Faculty Profiles - UCHIHASHI Takayuki

Inner lumen proteins stabilize doublet microtubules in cilia and flagella. Reviewed International journal

Mikito Owa, Takayuki Uchihashi, Haru-Aki Yanagisawa, Takashi Yamano, Hiro Iguchi, Hideya Fukuzawa, Ken-Ichi Wakabayashi, Toshio Ando, Masahide Kikkawa

Nature communications Vol. 10 ( 1 ) page： 1143 - 1143 2019.3

Language：English

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

DOI： 10.1038/s41467-019-09051-x

A ring-shaped hemoprotein trimer thermodynamically controlled by the supramolecular heme-heme pocket interaction

Oohora Koji, Kajihara Ryota, Fujimaki Nishiki, Uchihashi Takayuki, Hayashi Takashi

CHEMICAL COMMUNICATIONS Vol. 55 ( 11 ) page： 1544 - 1547 2019.2

Microtubule self-healing and defect creation investigated by in-line force measurements during high-speed atomic force microscopy imaging Reviewed

Christian Ganser, Takayuki Uchihashi

Nanoscale Vol. 11 ( 1 ) page： 125 - 135 2019.1

Single-unit imaging of membrane protein-embedded nanodiscs from two oriented sides by high-speed atomic force microscopy Reviewed

T. Haruyama, Y. Sugano, N. Kodera, T. Uchihashi, T. Ando, Y. Tanaka, H. Konno, T. Tsukazaki

Structure Vol. 27 ( 1 ) page： 152 - + 2019.1

Metastable asymmetrical structure of a shaftless V1 motor Reviewed

Shintaro Maruyama, Kano Suzuki, Motonori Imamura, Hikaru Sasaki, Hideyuki Matsunami, Kenji Mizutani, Yasuko Saito, Fabiana L. Imai, Yoshiko Ishizuka-Katsura, Tomomi Kimura-Someya, Mikako Shirouzu, Takayuki Uchihashi, Toshio Ando, Ichiro Yamato, Takeshi Murata

Science Advances Vol. 5 ( 1 ) page： eaau8149 - eaau8149 2019.1

Language：English Publishing type：Research paper (scientific journal) Publisher：American Association for the Advancement of Science (AAAS)

V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft. The nucleotide-free A3B3 complex of <italic>Enterococcus hirae</italic>, composed of three identical A1B1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-free mutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3 forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

DOI： 10.1126/sciadv.aau8149

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 Invited

内橋貴之

高分子 Vol. 68 ( 01 ) page： 564 - 568 2019

Real-time Nanoscale Visualization of Biological Molecules at Work with High-speed Atomic Force Microscopy

Uchihashi Takayuki

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) page： 253 - 256 2019

Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

Watanabe Hiroki, Uchihashi Takayuki

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) page： 281 - 285 2019

Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

Hiroki Watanabe, Takayuki Uchihashi

2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO) page： 281 - 285 2019

Language：English Publishing type：Research paper (international conference proceedings) Publisher：IEEE

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 similar to 20 mu m and similar to 3 mu m 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 similar to 50 mu m and similar to 32 mu m, 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.

Direct Observation and Manipulation of Supramolecular Polymerization by High-Speed Atomic Force Microscopy

Versatility of RNA-Binding Proteins in Living Cells Reviewed

Riki Kurokawa, Akihide Takeuchi, Nobuyuki Shiina, Masato Katahira, Takefumi Yamashita, Yoko Matsuno, Keisuke Hitachi, Shinsuke Ishigaki, Nesreen Haamad, Ryoma Yoneda, Naomi Ueda, Kei Iida, Motoyasu Hosokawa, Masatoshi Hagiwara, Mamiko Iida, Tsukasa Mashima, Yudai Yamaoki, Masatomo So, Takashi Nagata, Gen Sobue, Keiko Kondo, Hiroki Watanabe, Takayuki Uchihashi

Biomedical Sciences Vol. 5 ( 1 ) page： 7 - 7 2019

Fukui Tomoya, Uchihashi Takayuki, Sasaki Norihiko, Watanabe Hiroki, Takeuchi Masayuki, Sugiyasu Kazunori

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Vol. 57 ( 47 ) page： 15465 - 15470 2018.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

Despite recent advances in mechanistic understanding and controlled-synthesis methodologies regarding synthetic supramolecular assemblies, it has remained challenging to capture the molecular-level phenomena in real time, thus hindering further progress in this research field. In this study, we applied high-speed atomic-force microscopy (AFM), which has extraordinary spatiotemporal resolution (1 nm and sub-100 ms), to capture dynamic events occurring during synthetic molecular self-assembly. High-speed AFM permitted the visualization of unique dynamic behavior, such as seeded growth and self-repair in real time. Furthermore, scanning-probe AFM permitted the site-specific manipulation and functionalization of a molecular self-assembly. This powerful combination of bottom-up and top-down approaches at the molecular level should enable targeted syntheses of unprecedented functional nanoarchitectures.

DOI： 10.1002/anie.201809165

Monitoring Thermoresponsive Morphological Changes in Individual Hydrogel Microspheres. Reviewed International journal

Shusuke Matsui, Yuichiro Nishizawa, Takayuki Uchihashi, Daisuke Suzuki

ACS omega Vol. 3 ( 9 ) page： 10836 - 10842 2018.9

Language：English Publishing type：Research paper (scientific journal)

Real-time morphology/structure changes in individual hydrogel microspheres (microgels) were directly visualized at high spatiotemporal resolution using high-speed atomic force microscopy (HS-AFM) under temperature control ranging from room temperature to ∼40 °C. The recorded HS-AFM movies demonstrate that the size and morphology of thermoresponsive poly(N-isopropyl acrylamide)-based microgels change with increasing temperature at the individual microgel level. Specifically, the height of the microgels gradually decreases and domain structures appeared even below the volume phase transition temperature. Moreover, the domain structure is retained, even after the microgels have fully collapsed. The present study thus demonstrates that temperature-controlled HS-AFM is a useful tool for monitoring stimulus-responsiveness of microgels. In the near future, it should furthermore be possible to extend this temperature-controlled HS-AFM to other stimulus-responsive materials, including autonomously oscillating microgels.

DOI： 10.1021/acsomega.8b01770

Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling

Oohora Koji, Fujimaki Nishiki, Kajihara Ryota, Watanabe Hiroki, Uchihashi Takayuki, Hayashi Takashi

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Vol. 140 ( 32 ) page： 10145 - 10148 2018.8

Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time

Mori Tetsuya, Sugiyama Shogo, Byrne Mark, Johnson Carl Hirschie, Uchihashi Takayuki, Ando Toshio

NATURE COMMUNICATIONS Vol. 9 ( 1 ) page： 3245 2018.8

Scanning probe microscopy

Takahashi T., Fukui K.I., Kageshima M., Komeda T., Nakajima K., Nakayama T., Sumitomo K., Uchihashi T.

Japanese Journal of Applied Physics Vol. 57 ( 8 ) 2018.8

Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

Takayuki Umakoshi, Hikari Udaka, Takayuki Uchihashi, Toshio Ando, Miho Suzuki, Takeshi Fukuda

COLLOIDS AND SURFACES B-BIOINTERFACES Vol. 167 page： 267 - 274 2018.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates. (C) Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2018.04.015

Translating MOF chemistry into supramolecular chemistry: Soluble coordination nanofibers showing efficient photon upconversion

Masanori Hosoyamada, Nobuhiro Yanai, Keisuke Okumura, Takayuki Uchihashi, Nobuo Kimizuka

Chemical Communications Vol. 54 ( 50 ) page： 6828 - 6831 2018.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry

A method for synthesizing coordination nanofibers by extracting the structural motifs of metal-organic frameworks (MOFs) is demonstrated. In these soluble nanofibers, multiple chromophores with largely different sizes and shapes can be arranged at desired compositions, and excited triplet energy migrates among the densely assembled chromophore arrays, showing an efficient photon upconversion even at very low concentration.

DOI： 10.1039/c8cc01594e

Dynamic structural states of CIpB involved in its disaggregation function

Takayuki Uchihashi, Yo-hei Watanabe, Yosuke Nakazaki, Takashi Yamasaki, Hiroki Watanabe, Takahiro Maruno, Kentaro Ishii, Susumu Uchiyama, Chihong Song, Kazuyoshi Murata, Ryota Iino, Toshio Ando

NATURE COMMUNICATIONS Vol. 9 ( 1 ) page： 2147 2018.6

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

The ATP-dependent bacterial protein disaggregation machine, CIpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of CIpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize CIpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable CIpB to disentangle protein aggregates.

DOI： 10.1038/s41467-018-04587-w

Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation Reviewed

Takahiro Maruno, Hiroki Watanabe, Saki Yoneda, Takayuki Uchihashi, Satoru Adachi, Kunihito Arai, Taichi Sawaguchi, Susumu Uchiyama

Journal of Pharmaceutical Sciences Vol. 107 ( 6 ) page： 1521 - 1529 2018.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier B.V.

This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO−], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m2, which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1–2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.

DOI： 10.1016/j.xphs.2018.01.021