Faculty Profiles - UCHIHASHI Takayuki

Papers - UCHIHASHI Takayuki

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Construction of a cyclic regular-triangle trimer of cytochrome c555 with a central hole using sortase A.

Novientri G, Fujiwara K, Mashima T, Matsuura H, Ogata H, Uchihashi T, Fujii S, Sambongi Y, Hirota S

Chemistry (Weinheim an der Bergstrasse, Germany) page： e202404736 2025.3

Single-Molecule Imaging of Wood Xylans on Surfaces and Their Interaction with GH11 Xylanase Open Access

Schaubeder J.B., Ganser C., Palasingh C., Eibinger M., Nypelö T., Uchihashi T., Spirk S.

Biomacromolecules Vol. 26 ( 3 ) page： 1639 - 1646 2025.3

Language：English Publisher：Biomacromolecules

The knowledge of the molecular properties and arrangements of biopolymers in both solid and solution state are essential in the design of sustainable materials and biomedicine as they are decisive for mechanical strength, flexibility, and biodegradability. However, the structure of most biopolymers at charged interfaces can vary considerably, and their time-dependent visualization in liquid-state still remains challenging. In this work, we employed high-speed atomic force microscopy (HS-AFM) to visualize single xylan macromolecules from alkali-extracted birch and beechwood. On negatively charged mica surfaces, they appeared as individual macromolecules but assembled into aggregates on 3-aminopropyltriethoxysilane (APTES) surfaces (AP-mica). Hence, we further investigated the susceptibility to enzymatic degradation using an endoxylanase, which showed that the individual xylan macromolecules remained intact, while larger assemblies on AP-mica degraded over time. We demonstrate that HS-AFM is a powerful tool for understanding the molecular properties and degradation mechanisms of biopolymers. Moreover, by identifying alignment-dependent binding sites, strategies can be developed to ensure the biodegradability of composite materials by intelligent interface design.

DOI： 10.1021/acs.biomac.4c01446

Open Access

Scopus

PubMed

Dynamic Oligomerization Processes of Bacillus subtilis ClpP Protease Induced by ADEP1 Studied with High-Speed Atomic Force Microscopy Open Access

Ishikawa, F; Takahashi, K; Takaya, A; Tanabe, G; Homma, M; Uchihashi, T

ACS OMEGA Vol. 10 ( 7 ) page： 7381 - 7388 2025.2

Language：English Publisher：ACS Omega

Bacterial ClpPs are a highly conserved family of serine proteases that associate with members of the AAA+ ATPase (ATPase associated with diverse cellular activities) family to degrade protein substrates. The antibiotic A54556 factor (ADEP1) induces uncontrolled proteolysis by forming an ATPase-independent ClpP-ADEP complex. Cryo-EM analysis of Bacillus subtilis ClpP (Bs-ClpP) has demonstrated that ADEP1 binding shifts the protease to an active extended conformation and opens its axial entry pores. However, the dynamic oligomerization processes of Bs-ClpP induced by ADEP1 remain unclear. In this study, we used a combination of biochemical studies and high-speed atomic force microscopy (HS-AFM) to reveal how ADEP1 affects the oligomerization states and protease activity of Bs-ClpP, inducing the active extended state and protease activity of Bs-ClpP. HS-AFM observations demonstrated that the Bs-ClpP tetradecamer (2R state) forms via a progression from monomers to oligomers and then from oligomers to heptamers (R state) in the presence of ADEP1. Our results suggest that ADEP1 binding to monomeric Bs-ClpP triggers conformational changes that facilitate Bs-ClpP oligomerization (R and 2R states) and activation.

DOI： 10.1021/acsomega.4c11303

A look beyond topography: Transient phenomena of <i>Escherichia coli</i> cell division captured with high-speed in-line force mapping Open Access

Ganser, C; Nishiguchi, S; Chan, FY; Uchihashi, T

SCIENCE ADVANCES Vol. 11 ( 5 ) page： eads3010 2025.1

Language：English Publisher：Science Advances

Life on the nanoscale has been made accessible in recent decades by the development of fast and noninvasive techniques. High-speed atomic force microscopy (HS-AFM) is one such technique that shed light on single protein dynamics. Extending HS-AFM to effortlessly incorporate mechanical property mapping while maintaining fast imaging speed allows a look deeper than topography and reveal details of nanoscale mechanisms that govern life. Here, we present high-speed in-line force mapping (HS-iFM) to record mechanical properties and topography maps with high spatiotemporal resolution. Using HS-iFM, a comprehensive study of the nanoscale mechanical properties of living Escherichia coli revealed localized stiffening and details during cell division, formation and diffusion of pores in the membrane, and the impact of depressurization of a cell. The frame time was as low as 15 seconds with a spatial resolution of 5.5 nanometers per pixel in topography and 22 nanometers per pixel in force maps, allowing the capture of transient phenomena on bacterial surfaces in striking detail.

DOI： 10.1126/sciadv.ads3010

Nanoscale Visualization of Drosophila E-cadherin Ectodomain Fragments and Their Interactions Using DNA Origami Nanoblocks Open Access

Oda H., Nishiguchi S., Song C., Murata K., Uchihashi T., Suzuki Y.

Journal of Molecular Biology Vol. 437 ( 2 ) page： 168875 2025.1

Language：English Publisher：Journal of Molecular Biology

The adhesive function of cell surface proteins can be visually assessed through direct observation; however, the underlying structures that mediate adhesion typically remain invisible at the nanoscale level. This hinders knowledge on the diversity of molecular architectures responsible for cell–cell adhesion. Drosophila E-cadherin (DE-cadherin), a classical cadherin with a unique domain structure, demonstrates adhesive function; however, it lacks a structural model that explains its adhesion mechanism. Here, we present a novel application of DNA origami technology to create a cell-free, flat environment in which full DE-cadherin ectodomains are anchored using SNAP-tags and biotin-streptavidin interactions. DNA origami was assembled into a 120 nm long block, bearing 5 or 14 biotin:streptavidin sites that were evenly spaced on one lateral face. DE-cadherin ectodomain fragments were attached via biotinylated SNAP-tags. These decorated DNA origami nanoblocks were subjected to transmission electron and high-speed atomic force microscopy, which revealed a hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain, suggesting a role in mechanical flexibility. We also observed interactions between DE-cadherin ectodomains via their membrane-distal portions on single DNA origami nanoblocks. We reconstituted an adhesion-like process via pairing DNA origami nanoblocks using DE-cadherin ectodomain interactions. Homophilic associations of functional DE-cadherin ectodomains between the paired DNA origami nanoblocks were visualized at the nanoscale, displaying strand-like molecular configurations, likely representing the extracellular cadherin repeats without regular arrays of structural elements. This study introduces a DNA origami-based platform for reconstituting and visualizing cadherin ectodomain interactions, with potential applications for a broader range of adhesion molecules.

DOI： 10.1016/j.jmb.2024.168875

Scopus

PubMed

AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex Open Access

Yui Kanaoka, Takaharu Mori, Wataru Nagaike, Seira Itaya, Yuto Nonaka, Hidetaka Kohga, Takamitsu Haruyama, Yasunori Sugano, Ryoji Miyazaki, Muneyoshi Ichikawa, Takayuki Uchihashi, Tomoya Tsukazaki

Nature Communications Vol. 16 ( 1 ) page： 225 2025.1

Deformation Behavior of Microparticle-Based Polymer Films Visualized by AFM Equipped with a Stretching Device

Yuichiro Nishizawa, Masataka Uchida, Natsuki Watanabe, Feng-Yueh Chan, Christian Ganser, Takeshi Kawasaki, Yuma Sasaki, Daisuke Suzuki, Takayuki Uchihashi

ACS Applied Materials & Interfaces Vol. 16 ( 45 ) page： 63073 - 63082 2024.10

Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid β Fibrils Open Access

Maho Yagi-Utsumi, Yui Kanaoka, Shogo Miyajima, Satoru G. Itoh, Katsuhiko Yanagisawa, Hisashi Okumura, Takayuki Uchihashi, Koichi Kato

Journal of the american chemical society Vol. 146 ( 46 ) page： 31518 - 31528 2024.10

Elastomer Particle Monolayers Formed by the Compression of Poly(methyl acrylate) Microparticles at an Air/Water Interface. Reviewed

Sasaki Y, Nishizawa Y, Watanabe N, Uchihashi T, Suzuki D

Macromolecular rapid communications page： e2400604 2024.9

Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor. Reviewed International journal

Norihiro Takekawa, Tatsuro Nishikino, Jun-Ichi Kishikawa, Mika Hirose, Miki Kinoshita, Seiji Kojima, Tohru Minamino, Takayuki Uchihashi, Takayuki Kato, Katsumi Imada, Michio Homma

mBio page： e0126124 2024.9

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

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.

DOI： 10.1128/mbio.01261-24

PubMed

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4. Reviewed

Tanaka T, Hososhima S, Yamashita Y, Sugimoto T, Nakamura T, Shigemura S, Iida W, Sano FK, Oda K, Uchihashi T, Katayama K, Furutani Y, Tsunoda SP, Shihoya W, Kandori H, Nureki O

Molecular cell 2024.9

Nanoscale visualization of<i>Drosophila</i>E-cadherin ectodomain fragments and their interactions using DNA origami nanoblocks

Hiroki Oda, Shigetaka Nishiguchi, Chihong Song, Kazuyoshi Murata, Takayuki Uchihashi, Yuki Suzuki

2024.9

Publisher：Cold Spring Harbor Laboratory

Abstract

The adhesive function of cell surface proteins can be visually assessed through direct observation; however, the underlying structures that mediate adhesion typically remain invisible at the nanoscale level. This hinders knowledge on the diversity of molecular architectures responsible for cell-cell adhesion.DrosophilaE-cadherin (DE-cadherin), a classical cadherin with a unique domain structure, demonstrates adhesive function; however, it lacks a structural model that explains its adhesion mechanism. In this study, we present a novel application of DNA origami technology to create a cell-free, flat environment in which full DE-cadherin ectodomains are anchored using SNAP-tags and biotin-streptavidin interactions. DNA origami was assembled into a 120 nm long block, bearing 5 or 14 biotin:streptavidin sites that were evenly spaced on one lateral face. DE-cadherin ectodomain fragments were attached via biotinylated SNAP-tags. These decorated DNA origami nanoblocks were subjected to transmission electron and high-speed atomic force microscopy, which revealed a hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain, suggesting a role in mechanical flexibility. We also observed interactions between DE-cadherin ectodomains via their membrane-distal portions on single DNA origami nanoblocks. We reconstituted an adhesion-like process via pairing DNA origami nanoblocks using DE-cadherin ectodomain interactions. Homophilic associations of functional DE-cadherin ectodomains between the paired DNA origami nanoblocks were visualized at the nanoscale, displaying strand-like molecular configurations, likely representing the extracellular cadherin repeats without regular arrays of structural elements. This study introduces a DNA origami-based platform for reconstituting and visualizing cadherin ectodomain interactions, with potential applications for a broader range of adhesion molecules.

Highlights

DNA origami technology was applied to perform a structure-function study of cadherin.

DNA origami nanoblocks decorated with DE-cadherin ectodomains were observed by TEM/HS-AFM.

A hinge-like site that separated the membrane-distal and -proximal portions of the DE-cadherin ectodomain was revealed.

An adhesion-like process was mimicked via pairing two nanoblocks using DE-cadherin ectodomain interactions.

Homophilic associations of DE-cadherin ectodomains between the nanoblocks were visualized at the nanoscale level.

DOI： 10.1101/2024.09.02.610731

Protein degradation by a component of the chaperonin-linked protease ClpP

Ishikawa F., Homma M., Tanabe G., Uchihashi T.

Genes to Cells Vol. 29 ( 9 ) page： 695 - 709 2024.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Genes to Cells

In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.

DOI： 10.1111/gtc.13141

Scopus

PubMed

Construction of ligand-binding controlled hemoprotein assemblies utilizing 3D domain swapping Reviewed

Mashima, T; Yamanaka, M; Yoshida, A; Kobayashi, N; Kanaoka, Y; Uchihashi, T; Hirota, S

CHEMICAL COMMUNICATIONS Vol. 60 ( 70 ) page： 9440 - 9443 2024.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Chemical Communications

Association-controllable hemoprotein assemblies were constructed from a fusion protein containing two c-type cytochrome units using 3D domain swapping. The hemoprotein assembly exhibited a dynamic exchange between cyclic and linear structures and could be regulated by carbon monoxide (CO) and imidazole binding.

DOI： 10.1039/d4cc03129f

Web of Science

Scopus

PubMed

Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes Reviewed

Yamada H., Osaka H., Tatsumi N., Araki M., Abe T., Kaihara K., Takahashi K., Takashima E., Uchihashi T., Naruse K., Takei K.

Cells Vol. 13 ( 16 ) 2024.8

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

Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.

DOI： 10.3390/cells13161373

Scopus

PubMed

Photoresponsive Supramolecular Polymers Capable of Intrachain Folding and Interchain Aggregation Reviewed

Kenta Tamaki, Sougata Datta, Hiroki Hanayama, Christian Ganser, Takayuki Uchihashi, Shiki Yagai

Journal of the American Chemical Society Vol. 146 ( 32 ) page： 22166 - 22171 2024.7

Structural basis for the interaction between the bacterial cell division proteins FtsZ and ZapA

Junso Fujita, Kota Hibino, Gota Kagoshima, Natsuki Kamimura, Yuki Kato, Ryo Uehara, Keiichi Namba, Takayuki Uchihashi, Hiroyoshi Matsumura

2024.7

Authorship：Corresponding author Publisher：Cold Spring Harbor Laboratory

Abstract

Cell division in most bacteria is regulated by the tubulin homolog FtsZ as well as ZapA, a FtsZ-associated protein. However, how FtsZ and ZapA function coordinately has remained elusive. Here we report the cryo-electron microscopy structure of the ZapA-FtsZ complex. The complex forms an asymmetric ladder-like structure, in which the double antiparallel FtsZ protofilament on one side and a single protofilament on the other side are tethered by ZapA tetramers. In the complex, the extensive interactions of FtsZ with ZapA cause a structural change of the FtsZ protofilament, and the formation of the double FtsZ protofilament increases electrostatic repulsion. High-speed atomic force microscopy analysis revealed cooperative interactions of ZapA with FtsZ at a molecular level. Our findings not only provide a structural basis for the interaction between FtsZ and ZapA but also shed light on how ZapA binds to FtsZ protofilaments without disturbing FtsZ dynamics to promote cell division.

DOI： 10.1101/2024.07.18.604045

The Positively Charged Cluster in the N-terminal Disordered Region may Affect Prion Protein Misfolding: Cryo-EM Structure of Hamster PrP(23–144) Fibrils Reviewed International coauthorship

Lee C.H., Saw J.E., Chen E.H.L., Wang C.H., Uchihashi T., Chen R.P.Y.

Journal of Molecular Biology Vol. 436 ( 11 ) page： 168576 2024.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Molecular Biology

Prions, the misfolding form of prion proteins, are contagious proteinaceous macromolecules. Recent studies have shown that infectious prion fibrils formed in the brain and non-infectious fibrils formed from recombinant prion protein in a partially denaturing condition have distinct structures. The amyloid core of the in vitro-prepared non-infectious fibrils starts at about residue 160, while that of infectious prion fibrils formed in the brain involves a longer sequence (residues ∼90–230) of structural conversion. The C-terminal truncated prion protein PrP(23–144) can form infectious fibrils under certain conditions and cause disease in animals. In this study, we used cryogenic electron microscopy (cryo-EM) to resolve the structure of hamster sHaPrP(23-144) fibrils prepared at pH 3.7. This 2.88 Å cryo-EM structure has an amyloid core covering residues 94–144. It comprises two protofilaments, each containing five β-strands arranged as a long hairpin plus an N-terminal β-strand. This N-terminal β-strand resides in a positively charged cluster region (named PCC2; sequence 96–111), which interacts with the turn region of the opposite protofilaments’ hairpin to stabilize the fibril structure. Interestingly, this sHaPrP(23–144) fibril structure differs from a recently reported structure formed by the human or mouse counterpart at pH 6.5. Moreover, sHaPrP(23–144) fibrils have many structural features in common with infectious prions. Whether this structure is infectious remains to be determined. More importantly, the sHaPrP(23–144) structure is different from the sHaPrP(108–144) fibrils prepared in the same fibrillization buffer, indicating that the N-terminal disordered region, possibly the positively charged cluster, influences the misfolding pathway of the prion protein.

DOI： 10.1016/j.jmb.2024.168576

Scopus

PubMed

How Resilient is Wood Xylan to Enzymatic Degradation in a Matrix with Kraft Lignin? Reviewed International coauthorship

Schaubeder, JB; Ganser, C; Nypelo, T; Uchihashi, T; Spirk, S

BIOMACROMOLECULES Vol. 25 ( 6 ) page： 3532 - 3541 2024.5

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

Despite the potential of lignocellulose in manufacturing value-added chemicals and biofuels, its efficient biotechnological conversion by enzymatic hydrolysis still poses major challenges. The complex interplay between xylan, cellulose, and lignin in fibrous materials makes it difficult to assess underlying physico- and biochemical mechanisms. Here, we reduce the complexity of the system by creating matrices of cellulose, xylan, and lignin, which consists of a cellulose base layer and xylan/lignin domains. We follow enzymatic degradation using an endoxylanase by high-speed atomic force microscopy and surface plasmon resonance spectroscopy to obtain morphological and kinetic data. Fastest reaction kinetics were observed at low lignin contents, which were related to the different swelling capacities of xylan. We demonstrate that the complex processes taking place at the interfaces of lignin and xylan in the presence of enzymes can be monitored in real time, providing a future platform for observing phenomena relevant to fiber-based systems.

DOI： 10.1021/acs.biomac.4c00185

Web of Science

Scopus

PubMed

Key role of Pro230 in the hinge region on the IgG architecture and function

Yuuki Koseki, Yuki Yamaguchi, Michihiko Aoyama, Minoru Tada, Akinobu Senoo, Akiko Ishii-Watabe, Takayuki Uchihashi, Susumu Uchiyama, Koichi Kato, Saeko Yanaka, Jose M.M. Caaveiro

2024.5

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Papers - UCHIHASHI Takayuki