Takashi Tanii

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 microM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro‐modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 μm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 μm for the latter. Axon formation was evaluated by tau‐1/MAP2 immunostaining and live‐cell imaging of constitutively‐active kinesin‐1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.

Single-molecule study on the decay process of the football-shaped GroEL-GroES complex using zero-mode waveguides

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)2 complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the “football cycle ” and the “bullet cycle,” in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3–5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.

Array of bright silicon-vacancy centers in diamond fabricated by low-energy focused ion beam implantation

Among promising color centers for single-photon sources in diamond, the negatively charged silicon-vacancy (SiV−) has 70% of its emission to the zero-phonon line (ZPL), in contrast to the negatively charged nitrogen vacancy (NV−), which has a broad spectrum. Fabricating single centers of useful defect complexes with high yield and excellent grown-in defect properties by ion implantation has proven to be challenging. We have fabricated bright single SiV− centers by 60-keV focused ion beam implantation and subsequent annealing at 1000 °C with high positioning accuracy and a high yield of 15%.

Enhancement of field emission characteristics of tungsten emitters by single-walled carbon nanotube modification

We present a simple method for improving the field emission performance of tungsten-tip electron sources based on single-walled carbon nanotube (SWCNT) modification. By coating a sandwich-like thin film of Al–Fe–Al (with Fe as a catalyst) on a tungsten tip, SWCNTs were synthesized at 600 °C in a chemical vapor deposition (CVD) reactor. The influence of CNT modification on the electron emission characteristics of the emitters was investigated by means of a triode structure. We have found that CNT-modified tungsten tips exhibit low threshold-voltage for electron emission, and improved emission-current stability, compared with nonmodified and Al–Fe–Al-coated needles.

Green Tea Epigallocatechin Gallate Exhibits Anticancer Effect in Human Pancreatic Carcinoma Cells via the Inhibition of Both Focal Adhesion Kinase and Insulin-Like Growth Factor-I Receptor

The exact molecular mechanism by which epigallocatechin gallate (EGCG) suppresses human pancreatic cancer cell proliferation is unclear. We show here that EGCG-treated pancreatic cancer cells AsPC-1 and BxPC-3 decrease cell adhesion ability on micro-pattern dots, accompanied by dephosphorylations of both focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) whereas retained the activations of mitogen-activated protein kinase and mammalian target of rapamycin. The growth of AsPC-1 and BxPC-3 cells can be significantly suppressed by EGCG treatment alone in a dose-dependent manner. At a dose of 100 μM which completely abolishes activations of FAK and IGF-1R, EGCG suppresses more than 50% of cell proliferation without evidence of apoptosis analyzed by PARP cleavage. Finally, the MEK1/2 inhibitor U0126 enhances growth-suppressive effect of EGCG. Our data suggests that blocking FAK and IGF-1R by EGCG could prove valuable for targeted therapy, which can be used in combination with other therapies, for pancreatic cancer.

Effect of the C-terminal Truncation on the Functional Cycle of Chaperonin GroEL IMPLICATION THAT THE C-TERMINAL REGION FACILITATES THE TRANSITION FROM THE FOLDING-ARRESTED TO THE FOLDING-COMPETENT STATE

To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by ∼2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of ∼3- and ∼5-s duration ( Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434 g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an ∼5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.

In-situ guidance of individual neuronal processes by wet femtosecond-laser processing of self-assembled monolayers

In-situ guidance of neuronal processes (neurites) is demonstrated by applying wet femtosecond-laser processing to an organosilane self-assembled monolayer (SAM) template. By scanning focused laser beam between cell adhesion sites, on which primary neurons adhered and extended their neurites, we succeeded in guiding the neurites along the laser-scanning line. This guidance was accomplished by multiphoton laser ablation of cytophobic SAM layer and subsequent adsorption of cell adhesion molecule, laminin, onto the ablated region. This technique allows us to arbitrarily design neuronal networks in vitro.

Measuring Relative-Story Displacement and Local Inclination Angle Using Multiple Position-Sensitive Detectors

We propose a novel sensor system for monitoring the structural health of a building. The system optically measures the relative-story displacement during earthquakes for detecting any deformations of building elements. The sensor unit is composed of three position sensitive detectors (PSDs) and lenses capable of measuring the relative-story displacement precisely, even if the PSD unit was inclined in response to the seismic vibration. For verification, laboratory tests were carried out using an Xθ-stage and a shaking table. The static experiment verified that the sensor could measure the local inclination angle as well as the lateral displacement. The dynamic experiment revealed that the accuracy of the sensor was 150 μm in the relative-displacement measurement and 100 μrad in the inclination angle measurement. These results indicate that the proposed sensor system has sufficient accuracy for the measurement of relative-story displacement in response to the seismic vibration.

Fabrication of adenosine triphosphate-molecule recognition chip by means of bioluminous enzyme luciferase

We have succeeded in detecting the adenosine triphosphate (ATP) concentration in a solution quantitatively using an ATP-molecule recognition chip. The ATP-molecule recognition chip is composed of a silicon photodiode on which bioluminous enzyme luciferase is immobilized. When the chip was immersed in an ATP-containing solution, the luciferase emitted light and the photoinduced current detected by the photodiode was in proportion to the ATP concentration. We found that the photoinduced current fits the Michaelis-Menten plot. These results indicate that the luciferase is successfully immobilized on the silicon chip without losing the bioluminous activity and that the proposed device enables us to detect the ATP concentration in a solution by measuring the photoinduced current.