研究者詳細 - 内橋　貴之

高速原子間力顕微鏡による生体・合成高分子の動態イメージング招待有り査読有り

高分子 68 巻 ( 10 ) 頁： 564 - 568 2019年10月

Protein uptake into individual hydrogel microspheres visualized by high-speed atomic force microscopy 査読有り国際誌

Matsui, S; Hosho, K; Minato, H; Uchihashi, T; Suzuki, D

CHEMICAL COMMUNICATIONS 55 巻 ( 68 ) 頁： 10064 - 10067 2019年9月

記述言語：英語出版者・発行元：Chemical Communications

The moment of protein uptake into hydrogel microspheres (microgels) was directly monitored at the nanoscale by high-speed atomic force microscopy. The dynamic morphological changes in the microgels during protein uptake are largely different depending on the type of protein, and the properties and structure of the microgels, which would affect the colloidal stability of protein-microgel hybrids both in vitro and in vivo.

DOI： 10.1039/c9cc05116c

Evaluation of thermoresponsive structural changes in hydrogel microspheres by high-speed atomic force microscopy

Nishizawa, Y; Matsui, S; Urayama, K; Kureha, T; Sibayama, M; Uchihashi, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 巻 2019年8月

Assembly of rod-shaped hydrogel microspheres at the air/water interface

Honda, K; Sazuka, Y; Iizuka, K; Matsui, S; Uchihashi, T; Kureha, T; Shibayama, M; Watanabe, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 巻 2019年8月

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III 査読有り国際誌 Open Access

Yogo, R; Yamaguchi, Y; Watanabe, H; Yagi, H; Satoh, T; Nakanishi, M; Onitsuka, M; Omasa, T; Shimada, M; Maruno, T; Torisu, T; Watanabe, S; Higo, D; Uchihashi, T; Yanaka, S; Uchiyama, S; Kato, K

SCIENTIFIC REPORTS 9 巻 ( 1 ) 頁： 11957 - 11957 2019年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

DOI： 10.1038/s41598-019-48323-w

Atomically Controlled Surfaces, Interfaces and Nanostructures/Scanning Probe Microscopy FOREWORD Open Access

Takahashi, T; Fujita, D; Fukidome, H; Fukui, K; Hibino, H; Kageshima, M; Komeda, T; Nakajima, K; Nakamura, M; Nakayama, T; Nohira, H; Sasakawa, K; Sasaki, N; Sumitomo, K; Uchihashi, T; Watanabe, T; Yamada, Y

JAPANESE JOURNAL OF APPLIED PHYSICS 58 巻 ( SI ) 2019年8月

Development of wide-area tip-scanning high-speed atomic force microscopy

Watanabe H., Uchihashi T.

2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings 頁： 281 - 285 2019年8月

記述言語：日本語出版者・発行元：2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings

Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to ∼20 μm and ∼3 μm in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to ∼50 μm and ∼32 μm, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

DOI： 10.1109/3M-NANO46308.2019.8947429

Real-time nanoscale visualization of biological molecules at work with high-speed atomic force microscopy

Uchihashi T.

2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings 頁： 253 - 256 2019年8月

記述言語：日本語出版者・発行元：2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings

Atomic force microscopy (AFM) is a powerful tool to visualize biological molecules at nm resolution in liquid conditions and thus has been applied to a wide variety of biological molecules. One of the most coveted functions of AFM is 'fast recording' because it should allow us to directly visualize dynamic processes of biological molecules at work. We have been challenging to break this limitation over 15 years and established fast imaging of dynamic molecular processes. The most advanced high-speed AFM can now capture successive images at 30-60 ms/frame. Further, the interaction force between the tip and the sample, which significantly influences on soft biological molecules, is greatly reduced without deterioration of the scanning performance. In this short paper, typical applications of HS-AFM on biological and artificial molecules will be demonstrated.

DOI： 10.1109/3M-NANO46308.2019.8947375

Dynamic structural states of ClpB involved in its disaggregation function (vol 9, 2147, 2018) 査読有り国際誌 Open Access

Uchihashi, T; Watanabe, YH; Nakazaki, Y; Yamasaki, T; Watanabe, H; Maruno, T; Ishii, K; Uchiyama, S; Song, CH; Murata, K; Iino, R; Ando, T

NATURE COMMUNICATIONS 10 巻 ( 1 ) 頁： 3079 - 3079 2019年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

The original version of this Article did not acknowledge Ryota Iino as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article.

DOI： 10.1038/s41467-019-11204-x

Dynamics of Inter-Molecular Interactions Between Single Aβ<sub>42</sub> Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy 査読有り国際誌

Feng, L; Watanabe, H; Molino, P; Wallace, GG; Phung, SL; Uchihashi, T; Higgins, MJ

JOURNAL OF MOLECULAR BIOLOGY 431 巻 ( 15 ) 頁： 2687 - 2699 2019年7月

記述言語：英語出版者・発行元：Journal of Molecular Biology

Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ42 oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ42 species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ42 species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ42 species. The findings revealed that smaller Aβ42 species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to “permissive” binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.

DOI： 10.1016/j.jmb.2019.04.044

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses 査読有り国際誌 Open Access

Krayukhina, E; Yokoyama, M; Hayashihara, KK; Maruno, T; Noda, M; Watanabe, H; Uchihashi, T; Uchiyama, S

JOURNAL OF PHARMACEUTICAL SCIENCES 108 巻 ( 7 ) 頁： 2278 - 2287 2019年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Pharmaceutical Sciences

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

DOI： 10.1016/j.xphs.2019.02.002

High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit Open Access

Sumino A., Sumikama T., Uchihashi T., Oiki S.

SCIENCE ADVANCES 5 巻 ( 7 ) 2019年7月

記述言語：日本語出版者・発行元：Science Advances

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

DOI： 10.1126/sciadv.aax0495

Open Access

High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit 査読有り Open Access

A. Sumino, T. Sumikama, T. Uchihashi, S. Oiki

Science Advances 5 巻 ( 7 ) 頁： 1 - 10 2019年7月

Non-Thermoresponsive Decanano-sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature-Controlled High-Speed Atomic Force Microscopy 査読有り国際誌

Nishizawa, Y; Matsui, S; Urayama, K; Kureha, T; Shibayama, M; Uchihashi, T; Suzuki, D

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 58 巻 ( 26 ) 頁： 8809 - 8813 2019年6月

記述言語：英語出版者・発行元：Angewandte Chemie - International Edition

Despite the tremendous efforts devoted to the structural analysis of hydrogel microspheres (microgels), many details of their structures remain unclear. Reported in this study is that thermoresponsive poly(N-isopropyl acrylamide) (pNIPAm)-based microgels exhibit not only the widely accepted core–shell structures, but also inhomogeneous decanano-sized non-thermoresponsive spherical domains within their dense cores, which was revealed by temperature-controlled high-speed atomic force microscopy (TC-HS-AFM). Based on a series of experiments, it is concluded that the non-thermoresponsive domains are characteristic for pNIPAm microgels synthesized by precipitation polymerization, and plausible structures for microgels prepared by other polymerization techniques are proposed.

DOI： 10.1002/anie.201903483

Non‐Thermoresponsive Decanano‐sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature‐Controlled High‐Speed Atomic Force Microscopy 査読有り

Yuichiro Nishizawa, Shusuke Matsui, Kenji Urayama, Takuma Kureha, Mitsuhiro Shibayama, Takayuki Uchihashi, Daisuke Suzuki

Angewandte Chemie 131 巻 ( 26 ) 頁： 8901 - 8905 2019年6月

Hydrogel Microellipsoids that Form Robust String-Like Assemblies at the Air/Water Interface 査読有り国際誌

Honda, K; Sazuka, Y; Iizuka, K; Matsui, S; Uchihashi, T; Kureha, T; Shibayama, M; Watanabe, T; Suzuki, D

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 58 巻 ( 22 ) 頁： 7294 - 7298 2019年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Angewandte Chemie - International Edition

Soft colloidal particles such as hydrogel microspheres assemble at air/water or oil/water interfaces, where the soft colloids are highly deformed and their surface polymer chains are highly entangled with each other. Herein, we report the formation of robust one-dimensional, string-like colloidal assemblies through self-organization of hydrogel microspheres with shape anisotropy at the air/water interface of sessile droplets. Shape-anisotropic hydrogel microspheres were synthesized via two-step polymerization, whereby a hydrogel shell was formed onto preformed rigid microellipsoids. The shape anisotropy of the hydrogel microspheres was confirmed by transmission electron microscopy and high-speed atomic force microscopy as well as by light-scattering measurements. The present findings are crucial for the understanding of natural self-organization phenomena, where “softness” influences microscopic assembled structures such as those of Nostoc bacteria.

DOI： 10.1002/anie.201901611