Nanoscale dynamics and localization of single endogenous mRNAs in stress granules.

Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy.

Fully automated immunoassay for cholesterol uptake capacity to assess high-density lipoprotein function and cardiovascular disease risk.

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification.

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.

Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy.

Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores.

Spontaneously Blinking Fluorophores Based on Nucleophilic Addition/Dissociation of Intracellular Glutathione for Live-Cell Super-resolution Imaging.

Single-molecule localization microscopy (SMLM) allows the reconstruction of super-resolution images but generally requires prior intense laser irradiation and in some cases additives to induce blinking of conventional fluorophores. We previously introduced a spontaneously blinking rhodamine fluorophore based on an intramolecular spirocyclization reaction for live-cell SMLM under physiological conditions. Here, we report a novel principle of spontaneous blinking in living cells, which utilizes reversible ground-state nucleophilic attack of intracellular glutathione (GSH) upon a xanthene fluorophore.

Activatable fluorescent probes for hydrolase enzymes based on coumarin-hemicyanine hybrid fluorophores with large Stokes shifts.

We show that the equilibrium of intramolecular spirocyclization of coumarin-hemicyanine hybrid fluorophores can be finely tuned by means of chemical modifications. We used this scaffold to develop activatable fluorescent probes with large Stokes shifts for γ-glutamyltranspeptidase and esterase.

A novel liver-specific fluorescent anti-cancer drug delivery system using indocyanine green.

Indocyanine green (ICG) accumulates only in hepatocytes and their malignant counterpart, hepatocellular carcinoma (HCC). We have developed ICG-conjugated anti-cancer drugs and noted their significant accumulation in HCC cells both in vitro and in vivo. ICG-conjugated gemcitabine was less toxic to normal cells and it had superior anti-tumor action compared to gemcitabine alone in a subcutaneous tumor xenograft.

Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET Pairs in a Water Matrix.

We prepared a pair with a visible-absorbing donor dye and a near-infrared fluorescing acceptor dye. The donor and the acceptor were covalently linked close enough for Förster resonance energy transfer to occur. Under cryogenic conditions at 1.

A green-light-emitting, spontaneously blinking fluorophore based on intramolecular spirocyclization for dual-colour super-resolution imaging.

We have developed the first green-light-emitting, spontaneously blinking fluorophore (SBF), HEtetTFER. In combination with our near-infrared-light-emitting SBF (HMSiR), HEtetTFER allows dual-colour single-molecule localization microscopy (SMLM) in buffer solution without any additive and without photoactivation.

Long time-lapse nanoscopy with spontaneously blinking membrane probes.

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds.

A guide to use photocontrollable fluorescent proteins and synthetic smart fluorophores for nanoscopy.

Recent advances in nanoscopy, which breaks the diffraction barrier and can visualize structures smaller than the diffraction limit in cells, have encouraged biologists to investigate cellular processes at molecular resolution. Since nanoscopy depends not only on special optics but also on 'smart' photophysical properties of photocontrollable fluorescent probes, including photoactivatability, photoswitchability and repeated blinking, it is important for biologists to understand the advantages and disadvantages of fluorescent probes and to choose appropriate ones for their specific requirements. Here, we summarize the characteristics of currently available fluorescent probes based on both proteins and synthetic compounds applicable to nanoscopy and provide a guideline for selecting optimal probes for specific applications.

A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction.

The effect of linker length on binding affinity of a photoswitchable molecular glue for DNA.

Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external light stimuli to reversibly control DNA hybridization. To optimize the structure of PMG, we synthesized a series of PMGs and evaluated the effect of changing the methylene linker length on the binding affinity and photoresponse.

Synthesis of dimeric 2-amino-1,8-naphthyridine and related DNA-binding molecules.

The synthetic protocols for dimeric 2-amino-1,8-naphthyridine and related compounds are described in this unit. These compounds represent a novel class of compounds that bind selectively to mismatched base pairs. The compounds consist of two main components: the heterocycles and a linker.

Reversible control of DNA hybridization by photoresponsive ligands.

Mismatch binding ligands (MBL) can act as a molecular glue which brings two single stranded DNAs (ssDNA) together to form a double stranded DNA (dsDNA). We here demonstrate that photoresponsive MBL possessing a photochromic azobenzene chromophore can control duplex stability reversibly in response to external light stimuli. Synthesis and evaluation of a series of NCDA derivatives reveals significant correlation between the linker structures in MBLs and the molecular glue function.

Control of DNA hybridization by photoswitchable mismatch binding ligands.

We herein demonstrate that mismatch binding ligands (MBL) can function as a molecular glue which brings two single stranded DNA (ssDNA) together to form the double stranded DNA (dsDNA). Incorporation of a photoisomerizable azobenzene linkage provides further ability of reversibly controlling duplex stability with light.