Shin Mizukami

Covalent Protein Labeling Based on Noncatalytic β-Lactamase and a Designed FRET Substrate

Techniques for labeling proteins with small molecules have attracted the attention of many life scientists. We have developed a novel protein labeling system that combines a genetically modified, noncatalytic beta-lactamase variant and specific mechanism-based fluorescent probes. Rational design of the tag protein and the labeling probes enables highly specific incorporation of the fluorogen. The feasibility of our approach was confirmed by gel electrophoresis, mass spectrometry, fluorescence spectroscopy, and fluorescence microscopic imaging. Labeling techniques that satisfy the dual criteria of specificity and fluorogenicity have rarely been reported. As a consequence, this method could be a broadly useful research tool in the field of life science.

Gilbert damping in perpendicularly magnetized Pt/Co/Pt films investigated by all-optical pump-probe technique

To investigate the correlation between perpendicular magnetic anisotropy and intrinsic Gilbert damping, time-resolved magneto-optical Kerr effect was measured in Pt/Co(dCo)/Pt films. These films showed perpendicular magnetization at dCo=1.0 nm and a perpendicular magnetic anisotropy energy Kueff that was inversely proportional to dCo. With an analysis based on the Landau–Lifshitz–Gilbert equation, the intrinsic Gilbert damping constant α was evaluated by parameter-fitting of frequency and lifetime expressions to experimental data of angular variations in spin precession frequency and life-times. The α values increased significantly with decreasing dCo but not inversely proportional to dCo.

Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase‐3, such as DEVD‐MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase‐3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase‐3‐like activity within HeLa cells treated with etoposide. The caspase‐3‐like activity was increased in the cytosol at first, then expanded to the whole cell.

Design and Synthesis of Coumarin-Based Zn2+ Probes for Ratiometric Fluorescence Imaging

The physiological roles of free Zn(2+) have attracted great attention. To clarify those roles, there has been a need for ratiometric fluorescent Zn(2+) probes for practical use. We report the rational design and synthesis of a series of ratiometric fluorescent Zn(2+) probes. The structures of the probes are based on the 7-hydroxycoumarin structure. We focused on the relationship between the electron-donating ability of the 7-hydroxy group and the excitation spectra of 7-hydroxycoumarins, and exploited that relationship in the design of the ratiometric probes; as a result, most of the synthesized probes showed ratiometric Zn(2+)-sensing properties. Then, we designed and synthesized ratiometric Zn(2+) probes that can be excited with visible light, by choosing adequate substituents on coumarin dyes. Since one of the probes could permeate living cell membranes, we introduced the probe to living RAW264 cells and observed the intracellular Zn(2+) concentration via ratiometric fluorescence microscopy. As a result, the ratio value of the probe changed quickly in response to intracellular Zn(2+) concentration.

Dynamic visualization of RANKL and Th17-mediated osteoclast function

Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static - bone resorptive (R) to moving - nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.

Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and 19F MRI

Dynamic duo: Magnetic resonance imaging (MRI) can visualize deep regions of living bodies, whereas fluorescence measurement offers excellent sensitivity. These methods thus offer signal enhancement potential for detecting enzyme activities. A dual-mode off/on probe to detect caspase-3 activity by fluorescence and (19)F MRI is presented.

Lanthanide-Based Protease Activity Sensors for Time-Resolved Fluorescence Measurements

Lanthanide-based luminescent sensors are noteworthy because their long-lifetime luminescence enables time-resolved fluorescence measurements. After exploring suitable antenna groups, we designed and synthesized lanthanide-based luminogenic sensors detecting protease activities. This sensor yielded strong luminescence on addition of proteases such as calpain I and leucine aminopeptidase (LAP). Since the luminescence lifetimes of the probes were very long, the sensors could be applied to time-resolved measurements that exclude background fluorescence signals derived from proteins or other impurities. This sensing principle could be applicable to general time-resolved fluorescence assays for other proteases.

Photoactive Yellow Protein-Based Protein Labeling System with Turn-On Fluorescence Intensity

Protein labeling provides significant information about protein function. In this research, we developed a novel protein labeling technique by utilizing photoactive yellow protein (PYP). PYP is a small protein (14 kDa) derived from purple bacteria and binds to 7-hydroxycoumarin-3-carboxylic acid as well as to a natural ligand, 4-hydroxycinnamic acid, through a thioester bond with Cys69. Based on the structure and fluorescence property of this coumarin derivative, we designed two fluorescent probes that bind to PYP. One has an azido moiety, which allows stepwise labeling by click chemistry, and the other is a fluorogenic probe. The live-cell imaging and specific labeling of PYP were achieved by using both probes. The flexibility of the probe design and the small size of the tag protein are great advantages of this system against the existing methods. This novel labeling technique can be used in a wide variety of applications for biological research.

In Vivo Fluorescence Imaging of Bone-Resorbing Osteoclasts

Osteoclasts are giant polykaryons responsible for bone resorption. Because an enhancement or loss of osteoclast function leads to bone diseases such as osteoporosis and osteopetrosis, real-time imaging of osteoclast activity in vivo can be of great help for the evaluation of drugs. Herein, pH-activatable chemical probes BAp-M and BAp-E have been developed for the detection of bone-resorbing osteoclasts in vivo. Their acid dissociation constants (pK(a)) were determined as 4.5 and 6.2 by fluorometry in various pH solutions. These pK(a) values should be appropriate to perform selective imaging of bone-resorbing osteoclasts, because synthesized probes cannot fluoresce intrinsically at physiological pH and the pH in the resorption pit is lowered to about 4.5. Furthermore, BAp-M and BAp-E have a bisphosphonate moiety that enabled the probes to localize on bone tissues. The hydroxyapatite (HA) binding assay in vitro was, therefore, performed to confirm the tight binding of the probes to the bone tissues. Our probes showed intense fluorescence at low pH values but no fluorescence signal under physiological pH conditions on HA. Finally, we applied the probes to in vivo imaging of osteoclasts by using intravital two-photon microscopy. As expected, the fluorescence signals of the probes were locally observed between the osteoclasts and bone tissues, that is, in resorption pits. These results indicate that our pH-activatable probes will prove to be a powerful tool for the selective detection of bone-resorbing osteoclasts in vivo, because this is the first instance where in vivo imaging has been conducted in a low-pH region created by bone-resorbing osteoclasts.

Fast magnetization precession observed in L10-FePt epitaxial thin film

Fast magnetization precession is observed in L10-FePt alloy epitaxial thin films excited and detected by all-optical means. The precession frequency varies from 45 to 65 GHz depending on the applied magnetic field strength and direction, which can be explained by a uniform precession model taking account of first- and second-order uniaxial magnetic anisotropy. The lowest effective Gilbert damping constant has a minimum value of 0.055, which is about half that in Co/Pt multilayers and is comparable to Ni/Co multilayers with perpendicular magnetic anisotropy.