stimuli-responsive supramolecular chemosensors. a focus review.

In current chemistry, supramolecular materials that respond to a wide variety of external stimuli, such as solvents, temperature, light excitation, pH, and mechanical forces (pressure, stress, strain, and tension), have attracted considerable attention; for example, we have developed cyclodextrins, cucurbiturils, pillararenes, calixarenes, crown ether-based chemical sensors, or chemosensors. These supramolecular chemosensors have potential applications in imaging, probing, and cancer detection. Recently, we focused on pressure, particularly solution-state hydrostatic pressure, from the viewpoint of cancer therapy.

Quantitative modeling of regular retinal microglia distribution.

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia's morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated.

Discovery of an F-actin-binding small molecule serving as a fluorescent probe and a scaffold for functional probes.

Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. There is a growing demand for practical and accessible tools for dissecting the role of the actin cytoskeleton in cellular function, and the discovery of a new actin-binding small molecule is an important advance in the field, offering the opportunity to design and synthesize of new class of functional molecules. Here, we found an F-actin–binding small molecule and introduced two powerful tools based on a new class of actin-binding small molecule: One enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green light–controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor.

Adenosine 5'-triphosphate (ATP) works as an extracellular signaling molecule for cells in the brain, such as neurons and glia. Cellular communication via release of ATP is involved in a range of processes required for normal brain functions, and aberrant communication is associated with brain disorders. To investigate the mechanisms underlying these cellular processes, various techniques have been developed for the measurement of extracellular ATP.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor.

Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, FoF-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue.

Synaptic weight set by Munc13-1 supramolecular assemblies.

The weight of synaptic connections, which is controlled not only postsynaptically but also presynaptically, is a key determinant in neuronal network dynamics. The mechanisms controlling synaptic weight, especially on the presynaptic side, remain elusive. Using single-synapse imaging of the neurotransmitter glutamate combined with super-resolution imaging of presynaptic proteins, we identify a presynaptic mechanism for setting weight in central glutamatergic synapses.

Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model.

GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of β-hexosaminidase A, which comprises an αβ heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring β-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit β (HexB), which we have termed mod2B, composed of homodimeric β subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance.

Novel Hexosaminidase-Targeting Fluorescence Probe for Visualizing Human Colorectal Cancer.

Precise tumor diagnosis and evaluation of disease extent are crucial for treatment of solid cancers. In order to complement the limited ability of the unaided human eye to discriminate tumor tissue and normal tissue, we have developed a series of fluorescence probes activatable specifically in cancer tissues. Here, we describe the design, synthesis, and application of a new fluorescence probe targeting hexosaminidase (HMRef-βGlcNAc), which is located in lysosomes and is overexpressed in several carcinomas, including colorectal cancer.

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain.

Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo.

Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on β-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-βGal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation.

High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission.

Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high-throughput screening system, designated hybrid-type fluorescence indicator development (HyFInD), to identify optimal position-specific fluorophore labeling in hybrid-type sensors consisting of combinations of ligand-binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine-reactive fluorescence probes with a set of cysteine-scanning mutants of the 274 amino acid S1S2 domain of AMPA-type glutamate receptor GluA2 subunit.

High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.

Overexpression of growth factor receptors in cancers, e.g., human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness.

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.

Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane.

Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics.

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization.

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme.

Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe.

Objectives: Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer.

Methods: Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry.

Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase-activated fluorescent probe.

The ability of the unaided human eye to detect small cancer foci or accurate borders between cancer and normal tissue during surgery or endoscopy is limited. Fluorescent probes are useful for enhancing visualization of small tumors but are typically limited by either high background signal or the requirement for administration hours to days before use. We synthesized a rapidly activatable, cancer-selective fluorescence imaging probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), with intramolecular spirocyclic caging for complete quenching.

β-Galactosidase fluorescence probe with improved cellular accumulation based on a spirocyclized rhodol scaffold.

We identified a rhodol bearing a hydroxymethyl group (HMDER) as a suitable scaffold for designing fluorescence probes for various hydrolases. HMDER shows strong fluorescence at physiological pH, but phenolic O-alkylation of HMDER results in a strong preference for the spirocyclic form, which has weak fluorescence. As a proof of concept, we utilized this finding to develop a new fluorescence probe for β-galactosidase.

Intraperitoneal photodynamic therapy mediated by a fullerene in a mouse model of abdominal dissemination of colon adenocarcinoma.

Unlabelled: Functionalized fullerenes represent a new class of photosensitizer (PS) that is being investigated for photodynamic therapy (PDT) of various diseases, including cancer. We tested the hypothesis that fullerenes could be used to mediate PDT of intraperitoneal (IP) carcinomatosis in a mouse model. In humans this form of cancer responds poorly to standard treatment and manifests as a thin covering of tumor nodules on intestines and on other abdominal organs.

Fluorescence imaging of tumors with "smart" pH-activatable targeted probes.

One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background originated from non-target tissues. In this study, a "smart" activatable strategy for specific tumor imaging is proposed in which pH-activatable targeted probes specifically detect tumors after binding to the target cell surface proteins, internalization, and eventual acidic pH activation within the acidic organelles. We successfully visualized submillimeter-sized tumors using this strategy in two different tumor mouse models.

Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes.

A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe.