研究者詳細 - 内橋　貴之

論文 - 内橋　貴之

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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) 頁： e202404736 2025年3月

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 26 巻 ( 3 ) 頁： 1639 - 1646 2025年3月

記述言語：英語出版者・発行元：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

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 10 巻 ( 7 ) 頁： 7381 - 7388 2025年2月

記述言語：英語出版者・発行元：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 11 巻 ( 5 ) 頁： eads3010 2025年1月

記述言語：英語出版者・発行元：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 437 巻 ( 2 ) 頁： 168875 2025年1月

記述言語：英語出版者・発行元：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

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

Kanaoka Y., Mori T., Nagaike W., Itaya S., Nonaka Y., Kohga H., Haruyama T., Sugano Y., Miyazaki R., Ichikawa M., Uchihashi T., Tsukazaki T.

Nature Communications 16 巻 ( 1 ) 頁： 225 2025年1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

Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins.

DOI： 10.1038/s41467-024-54875-x

その他リンク： https://www.nature.com/articles/s41467-024-54875-x

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

Nishizawa, Y; Uchida, M; Watanabe, N; Chan, FY; Ganser, C; Kawasaki, T; Sasaki, Y; Suzuki, D; Uchihashi, T

ACS APPLIED MATERIALS & INTERFACES 16 巻 ( 45 ) 頁： 63073 - 63082 2024年10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：ACS Applied Materials and Interfaces

Understanding the structural changes and property alterations at the nanoscale and microscopic levels is critical to clarifying the deformation behavior and mechanical properties of polymer materials. Especially, in latex films composed of polymer nanoparticles, it is widely accepted that the remaining interfaces between microparticles in the film affect their brittleness. However, detailed information on nanoscale changes of latex films during deformation remains unclear due to technical difficulties in analyzing the microstructures under mechanical stress. In this study, we employed atomic force microscopy equipped with a uniaxial stretching device to visualize the surface structures of films composed of slightly cross-linked microparticles under elongation strain. The observations revealed that the latex film deforms in a nonaffine manner, which is attributed to the concurrent deformation of individual microparticles and the pull-out of interpenetration between them. Furthermore, by introducing a load-strain measurement mechanism to the stretching device, we compared the relationships between nanostructural changes, local property changes, and macroscopic deformation of microparticle-based films. The results suggest that loads are dominated by the deformation of microparticles and dissipate as the interpenetration of surface polymer chains between microparticles is pulled out.

DOI： 10.1021/acsami.4c16013

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

Yagi-Utsumi M., Kanaoka Y., Miyajima S., Itoh S.G., Yanagisawa K., Okumura H., Uchihashi T., Kato K.

Journal of the American Chemical Society 146 巻 ( 46 ) 頁： 31518 - 31528 2024年10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 the American Chemical Society

Understanding the dynamic assembly process of amyloid β (Aβ) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer’s disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aβ fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aβ antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aβ monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aβ assembly during fibril formation and suggests novel strategies for Alzheimer’s therapy aimed at inhibiting the fibril elongation.

DOI： 10.1021/jacs.4c08841

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

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

Macromolecular rapid communications 頁： e2400604 2024年9月

Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor. 査読有り国際誌

Takekawa N, Nishikino T, Kishikawa J-i, Hirose M, Kinoshita M, Kojima S, Minamino T, Uchihashi T, Kato T, Imada K, Homma M

mBio 頁： e0126124 2024年9月

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

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年9月

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年9月

出版者・発行元：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 29 巻 ( 9 ) 頁： 695 - 709 2024年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Construction of ligand-binding controlled hemoprotein assemblies utilizing 3D domain swapping 査読有り

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

CHEMICAL COMMUNICATIONS 60 巻 ( 70 ) 頁： 9440 - 9443 2024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：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

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

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

Cells 13 巻 ( 16 ) 2024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：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

Photoresponsive Supramolecular Polymers Capable of Intrachain Folding and Interchain Aggregation 査読有り

Tamaki, K; Datta, S; Hanayama, H; Ganser, C; Uchihashi, T; Yagai, S

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 146 巻 ( 32 ) 頁： 22166 - 22171 2024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 the American Chemical Society

The competition between polymer chain folding and aggregation is a critical structuring process that determines the physical properties of synthetic and biopolymers. However, supramolecular polymer systems that exhibit both processes have not yet been reported. We herein introduce a system in which folded supramolecular polymers spontaneously undergo interchain aggregation due to a rearrangement in internal molecular order, converting them into crystalline aggregates. These folded supramolecular polymers slowly crystallize over the course of half a day, due to their characteristic higher-order structures. However, the photoisomerization of the trans-azobenzene incorporated into the monomer to the cis isomer leads to unfolding of the polymer, accelerating the intrachain and interchain molecular ordering to a few hours. The intermediate structures visualized by AFM demonstrate that the unfolding is coupled with interchain aggregation.

DOI： 10.1021/jacs.4c07878

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年7月

担当区分：責任著者出版者・発行元：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 査読有り国際共著

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

Journal of Molecular Biology 436 巻 ( 11 ) 頁： 168576 2024年6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 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

How Resilient is Wood Xylan to Enzymatic Degradation in a Matrix with Kraft Lignin? 査読有り国際共著

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

BIOMACROMOLECULES 25 巻 ( 6 ) 頁： 3532 - 3541 2024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：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

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年5月

Tenth International AFMBioMed Conference on AFM in Life Sciences and Medicine, August 30–September 2, 2022, Nagoya-Okasaki, Japan 招待有り国際共著

Uchihashi T., Rico F., Pellequer J.L.

Journal of Molecular Recognition 37 巻 ( 3 ) 2024年5月

Identification of potential C1-binding sites in the immunoglobulin C<inf>L</inf> domains 査読有り

Yanaka S., Kodama A., Nishiguchi S., Fujita R., Shen J., Boonsri P., Sung D., Isono Y., Yagi H., Miyanoiri Y., Uchihashi T., Kato K.

International Immunology 36 巻 ( 8 ) 頁： 405 - 411 2024年4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：International Immunology

Immunoglobulin G (IgG) molecules that bind antigens on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single-molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centred on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

DOI： 10.1093/intimm/dxae017

Compressed sensing reconstruction of cell mechanical images obtained from atomic force microscopy 査読有り

Miyata, Y; Chan, FY; Uchihashi, T; Okajima, T

JAPANESE JOURNAL OF APPLIED PHYSICS 63 巻 ( 4 ) 2024年4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：Japanese Journal of Applied Physics

Compressed sensing (CS), a technique in signal processing that reconstructs sparse signals from a limited sampling number, has been valuable in topographic images obtained from atomic force microscopy (AFM). However, how CS is effective in reconstructing AFM mechanical images remains unclear. We investigated the reconstruction of topographic and mechanical images of living cells, such as developing embryos obtained from AFM mapping experiments using CS. The results showed that both topographic and mechanical images of embryonic cells in the different developmental stages were well reconstructed at a spatial resolution higher than the original AFM images. These results suggested that the CS approach enabled the cell mechanical properties, together with cell surface morphology, using AFM mapping measurements to be faster than the conventional AFM methods without reducing the spatial resolution.

DOI： 10.35848/1347-4065/ad34de

Application of high-speed atomic force microscopy in visualizing the dynamics of synthetic polymers 招待有り査読有り

Uchihashi, T; Nishizawa, Y

JAPANESE JOURNAL OF APPLIED PHYSICS 63 巻 ( 4 ) 2024年4月

担当区分：責任著者記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：Japanese Journal of Applied Physics

High-speed atomic force microscopy (HS-AFM) is a technique that enables real-time imaging of nanoscale phenomena in solution. It was originally developed to visualize biomolecules, whose dynamics in solution significantly affect the manifestation of their functions, and has contributed to the understanding of molecular mechanisms based on the observation of single-molecule dynamics of proteins. In recent years, its application has broadened to include not only biomolecules, but also the structural dynamics of supramolecular assemblies that associate and dissociate in solution, as well as the evaluation of synthetic molecules such as polymer gels that swell in solution. In this paper, we review some of our recent studies on the application of HS-AFM to supramolecular polymers and hydrogel particles.

DOI： 10.35848/1347-4065/ad33f2

FliH and FliI help FlhA bring strict order to flagellar protein export in <i>Salmonella</i> 国際誌

Kinoshita, M; Minamino, T; Uchihashi, T; Namba, K

COMMUNICATIONS BIOLOGY 7 巻 ( 1 ) 頁： 366 - 366 2024年3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：Communications Biology

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368–372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.

DOI： 10.1038/s42003-024-06081-0

<i>In Situ</i> Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope 査読有り

Yang, KS; Chan, FY; Watanabe, H; Yoshioka, S; Inouye, Y; Uchihashi, T; Ishitobi, H; Verma, P; Umakoshi, T

NANO LETTERS 24 巻 ( 9 ) 頁： 2805 - 2811 2024年2月

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product 査読有り

Tanaka, Y; Uchihashi, T; Nakamura, A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 752 巻頁： 109854 2024年2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：Archives of Biochemistry and Biophysics

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 μM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 μM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 μM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

DOI： 10.1016/j.abb.2023.109854

Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy. 査読有り

Yanaka S, Watanabe H, Yogo R, Kongsema M, Kondo S, Yagi H, Uchihashi T, Kato K

Biological & pharmaceutical bulletin 47 巻 ( 1 ) 頁： 334 - 338 2024年1月

[Protein degradation in bacteria: focus on the ClpP protease]. 査読有り

Ishikawa F, Homma M, Tanabe G, Uchihashi T

Nihon saikingaku zasshi. Japanese journal of bacteriology 79 巻 ( 1 ) 頁： 1 - 13 2024年

担当区分：責任著者記述言語：日本語出版者・発行元：日本細菌学会

<p>細胞内のタンパク質は生まれ（合成され），働いて，死んで（分解されて）いく。タンパク質の一生の中で，生まれることも重要であるが，どのように死んでいくかもまた等しく重要な生物の営みである。細胞外に分泌されるプロテアーゼは，外のタンパク質を分解することによって栄養物として取り込むために利用される。細胞内の不要になった，あるいは害になるようなタンパク質を分解するプロテアーゼも存在する。真核生物では，プロテアソームと呼ばれる巨大酵素複合体がそのタンパク質の分解を主に行う。原核生物である細菌も類似のシステムをもつ。細菌の細胞の中で，そのタンパク質の死に関与するATP依存的なプロテアーゼClpの複雑な分解系の制御と作動機構について，特に，ClpXPを中心に，また，細菌に対する抗生物質のターゲットとしての側面について解説したい。</p>

DOI： 10.3412/jsb.79.1

Phase separation-induced glass transition under critical miscible conditions 査読有り

Watanabe M., Shi D., Kiyama R., Maruyama K., Nishizawa Y., Uchihashi T., Gong J.P., Nonoyama T.

Materials Advances 2024年

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

Plasticizers have been widely utilized to adjust the glass transition temperature (Tg) of glassy polymeric materials. To optimize performance while minimizing volume, plasticizers with a strong affinity for the target polymer are typically chosen. If we consider a combination of a glassy polymer and a plasticizer with a critical miscibility condition, where the miscible/immiscible states are altered by changing the temperature, phase separation induced by temperature variations will trigger the glass transition. In this study, we report on a polymeric material synthesized from a blend of a high Tg polymer and a plasticizer, exhibiting a phase separation-induced glass transition around the upper critical solution temperature (UCST). It is expected from a crossover point of the Tg curve and the demixing curve in a thermodynamic phase diagram, corresponding to the Berghmann point. Poly(isobornyl acrylate) (PIBXA) with an original Tg of ∼100 °C and triethyl phosphate (TEP) were employed as the glassy polymer and plasticizer, respectively. When the TEP fraction was relatively small (∼10 wt%), the sample showed no phase separation and a decrease in Tg compared to that of the pristine PIBXA, following the conventional trend of plasticizer addition. Conversely, at 20 wt% or higher fractions, the samples displayed UCST-type phase separation and an abnormal increase in Tg with increasing plasticizer content. Furthermore, this miscible/immiscible transition can be predicted through an analysis of the temperature-corrected Hansen solubility parameter (HSP). This report proposes a novel role for plasticizers in adjusting Tg and prediction of objective combinations that satisfy the critical miscible condition.

DOI： 10.1039/d4ma00737a

Determination of the inhomogeneous deswelling of thermoresponsive poly(N-isopropyl methacrylamide) microgels by combining dynamic light scattering, high-speed atomic force microscopy, and electrophoresis Open Access

Nishizawa Y., Inui T., Uchihashi T., Suzuki D.

Polymer Journal 2024年

出版者・発行元：Polymer Journal

Poly(N-isopropyl methacrylamide) microgels with a uniform size of approximately 250 nm in their swollen state were synthesized via aqueous free-radical precipitation polymerization. The electrodynamic phenomena of the microgels were analyzed quantitatively using Ohshima’s equation, which explains the electrodynamic phenomena of natural and artificial soft colloids. The thermoresponsive deswelling of the microgels with increasing temperature was investigated by combining electrophoresis with dynamic light scattering and high-speed atomic force microscopy. Based on the obtained results, a plausible mechanism for the structural change during microgel deswelling is proposed, in which the core dwells first and the outermost surface dwells last.

DOI： 10.1038/s41428-024-00988-5

The two-step cargo recognition mechanism of heterotrimeric kinesin 査読有り

Jiang, XG; Ogawa, T; Yonezawa, K; Shimizu, N; Ichinose, S; Uchihashi, T; Nagaike, W; Moriya, T; Adachi, N; Kawasaki, M; Dohmae, N; Senda, T; Hirokawa, N

EMBO REPORTS 24 巻 ( 11 ) 頁： e56864 2023年11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：EMBO Reports

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

DOI： 10.15252/embr.202356864

Measuring mechanical properties with high-speed atomic force microscopy 招待有り査読有り

Ganser, C; Uchihashi, T

MICROSCOPY 73 巻 ( 1 ) 頁： 14 - 21 2023年10月

高速原子間力顕微鏡による高分子ゲル微粒子評価

西澤佑一朗, 内橋貴之

アンサンブル 25 巻 ( 4 ) 頁： 271 - 278 2023年10月

記述言語：日本語出版者・発行元：分子シミュレーション学会

<p><tt>高速原子間力顕微鏡（高速</tt>AFM<tt>）は，溶液中で生じるナノスケール現象を，サブ秒でリアルタイム撮影できる顕微鏡技術である．元々は，溶液中で機能を発揮するタンパク質を観察することを目的に開発され，生命科学分野でタンパク質の一分子動態解析に貢献してきた．一方で，</tt><tt>高速</tt>AFM <tt>技術自体の適用範囲は生体試料に限られているわけではないので，溶液中で会合解離する超分子や，</tt> <tt>溶液で膨潤する高分子ゲル等の合成分子の構造動態評価に対しても応用されつつある．本稿では，高速</tt>AFM <tt>の特徴と高分子分野での応用例について概説する</tt><tt>． </tt></p>

DOI： 10.11436/mssj.25.271

Elastic Polymer Coated Nanoparticles with Fast Clearance for <sup>19</sup>F MR Imaging 査読有り

Konishi Y., Minoshima M., Fujihara K., Uchihashi T., Kikuchi K.

Angewandte Chemie - International Edition 62 巻 ( 40 ) 頁： e202308565 2023年10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

19F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19F MRI imaging. Importantly, our 19F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.

DOI： 10.1002/anie.202308565

Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin: Multiple roles of Glu54 in Twin-peaked Rhodopsins 査読有り

Mannen K., Nagata T., Rozenberg A., Konno M., del Carmen Marín M., Bagherzadeh R., Béjà O., Uchihashi T., Inoue K.

Journal of Molecular Biology 頁： 168331 - 168331 2023年10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 Molecular Biology

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr–Ala–Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

DOI： 10.1016/j.jmb.2023.168331

Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication 査読有り国際共著国際誌

Cho, CR; Ganser, C; Uchihashi, T; Kato, K; Song, JJ

COMMUNICATIONS BIOLOGY 6 巻 ( 1 ) 頁： 993 - 993 2023年9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：Communications Biology

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

DOI： 10.1038/s42003-023-05373-1

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography 査読有り国際共著国際誌

Chen, EHL; Wang, CH; Liao, YT; Chan, FY; Kanaoka, Y; Uchihashi, T; Kato, K; Lai, LS; Chang, YW; Ho, MC; Chen, RPY

NATURE COMMUNICATIONS 14 巻 ( 1 ) 頁： 5464 - 5464 2023年9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 abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

DOI： 10.1038/s41467-023-41156-2

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status 査読有り

Yagi-Utsumi, M; Miura, H; Ganser, C; Watanabe, H; Hiranyakorn, M; Satoh, T; Uchihashi, T; Kato, K; Okazaki, KI; Aoki, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 24 巻 ( 16 ) 頁： 12865 - 12865 2023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：International Journal of Molecular Sciences

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

DOI： 10.3390/ijms241612865

Single microgel degradation governed by heterogeneous nanostructures 査読有り

Nishizawa, Y; Yokoi, H; Uchihashi, T; Suzuki, D

SOFT MATTER 19 巻 ( 27 ) 頁： 5068 - 5075 2023年7月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Soft Matter

Although the degradation of colloidal particles is one of the most attractive phenomena in the field of biological and environmental science, the degradation mechanism of single particles remains to be elucidated. In this study, in order to clarify the impact of the structure of a single particle on the oxidative degradation processes, thermoresponsive colloidal particles with chemical cleavage points were synthesized as a model, and their degradation behavior was evaluated using high-speed atomic force microscopy (HS-AFM) as well as conventional scattering techniques. The real-time observation of single-particle degradation revealed that the degradation behavior of microgels is governed by their inhomogeneous nanostructure, which originates from the polymerization method and their hydrophilicity. Our findings can be expected to advance the design of carriers for drug-delivery and the understanding of the formation processes of micro (nano)plastics.

DOI： 10.1039/d3sm00216k

Multistep, site-selective noncovalent synthesis of two-dimensional block supramolecular polymers 査読有り

Sasaki, N; Kikkawa, J; Ishii, Y; Uchihashi, T; Imamura, H; Takeuchi, M; Sugiyasu, K

NATURE CHEMISTRY 15 巻 ( 7 ) 頁： 922 - + 2023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 Chemistry

Although the principles of noncovalent bonding are well understood and form the basis for the syntheses of many intricate supramolecular structures, supramolecular noncovalent synthesis cannot yet achieve the levels of precision and complexity that are attainable in organic and/or macromolecular covalent synthesis. Here we show the stepwise synthesis of block supramolecular polymers from metal–porphyrin derivatives (in which the metal centre is Zn, Cu or Ni) functionalized with fluorinated alkyl chains. These monomers first undergo a one-dimensional supramolecular polymerization and cyclization process to form a toroidal structure. Subsequently, successive secondary nucleation, elongation and cyclization steps result in two-dimensional assemblies with concentric toroidal morphologies. The site selectivity endowed by the fluorinated chains, reminiscent of regioselectivity in covalent synthesis, enables the precise control of the compositions and sequences of the supramolecular structures, as demonstrated by the synthesis of several triblock supramolecular terpolymers. [Figure not available: see fulltext.].

DOI： 10.1038/s41557-023-01216-y

その他リンク： https://www.nature.com/articles/s41557-023-01216-y

High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha 査読有り国際共著

Chien, YC; Wang, YS; Sridharan, D; Kuo, CW; Chien, CT; Uchihashi, T; Kato, K; Angata, T; Meng, TC; Hsu, STD; Khoo, KH

JACS AU 3 巻 ( 7 ) 頁： 1864 - 1875 2023年6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：JACS Au

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

DOI： 10.1021/jacsau.3c00124

Closed-loop recycling of microparticle-based polymers 査読有り

Watanabe, T; Minato, H; Sasaki, Y; Hiroshige, S; Suzuki, H; Matsuki, N; Sano, K; Wakiya, T; Nishizawa, Y; Uchihashi, T; Kureha, T; Shibayama, M; Takata, T; Suzuki, D

GREEN CHEMISTRY 25 巻 ( 9 ) 頁： 3418 - 3424 2023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：Green Chemistry

Contemporary polymer science is shifting toward the development of recycling systems to curb global resource depletion and environmental contamination. However, most methods of polymer recycling require cleavage of chemical bonds, which diminishes the quality of the polymers during recycling. Here, we propose a recycling strategy for tough polymers based on microparticles, which allows materials recycling without loss of their properties (‘closed-loop’ recycling). The polymer microparticles can be used to generate tough polymer films by controlling the interparticle physical cross-linking, and subsequently recycled on demand by disassembling into individual microparticles without chemical reactions. Our “microparticle-based concept” for polymer recycling circumvents the infamous trade-off between mechanical stability and degradability of polymers and be expected to open new avenues for closed-loop recycling of polymer materials.

DOI： 10.1039/d3gc00090g

Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy 査読有り

Nishiguchi, S; Kasai, RS; Uchihashi, T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 120 巻 ( 18 ) 頁： e2302047120 2023年4月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Cadherin EGF LAG seven-pass G-type receptors (CELSR) cadherins, members of the cadherin superfamily, and adhesion G-protein-coupled receptors, play a vital role in cell–cell adhesion. The mutual binding of the extracellular domains (ectodomains) of CELSR cadherins between cells is crucial for tissue formation, including the establishment of planar cell polarity, which directs the proper patterning of cells. CELSR cadherins possess nine cadherin ectodomains (EC1–EC9) and noncadherin ectodomains. However, the structural and functional mechanisms of the binding mode of CELSR cadherins have not been determined. In this study, we investigated the binding mode of CELSR cadherins using single-molecule fluorescence microscopy, high-speed atomic force microscopy (HS-AFM), and bead aggregation assay. The fluorescence microscopy analysis results indicated that the trans-dimer of the CELSR cadherin constitutes the essential adhesive unit between cells. HS-AFM analysis and bead aggregation assay results demonstrated that EC1–EC8 entirely overlap and twist to form antiparallel dimer conformations and that the binding of EC1–EC4 is sufficient to sustain bead aggregation. The interaction mechanism of CELSR cadherin may elucidate the variation of the binding mechanism within the cadherin superfamily and physiological role of CELSR cadherins in relation to planar cell polarity.

DOI： 10.1073/pnas.2302047120

Creation of single molecular conjugates of metal-organic cages and DNA 査読有り国際誌

Nakajo T., Kusaka S., Hiraoka H., Nomura K., Matsubara N., Baba R., Yoshida Y., Nakamoto K., Honma M., Iguchi H., Uchihashi T., Abe H., Matsuda R.

Chemical Communications 59 巻 ( 33 ) 頁： 4974 - 4977 2023年4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：Chemical Communications

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

DOI： 10.1039/d3cc00460k

高速原子間力顕微鏡で明らかになった細胞間接着分子カドヘリンのダイマー形成過程招待有り査読有り

西口茂孝, 古田忠臣, 内橋貴之

生物物理 63 巻 ( 2 ) 頁： 104 - 106 2023年4月

担当区分：責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人日本生物物理学会

<p>多細胞動物のからだの形作りは，隣り合う細胞と細胞をつなぐカドヘリン間の結合によって制御されているが，その結合機構については不明な点が多かった．高速原子間力顕微鏡を用いて，溶液中におけるカドヘリンの結合過程を一分子のスケールで直接可視化することで，カドヘリンの結合機構を解析した．</p>

DOI： 10.2142/biophys.63.104

3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy 査読有り

Rashid M.R., Ganser C., Akter M., Nasrin S.R., Kabir A.M.R., Sada K., Uchihashi T., Kakugo A.

Chemistry Letters 52 巻 ( 2 ) 頁： 100 - 104 2023年2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：Chemistry Letters

Biomolecular motor, microtubule (MT) and kinesin based molecular swarm robots are important due to their applications in cooperative task achievements, while the structural details are still unknown. In this work, high-speed atomic force microscopy was used to observe the MT swarm ring structure at nanometerlevel resolution. MTs were observed to pile up in multiple layers to form swarm rings. The number of MTs involved in force generation was estimated which will specifically impact the understanding of the force-generation capability of swarms.

DOI： 10.1246/cl.220491

Disulfide Bond-mediated Oligomerization of a Green Fluorescent Protein in Solution 査読有り

Soon J.W., Oohora K., Uchihashi T., Hayashi T.

Chemistry Letters 52 巻 ( 2 ) 頁： 105 - 109 2023年2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：Chemistry Letters

A green fluorescent protein (GFP) variant with two cysteine residues added to the protein surface was modified with 2,2′- dipyridyl disulfide and oligomerization of the modified protein and the unmodified variant via the disulfide bond was achieved in solution. The fluorescence anisotropy decay of the obtained GFP oligomer is capable of efficient homo-FRET.

DOI： 10.1246/cl.220495

Biosynthesis of highly branched gold nanoparticles through structural engineering of fatty acids 査読有り

Yue Y., Yokota Y., Uchihashi T.

iScience 26 巻 ( 1 ) 頁： 105864 2023年1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：iScience

Biomimetic approaches have been used to develop inorganic nanomaterials with complex morphologies and functions. Fatty acids are among the most important and decomposable biomolecules in nature. However, the controlled synthesis of branched gold nanoparticles using these biomolecules has not been reported. Herein, we demonstrate a strategy to produce highly branched gold nanoparticles through structural engineering of fatty acids. Furthermore, we developed a method for tailoring fatty acid molecules by altering their aliphatic chains to facilitate the morphological evolution of gold nanoparticles from spherical to branched shape. It is found that the growth of the nanoparticles is sensitive to characteristics of fatty acids, such as saturation degrees. The growth of the nanoparticle is visualized by high-speed atomic force microscopy. The reaction mechanisms and growth processes of branched gold nanoparticles are proposed. This work may serve as a cornerstone to the design in a biomimetic fashion for the controllable synthesis of metallic nanomaterials.

DOI： 10.1016/j.isci.2022.105864

Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex 査読有り国際誌

Matsuo, Y; Uchihashi, T; Inada, T

NATURE COMMUNICATIONS 14 巻 ( 1 ) 頁： 79 - 79 2023年1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 collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

DOI： 10.1038/s41467-022-35608-4

Introduction of Session 2, "Advanced methods for retinal proteins" 招待有り

Uchihashi, T; Kandori, H

BIOPHYSICS AND PHYSICOBIOLOGY 20 巻 ( Supplemental ) 頁： n/a 2023年

支部だより

古谷祐詞, 内橋貴之, 矢木真穂

生物物理 63 巻 ( 5 ) 頁： 285 - 286 2023年

Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane 査読有り

Takahashi K., Nishikino T., Kajino H., Kojima S., Uchihashi T., Homma M.

Biophysics and physicobiology 20 巻 ( 2 ) 頁： n/a 2023年

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

The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor–stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the two-transmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF–fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.

DOI： 10.2142/biophysico.bppb-v20.0028

Clarification of Surface Deswelling of Thermoresponsive Microgels by Electrophoresis 査読有り

Nishizawa, Y; Inui, T; Namioka, R; Uchihashi, T; Watanabe, T; Suzuki, D

LANGMUIR 38 巻 ( 51 ) 頁： 16084 - 16093 2022年12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：Langmuir

Although many investigations of thermoresponsive microgels have been reported, their surface properties, which are crucial in colloid science, are still not fully understood. In this study, microgels with surface-localized charged groups were synthesized by precipitation polymerization, and their electrophoretic behaviors were analyzed using a modified version of Ohshima's equation to obtain two surface properties of the soft particles: the softness parameter and the surface charge density. This systematic evaluation allows us to discuss the thermoresponsiveness of the overall microgels and their surfaces separately. Furthermore, the validity of the surface properties obtained from electrophoresis was verified by comparing them with the results of seeded emulsion polymerization in the presence of the microgels and the force-indentation curves obtained via high-speed atomic force microscopy (HS-AFM).

DOI： 10.1021/acs.langmuir.2c02742

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface 査読有り国際共著

Uchiyama, T; Uchihashi, T; Ishida, T; Nakamura, A; Vermaas, JV; Crowley, MF; Samejima, M; Beckham, GT; Igarashi, K

SCIENCE ADVANCES 8 巻 ( 51 ) 頁： eade5155 2022年12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：Science Advances

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

DOI： 10.1126/sciadv.ade5155

Tip-scan high-speed atomic force microscopy with a uniaxial substrate stretching device for studying dynamics of biomolecules under mechanical stress 査読有り

Chan, FY; Kurosaki, R; Ganser, C; Takeda, T; Uchihashi, T

REVIEW OF SCIENTIFIC INSTRUMENTS 93 巻 ( 11 ) 頁： 113703 - 113703 2022年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Review of Scientific Instruments

High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

DOI： 10.1063/5.0111017

Scanning Probe Microscopy 招待有り

Nakajima K., Sumitomo K., Nakayama T., Ken-Ichi Fukui , Kageshima M., Kawai S., Komeda T., Takahashi T., Uchihashi T.

Japanese Journal of Applied Physics 61 巻 ( SL ) 2022年9月

Mechanism of the Irreversible Transition from Pentamer to Monomer at pH 2 in a Blue Proteorhodopsin

Sumikawa, M; Abe-Yoshizumi, R; Uchihashi, T; Kandori, H

BIOCHEMISTRY 61 巻 ( 18 ) 頁： 1936 - 1944 2022年8月

記述言語：英語出版者・発行元：Biochemistry

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λmax∼490 nm) and green-absorbing PRs (GPR; λmax∼525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different pKavalues of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.

DOI： 10.1021/acs.biochem.2c00328

Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy 査読有り国際誌

Nishiguchi, S; Furuta, T; Uchihashi, T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 119 巻 ( 30 ) 頁： e2208067119 2022年7月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Classical cadherins play key roles in cell–cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

DOI： 10.1073/pnas.2208067119

Protein Needles Designed to Self-Assemble through Needle Tip Engineering 査読有り

Kikuchi, K; Fukuyama, T; Uchihashi, T; Furuta, T; Maeda, YT; Ueno, T

SMALL 18 巻 ( 10 ) 頁： 2106401 - 2106401 2022年3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：Small

The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of β-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein–protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

DOI： 10.1002/smll.202106401

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/smll.202106401

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of C<sub>H</sub>1 Domain Deletion 査読有り国際誌

Yanaka, S; Nishiguchi, S; Yogo, R; Watanabe, H; Shen, JA; Yagi, H; Uchihashi, T; Kato, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 23 巻 ( 4 ) 2022年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH 1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH 1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

DOI： 10.3390/ijms23042090

Shape-selective one-step synthesis of branched gold nanoparticles on the crystal surface of redox-active Pd<SUP>II</SUP>-macrocycles 査読有り

Yamashita, Y; Tashiro, S; Ishii, Y; Uchihashi, T; Matsushita, N; Kubota, R; Shionoya, M

DALTON TRANSACTIONS 51 巻 ( 4 ) 頁： 1318 - 1324 2022年1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：Dalton Transactions

The synthesis of branched gold nanoparticles (AuNPs) with shape- and size-specific optical properties requires effective control of the particle formation mechanism using appropriate reducing agents and protective agents that prevent particle aggregation in solution. In this context, the heterogeneous synthesis of AuNPs using solid surfaces of graphene oxides and metal–organic frameworks has attracted much attention. These materials are characterized by their ability to immobilize and stabilize the particles grown on the surface without the need for additional protective agents. However, the shape- and size-selective synthesis of AuNPs using solid surfaces remains challenging. Herein, we report the shape-selective one-step synthesis of monodisperse branched AuNPs using a metal–macrocycle framework (MMF), a porous molecular crystal of PdII3-tris(phenylenediamine) macrocycle. Konpeito-Shaped branched AuNPs with uniform size were obtained on the surface of MMF by mixing HAuCl4·4H2O, l-ascorbic acid and MMF microcrystals. Spectroscopic and microscopic observations confirmed that MMF promoted the reduction of gold by its reductive activity as well as acted as a solid support to electrostatically immobilize the pseudo-seed particles for further growth on the crystal surface. In addition, the MMF also served as a substrate for in situ high-speed AFM imaging due to the effective immobilization of AuNPs on the surface, allowing direct visualization of the particle growth. Since the chemical structural features of MMF allow the growth of branched AuNPs via pseudo-seeding, this approach would provide new synthetic methods for obtaining a variety of gold nanostructures.

DOI： 10.1039/d1dt03973c

The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes 査読有り国際誌

Hamasaki E., Wakita N., Yasuoka H., Nagaoka H., Morita M., Takashima E., Uchihashi T., Takeda T., Abe T., Lee J.W., Iimura T., Saleem M.A., Ogo N., Asai A., Narita A., Takei K., Yamada H.

Frontiers in Cell and Developmental Biology 10 巻頁： 884509 - 884509 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Cell and Developmental Biology

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

DOI： 10.3389/fcell.2022.884509

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion 査読有り

Saeko Yanaka, Shigetaka Nishiguchi, Rina Yogo, Hiroki Watanabe, Jiana Shen, Hirokazu Yagi, Takayuki Uchihashi, and Koichi Kato

International Journal of Molecular Sciences 23 巻 ( 4 ) 頁： 2090 2021年12月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

DOI： 10.3390/ijms23042090.

Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin 査読有り

Sumikawa, M; Abe-Yoshizumi, R; Uchihashi, T; Kandori, H

JOURNAL OF PHYSICAL CHEMISTRY LETTERS 12 巻 ( 51 ) 頁： 12225 - 12229 2021年12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 Physical Chemistry Letters

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax ∼ 490 nm) and green-absorbing PR (GPR; λmax ∼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.

DOI： 10.1021/acs.jpclett.1c03355

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade 査読有り国際誌

Yagi-Utsumi, M; Aoki, K; Watanabe, H; Song, CH; Nishimura, S; Satoh, T; Yanaka, S; Ganser, C; Tanaka, S; Schnapka, V; Goh, EW; Furutani, Y; Murata, K; Uchihashi, T; Arakawa, K; Kato, K

SCIENTIFIC REPORTS 11 巻 ( 1 ) 頁： 21328 - 21328 2021年11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

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades’ response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol–gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

DOI： 10.1038/s41598-021-00724-6

Construction of ferritin hydrogels utilizing subunit-subunit interactions 査読有り

Yamanaka, M; Mashima, T; Ogihara, M; Okamoto, M; Uchihashi, T; Hirota, S

PLOS ONE 16 巻 ( 11 ) 頁： e0259052 - e0259052 2021年11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：PLoS ONE

Various proteins form nanostructures exhibiting unique functions, making them attractive as next-generation materials. Ferritin is a hollow spherical protein that incorporates iron ions. Here, we found that hydrogels are simply formed from concentrated apoferritin solutions by acid denaturation and subsequent neutralization. The water content of the hydrogel was approximately 80%. The apoferritin hydrogel did not decompose in the presence of 1 M HCl, 2-mercaptoethanol, or methanol but was dissolved in the presence of 1 M NaOH, by heating at 80C, or by treatment with trypsin or 6 M guanidine hydrochloride. The Young’s modulus of the hydrogel was 20.4 ± 12.1 kPa according to local indentation experimentes using atomic force microscopy, indicating that the hydrogel was relatively stiff. Transition electron microscopy measurements revealed that a fibrous network was constructed in the hydrogel. The color of the hydrogel became yellow-brown upon incubation in the presence of Fe3+ ions, indicating that the hydrogel adsorbed the Fe3+ ions. The yellow-brown color of the Fe3 +-adsorbed hydrogel did not change upon incubation in pure water, whereas it became pale by incubating it in the presence of 100 mM ethylenediaminetetraacetic acid (EDTA). The apoferritin hydrogel also adsorbed Co2+ and Cu2+ ions and released them in the presence of EDTA, while it adsorbed less Ni2+ ions; more Fe3+ ions adsorbed to the apoferritin hydrogel than other metal ions, indicating that the hydrogel keeps the iron storage characteristic of ferritin. These results demonstrate a new property of ferritin: the ability to form a hydrogel that can adsorb/desorb metal ions, which may be useful in designing future biomaterials.

DOI： 10.1371/journal.pone.0259052

Deformation of microtubules regulates translocation dynamics of kinesin 査読有り国際共著国際誌

Nasrin, SR; Ganser, C; Nishikawa, S; Kabir, AMR; Sada, K; Yamashita, T; Ikeguchi, M; Uchihashi, T; Hess, H; Kakugo, A

SCIENCE ADVANCES 7 巻 ( 42 ) 頁： eabf2211 2021年10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：Science Advances

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

DOI： 10.1126/sciadv.abf2211

Scanning Probe Microscopy FOREWORD 招待有り

Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

JAPANESE JOURNAL OF APPLIED PHYSICS 60 巻 ( SE ) 2021年9月

高速原子間力顕微鏡による一分子動態計測：最近の応用研究招待有り

内橋貴之

生化学 93 巻頁： 526 - 561 2021年8月

Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible β-Barrel Structure in Solution and Keeps This Structure in Dehydration 査読有り国際誌

Miyazawa, K; Itoh, SG; Watanabe, H; Uchihashi, T; Yanaka, S; Yagi-Utsumi, M; Kato, K; Arakawa, K; Okumura, H

JOURNAL OF PHYSICAL CHEMISTRY B 125 巻 ( 32 ) 頁： 9145 - 9154 2021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：Journal of Physical Chemistry B

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

DOI： 10.1021/acs.jpcb.1c04850

Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images 査読有り国際誌

Dasgupta, B; Miyashita, O; Uchihashi, T; Tama, F

FRONTIERS IN MOLECULAR BIOSCIENCES 8 巻頁： 704274 - 704274 2021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：Frontiers in Molecular Biosciences

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

DOI： 10.3389/fmolb.2021.704274

JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes 査読有り国際誌

Sakane, A; Yano, T; Uchihashi, T; Horikawa, K; Hara, Y; Imoto, I; Kurisu, S; Yamada, H; Takei, K; Sasaki, T

COMMUNICATIONS BIOLOGY 4 巻 ( 1 ) 頁： 551 - 551 2021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：Communications Biology

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

DOI： 10.1038/s42003-021-02080-7

Non-close-packed arrangement of soft elastomer microspheres on solid substrates 査読有り

Sasaki, Y; Hiroshige, S; Takizawa, M; Nishizawa, Y; Uchihashi, T; Minato, H; Suzuki, D

RSC ADVANCES 11 巻 ( 24 ) 頁： 14562 - 14567 2021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：RSC Advances

Unlike rigid microparticles, soft and deformable elastomer (rubber) microspheres were found to exhibit a non-close-packed arrangement on solid substrates after the evaporation of water from their dispersions. The microscopic observation revealed that individual microspheres are ordered in regular intervals at the air/water interface of a sessile droplet and remain fixed on the substrate without being affected by the capillary forces during evaporation due to their deformability. Moreover, using the Langmuir-Blodgett method, thin films of non-close-packed structures could be successfully generated over large areas. Our findings may potentially help to control the arranged structures of elastomer microspheres, which can be expected to improve the nano-science and technology for the precise control for e.g. surface patterning. This journal is

DOI： 10.1039/d1ra02688g

Nanostructure and thermoresponsiveness of poly(<i>N</i>-isopropyl methacrylamide)-based hydrogel microspheres prepared <i>via</i> aqueous free radical precipitation polymerization 査読有り

Nishizawa, Y; Minato, H; Inui, T; Saito, I; Kureha, T; Shibayama, M; Uchihashi, T; Suzuki, D

RSC ADVANCES 11 巻 ( 22 ) 頁： 13130 - 13137 2021年4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：RSC Advances

Thermoresponsive hydrogel microspheres (microgels) are smart materials that quickly respond to external stimuli, and their thermoresponsiveness can be tuned by varying the constituent chemical species. Although uniformly sized microgels can be preparedviaaqueous free radical precipitation polymerization, the nanostructure of the obtained microgels is complex and remains unclear so far. In the present study, the nanostructure and thermoresponsiveness of poly(N-isopropyl methacrylamide) (pNIPMAm)-based microgels, which have a volume-transition temperature of ∼43 °C, were evaluated mainly using temperature-controllable high-speed atomic force microscopy. These observations, which are characterized by high spatio-temporal resolution, revealed that the pNIPMAm microgels have a peculiar heterogeneous structure, for example a core-shell and non-thermoresponsive nanostructure in the core region, that originates from the precipitation polymerization process. Furthermore, it was found that the adsorption concentration of the microgels on the substrate is one of the keys for controlling their thermoresponsiveness. These findings can be expected to advance the design of new materials such as thermoresponsive nanosheets and stimuli-responsive coatings.

DOI： 10.1039/d1ra01650d

Dynamic Assembly/Disassembly of <i>Staphylococcus aureus</i> FtsZ Visualized by High-Speed Atomic Force Microscopy 査読有り国際誌

Fujita, J; Sugiyama, S; Terakado, H; Miyazaki, M; Ozawa, M; Ueda, N; Kuroda, N; Tanaka, S; Yoshizawa, T; Uchihashi, T; Matsumura, H

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 4 ) 頁： 1 - 10 2021年2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：International Journal of Molecular Sciences

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architec-tures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.

DOI： 10.3390/ijms22041697

Nanostructures, Thermoresponsiveness, and Assembly Mechanism of Hydrogel Microspheres during Aqueous Free-Radical Precipitation Polymerization 査読有り国際誌

Nishizawa, Y; Minato, H; Inui, T; Uchihashi, T; Suzuki, D

LANGMUIR 37 巻 ( 1 ) 頁： 151 - 159 2021年1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：Langmuir

Although techniques to produce uniformly sized hydrogel microspheres (microgels) by aqueous free-radical precipitation polymerization are well established, the details of the polymerization process remain mysterious. In the present study, the structural evolution and thermoresponsiveness of the developing microgels during the polymerization were evaluated by temperature-controlled high-speed atomic force microscopy. This analysis clarified that the swelling properties of the precursor microgels formed in the early stages of the polymerization are quite low due to the high incorporation of cross-linkers and that non-thermoresponsive deca-nanosized spherical domains are already present in the precursor microgels. Furthermore, we succeeded in tracking the formation of nuclei and their growth process, which has never been fully understood, in aqueous solution by real-time observations. These findings will help us to design functional microgels with the desired nanostructures via precipitation polymerization.

DOI： 10.1021/acs.langmuir.0c02654

Single-molecule dynamics observed by high-speed atomic force microscopy: Recent applications

Uchihashi T.

Seikagaku 93 巻 ( 4 ) 頁： 526 - 531 2021年

Structural insights into the mechanism of rhodopsin phosphodiesterase 査読有り国際誌

Ikuta, T; Shihoya, W; Sugiura, M; Yoshida, K; Watari, M; Tokano, T; Yamashita, K; Katayama, K; Tsunoda, SP; Uchihashi, T; Kandori, H; Nureki, O

NATURE COMMUNICATIONS 11 巻 ( 1 ) 頁： 5605 - 5605 2020年11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

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

DOI： 10.1038/s41467-020-19376-7

Novel <i>Babesia bovis</i> exported proteins that modify properties of infected red blood cells 査読有り国際誌

Hakimi, H; Templeton, TJ; Sakaguchi, M; Yamagishi, J; Miyazaki, S; Yahata, K; Uchihashi, T; Kawazu, S; Kaneko, O; Asada, M

PLOS PATHOGENS 16 巻 ( 10 ) 頁： e1008917 2020年10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：PLoS Pathogens

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV-III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

DOI： 10.1371/journal.ppat.1008917

Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution 査読有り国際誌

Maity, B; Li, ZP; Niwase, K; Ganser, C; Furuta, T; Uchihashi, T; Lu, DN; Ueno, T

PHYSICAL CHEMISTRY CHEMICAL PHYSICS 22 巻 ( 33 ) 頁： 18562 - 18572 2020年9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：Physical Chemistry Chemical Physics

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications. This journal is

DOI： 10.1039/d0cp02069a

Thermoresponsive structural changes of single poly(<i>N</i>-isopropyl acrylamide) hydrogel microspheres under densely packed conditions on a solid substrate 査読有り

Minato, H; Nishizawa, Y; Uchihashi, T; Suzuki, D

POLYMER JOURNAL 52 巻 ( 9 ) 頁： 1137 - 1141 2020年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Polymer Journal

The thermoresponsiveness of hydrogel microspheres (microgels) was visualized in situ at the nanoscale using temperature-controllable high-speed atomic force microscopy (TC-HS-AFM). The morphological changes of the microgels are strongly affected by their packing conditions and by their adsorption on a solid substrate.

DOI： 10.1038/s41428-020-0372-3

その他リンク： http://www.nature.com/articles/s41428-020-0372-3

Convergent evolution of processivity in bacterial and fungal cellulases 査読有り国際誌

Uchiyama, T; Uchihashi, T; Nakamura, A; Watanabe, H; Kaneko, S; Samejima, M; Igarashi, K

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 117 巻 ( 33 ) 頁： 19896 - 19903 2020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：Proceedings of the National Academy of Sciences of the United States of America

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, “traffic accidents” were observed, in which the two cellulases blocked each other’s progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

DOI： 10.1073/pnas.2011366117

Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA 査読有り国際誌

Hamad, N; Watanabe, H; Uchihashi, T; Kurokawa, R; Nagata, T; Katahira, M

CHEMICAL COMMUNICATIONS 56 巻 ( 64 ) 頁： 9134 - 9137 2020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：Chemical Communications

High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided. This journal is

DOI： 10.1039/d0cc03776a

Scanning Probe Microscopy FOREWORD

Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

JAPANESE JOURNAL OF APPLIED PHYSICS 59 巻 2020年8月

Assembly Mechanism of a Supramolecular MS-Ring Complex To Initiate Bacterial Flagellar Biogenesis in Vibrio Species 査読有り国際誌

Terashima, H; Hirano, K; Inoue, Y; Tokano, T; Kawamoto, A; Kato, T; Yamaguchi, E; Namba, K; Uchihashi, T; Kojima, S; Homma, M

JOURNAL OF BACTERIOLOGY 202 巻 ( 16 ) 2020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：Journal of Bacteriology

The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein and is an essential core structure that is a part of the rotor. The number and locations of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly or on the contributions of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component, FliG, facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating a requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed green fluorescent protein (GFP)-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was coexpressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to that of the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations. IMPORTANCE Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, which is embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization, and a supramolecular complex assembly.

DOI： 10.1128/JB.00236-20

ヘリオロドプシンおよびシゾロドプシンの構造から明らかになった微生物型ロドプシンの多様性(Structures of heliorhodopsin and schizorhodopsin elucidate the structural diversity of microbial rhodopsins)

Shihoya Wataru, Inoue Keiichi, Manish Singh, Higuchi Akimitsu, Konno Masae, Yoshizumi Rei, Uchihashi Takayuki, Kandori Hideki, Nureki Osamu

生物物理 60 巻 ( Suppl.1-2 ) 頁： S154 - S154 2020年8月

Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy 査読有り国際誌

Tokano, T; Kato, Y; Sugiyama, S; Uchihashi, T; Noguchi, T

JOURNAL OF PHYSICAL CHEMISTRY B 124 巻 ( 28 ) 頁： 5847 - 5857 2020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 Physical Chemistry B

Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

DOI： 10.1021/acs.jpcb.0c03892

Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy 査読有り国際誌

Ueno, T; Niwase, K; Tsubokawa, D; Kikuchi, K; Takai, N; Furuta, T; Kawano, R; Uchihashi, T

NANOSCALE 12 巻 ( 15 ) 頁： 8166 - 8173 2020年4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：Nanoscale

Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

DOI： 10.1039/d0nr01121e

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy 査読有り国際誌

Konno, H; Watanabe-Nakayama, T; Uchihashi, T; Okuda, M; Zhu, LW; Kodera, N; Kikuchi, Y; Ando, T; Taguchi, H

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 117 巻 ( 14 ) 頁： 7831 - 7836 2020年4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：Proceedings of the National Academy of Sciences of the United States of America

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

DOI： 10.1073/pnas.1916452117

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H<SUP>+</SUP> pumps 査読有り国際誌

Inoue, K; Tsunoda, SP; Singh, M; Tomida, S; Hososhima, S; Konno, M; Nakamura, R; Watanabe, H; Bulzu, PA; Banciu, HL; Andrei, AS; Uchihashi, T; Ghai, R; Béjà, O; Kandori, H

SCIENCE ADVANCES 6 巻 ( 15 ) 頁： eaaz2441 2020年4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：Science Advances

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

DOI： 10.1126/sciadv.aaz2441

Recent advances in bioimaging with high-speed atomic force microscopy 査読有り国際誌

Uchihashi T., Ganser C.

Biophysical Reviews 12 巻 ( 2 ) 頁： 363 - 369 2020年4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：Biophysical Reviews

Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

DOI： 10.1007/s12551-020-00670-z

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from <i>Serratia marcescens</i> 査読有り国際誌

Visootsat, A; Nakamura, A; Vignon, P; Watanabe, H; Uchihashi, T; Iino, R

JOURNAL OF BIOLOGICAL CHEMISTRY 295 巻 ( 7 ) 頁： 1915 - 1925 2020年2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 Biological Chemistry

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

DOI： 10.1074/jbc.RA119.012078

Induced-Fit Pathway Accelerated Binding of Agitoxin-2 to A K<SUP>+</SUP> Channel Imaged by HS-AFM

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

BIOPHYSICAL JOURNAL 118 巻 ( 3 ) 頁： 236A - 236A 2020年2月

Construction of a Hexameric Hemoprotein Sheet and Direct Observation of Dynamic Processes of Its Formation 査読有り

Oohora, K; Hirayama, S; Uchihashi, T; Hayashi, T

CHEMISTRY LETTERS 49 巻 ( 2 ) 頁： 186 - 190 2020年2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：Chemistry Letters

A two-dimensional sheet assembly of a hexameric hemoprotein was constructed. A single cysteine residue was introduced onto each subunit surface in the hexameric hemoprotein and modified with a maleimide-tethering tripeptide FGG tag to provide a hostguest interaction between one cucurbit[8]uril molecule (CB8) and two FGG tags. The addition of CB8 into the engineered protein forms the assembly as a sheet-like precipitate. High-speed atomic force microscopic measurements directly reveal the detailed structure and the dynamic process of formation.

DOI： 10.1246/cl.190855

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies 査読有り国際誌

Umakoshi, T; Fukuda, S; Iino, R; Uchihashi, T; Ando, T

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1864 巻 ( 2 ) 頁： 129325 2020年2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：Biochimica et Biophysica Acta - General Subjects

Background: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. Methods: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. Results: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. Conclusions: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. General significance: We achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

DOI： 10.1016/j.bbagen.2019.03.011

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation 査読有り国際誌

Yagi-Utsumi, M; Sikdar, A; Song, CH; Park, J; Inoue, R; Watanabe, H; Burton-Smith, RN; Kozai, T; Suzuki, T; Kodama, A; Ishii, K; Yagi, H; Satoh, T; Uchiyama, S; Uchihashi, T; Joo, K; Lee, J; Sugiyama, M; Murata, K; Kato, K

SCIENTIFIC REPORTS 10 巻 ( 1 ) 頁： 1540 - 1540 2020年1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

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

DOI： 10.1038/s41598-020-58371-2

Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(<i>N</i>-isopropylacrylamide) toward an Artificial Light-Harvesting System 査読有り国際誌

Hirayama, S; Oohora, K; Uchihashi, T; Hayashi, T

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 142 巻 ( 4 ) 頁： 1822 - 1831 2020年1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 the American Chemical Society

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

DOI： 10.1021/jacs.9b10080

Dynamics of Inter-Molecular Interactions Between Single Aβ<sub>42</sub> Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy (vol 431, pg 2687, 2019) 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 432 巻 ( 2 ) 頁： 633 - 633 2020年1月

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

The authors would like to correct the values of Koff (s−1) in ‘Table 1. Summary of properties’ for column of Aβ15–20nm and Aβ36nm, replacing 2.17 ± 0.87 with 1.82, 1.64 ± 0.64 with 1.39, respectively. Enclosed you will find correct version of Table 1. Also, the Koff (s−1) values appear in the sentence “A mean lifetime <t> = 0.55 ± 0.22 s, with dissociation rate constant koff (1/<t>) = 2.17 ± 0.87 s−1, for the Aβ15–20 nm ↔ Aβ15–20 nm compared to the Aβ15–20 nm ↔ Aβ36 nm with a mean lifetime = 0.72 ± 0.28 s (koff = 1.64 ± 0.64 s−1), indicating the latter to some extent forms more tightly bound complexes due to its slower dissociation rate.” on page 2693 in the article. The koff (1/<t>) values of 2.17 ± 0.87 s−1 and 1.64 ± 0.64 s−1 are now corrected to 1.82 s−1 and 1.39 s−1, respectively. The authors would like to apologise for any inconvenience caused. [Table presented]

DOI： 10.1016/j.jmb.2019.10.008

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge 査読有り国際誌

Tatebe, H; Lim, CT; Konno, H; Shiozaki, K; Shinohara, A; Uchihashi, T; Furukohri, A

NATURE COMMUNICATIONS 11 巻 ( 1 ) 頁： 370 - 370 2020年1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 human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

DOI： 10.1038/s41467-019-14025-0

On-Membrane Dynamic Interplay between Anti-GM1 IgG Antibodies and Complement Component C1q 査読有り国際誌

Yanaka, S; Yogo, R; Watanabe, H; Taniguchi, Y; Satoh, T; Komura, N; Ando, H; Yagi, H; Yuki, N; Uchihashi, T; Kato, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 巻 ( 1 ) 2020年1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：International Journal of Molecular Sciences

Guillain–Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc–Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain–Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

DOI： 10.3390/ijms21010147

Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone 査読有り国際誌

Cho, C; Jang, J; Kang, YJ; Watanabe, H; Uchihashi, T; Kim, SJ; Kato, K; Lee, JY; Song, JJ

NATURE COMMUNICATIONS 10 巻 ( 1 ) 頁： 5764 - 5764 2019年12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 fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3–H4 loading by utilizing ATP.

DOI： 10.1038/s41467-019-13743-9

Crystal structure of heliorhodopsin 査読有り国際誌

Shihoya, W; Inoue, K; Singh, M; Konno, M; Hososhima, S; Yamashita, K; Ikeda, K; Higuchi, A; Izume, T; Okazaki, S; Hashimoto, M; Mizutori, R; Tomida, S; Yamauchi, Y; Abe-Yoshizumi, R; Katayama, K; Tsunoda, SP; Shibata, M; Furutani, Y; Pushkarev, A; Béjá, O; Uchihashi, T; Kandori, H; Nureki, O

NATURE 574 巻 ( 7776 ) 頁： 132 - + 2019年10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

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

DOI： 10.1038/s41586-019-1604-6

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

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

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

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

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

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

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

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

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome Subunits 査読有り国際誌

Sekiguchi, T; Satoh, T; Kurimoto, E; Song, CH; Kozai, T; Watanabe, H; Ishii, K; Yagi, H; Yanaka, S; Uchiyama, S; Uchihashi, T; Murata, K; Kato, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 20 巻 ( 9 ) 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：International Journal of Molecular Sciences

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1–α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

DOI： 10.3390/ijms20092308

Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins 査読有り国際誌

Miyamoto, T; Hayashi, Y; Yoshida, K; Watanabe, H; Uchihashi, T; Yonezawa, K; Shimizu, N; Kamikubo, H; Hirota, S

ACS SYNTHETIC BIOLOGY 8 巻 ( 5 ) 頁： 1112 - 1120 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：ACS Synthetic Biology

Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

DOI： 10.1021/acssynbio.9b00019

Inner lumen proteins stabilize doublet microtubules in cilia and flagella 査読有り国際誌

Owa, M; Uchihashi, T; Yanagisawa, H; Yamano, T; Iguchi, H; Fukuzawa, H; Wakabayashi, K; Ando, T; Kikkawa, M

NATURE COMMUNICATIONS 10 巻 ( 1 ) 頁： 1143 - 1143 2019年3月

記述言語：英語出版者・発行元：Nature Communications

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, K; Kajihara, R; Fujimaki, N; Uchihashi, T; Hayashi, T

CHEMICAL COMMUNICATIONS 55 巻 ( 11 ) 頁： 1544 - 1547 2019年2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：Chemical Communications

Engineered cytochrome b562, a small hemoprotein, with an externally-attached heme moiety via a moderately long linker at a suitable position predominantly forms a thermodynamically stable ring-shaped trimer in dilute solution. In an equilibrium between supramolecular polymerization and depolymerization, the ring-shaped trimer is kinetically trapped even in a concentrated solution.

DOI： 10.1039/c8cc09314h

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

Ganser, C; Uchihashi, T

NANOSCALE 11 巻 ( 1 ) 頁： 125 - 135 2019年1月

記述言語：英語出版者・発行元：Royal Society of Chemistry ({RSC})

Microtubules are biopolymers composed of tubulin and play diverse roles in a wide variety of biological processes such as cell division, migration and intracellular transport in eukaryotic cells. To perform their functions, microtubules are mechanically stressed and, thereby, susceptible to structural defects. Local variations in mechanical properties caused by these defects modulate their biological functions, including binding and transportation of microtubule-associated proteins. Therefore, assessing the local mechanical properties of microtubules and analyzing their dynamic response to mechanical stimuli provide insight into fundamental processes. It is, however, not trivial to control defect formation, gather mechanical information at the same time, and subsequently image the result at a high temporal resolution at the molecular level with minimal delay. In this work, we describe the so-called in-line force curve mode based on high-speed atomic force microscopy. This method is directly applied to create defects in microtubules at the level of tubulin dimers and monitor the following dynamic processes around the defects. Furthermore, force curves obtained during defect formation provide quantitative mechanical information to estimate the bonding energy between tubulin dimers.

DOI： 10.1039/c8nr07392a

Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy 査読有り

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

STRUCTURE 27 巻 ( 1 ) 頁： 152 - + 2019年1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：Structure

Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

DOI： 10.1016/j.str.2018.09.005

Metastable asymmetrical structure of a shaftless V<sub>1</sub> motor 査読有り

Maruyama, S; Suzuki, K; Imamura, M; Sasaki, H; Matsunami, H; Mizutani, K; Saito, Y; Imai, FL; Ishizuka-Katsura, Y; Kimura-Someya, T; Shirouzu, M; Uchihashi, T; Ando, T; Yamato, I; Murata, T

SCIENCE ADVANCES 5 巻 ( 1 ) 頁： eaau8149 - eaau8149 2019年1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：Science Advances

V 1 -ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A 3 B 3 complex and a central DF shaft. The nucleotide-free A 3 B 3 complex of Enterococcus hirae, composed of three identical A 1 B 1 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 A 3 B 3 . The nucleotide-free mutant A 3 B 3 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 A 3 B 3 forms a metastable (stressed) asymmetric structure composed of unstable A 1 B 1 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招待有り

内橋貴之

高分子 68 巻 ( 01 ) 頁： 564 - 568 2019年

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) 頁： 281 - 285 2019年

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) 頁： 253 - 256 2019年

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) 頁： 281 - 285 2019年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：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.

Versatility of RNA-Binding Proteins in Living Cells 査読有り

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 5 巻 ( 1 ) 頁： 7 - 7 2019年

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

Fukui, T; Uchihashi, T; Sasaki, N; Watanabe, H; Takeuchi, M; Sugiyasu, K

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 57 巻 ( 47 ) 頁： 15465 - 15470 2018年11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

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 査読有り国際誌

Matsui, S; Nishizawa, Y; Uchihashi, T; Suzuki, D

ACS OMEGA 3 巻 ( 9 ) 頁： 10836 - 10842 2018年9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：ACS Omega

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, K; Fujimaki, N; Kajihara, R; Watanabe, H; Uchihashi, T; Hayashi, T

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 140 巻 ( 32 ) 頁： 10145 - 10148 2018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：Journal of the American Chemical Society

A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

DOI： 10.1021/jacs.8b06690

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

Mori, T; Sugiyama, S; Byrne, M; Johnson, CH; Uchihashi, T; Ando, T

NATURE COMMUNICATIONS 9 巻 ( 1 ) 頁： 3245 2018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：Nature Communications

The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA’s affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.

DOI： 10.1038/s41467-018-05438-4

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 57 巻 ( 8 ) 2018年8月

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

Umakoshi, T; Udaka, H; Uchihashi, T; Ando, T; Suzuki, M; Fukuda, T

COLLOIDS AND SURFACES B-BIOINTERFACES 167 巻頁： 267 - 274 2018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：Colloids and Surfaces B: Biointerfaces

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.

DOI： 10.1016/j.colsurfb.2018.04.015

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

Hosoyamada, M; Yanai, N; Okumura, K; Uchihashi, T; Kimizuka, N

CHEMICAL COMMUNICATIONS 54 巻 ( 50 ) 頁： 6828 - 6831 2018年6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：Chemical Communications

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

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

NATURE COMMUNICATIONS 9 巻 ( 1 ) 頁： 2147 2018年6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 ATP-dependent bacterial protein disaggregation machine, ClpB 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 ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB 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 ClpB to disentangle protein aggregates.

DOI： 10.1038/s41467-018-04587-w

Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation 査読有り

Maruno, T; Watanabe, H; Yoneda, S; Uchihashi, T; Adachi, S; Arai, K; Sawaguchi, T; Uchiyama, S

JOURNAL OF PHARMACEUTICAL SCIENCES 107 巻 ( 6 ) 頁： 1521 - 1529 2018年6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

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/m 2 , 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

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy

Shibata, M; Inoue, K; Ikeda, K; Konno, M; Singh, M; Kataoka, C; Abe-Yoshizumi, R; Kandori, H; Uchihashi, T

SCIENTIFIC REPORTS 8 巻 ( 1 ) 頁： 8262 2018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：Scientific Reports

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

DOI： 10.1038/s41598-018-26606-y

Structural properties determining low K<SUP>+</SUP> affinity of the selectivity filter in the TWIK1 K<SUP>+</SUP> channel

Tsukamoto, H; Higashi, M; Motoki, H; Watanabe, H; Ganser, C; Nakajo, K; Kubo, Y; Uchihashi, T; Furutani, Y

JOURNAL OF BIOLOGICAL CHEMISTRY 293 巻 ( 18 ) 頁： 6969 - 6984 2018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：Journal of Biological Chemistry

Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.

DOI： 10.1074/jbc.RA118.001817

Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an -Helix-Inserted Circular Permutant of Cytochrome <i>c</i><sub>555</sub>

Oda, A; Nagao, S; Yamanaka, M; Ueda, I; Watanabe, H; Uchihashi, T; Shibata, N; Higuchi, Y; Hirota, S

CHEMISTRY-AN ASIAN JOURNAL 13 巻 ( 8 ) 頁： 964 - 967 2018年4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：Chemistry - An Asian Journal

Highly-ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c555, and assembled BBP into a triangle-shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain-swapping hinge loop of cyt c555 and connecting the original N- and C-terminal α-helices with an α-helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X-ray crystallographic analysis of the BBP trimer, the N-terminal region of one BBP molecule interacted intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N- and C-terminal α-helices with an α-helical linker may be useful for constructing organized protein structures.

DOI： 10.1002/asia.201800252

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Terahara, N; Inoue, Y; Kodera, N; Morimoto, YV; Uchihashi, T; Imada, K; Ando, T; Namba, K; Minamino, T

SCIENCE ADVANCES 4 巻 ( 4 ) 頁： eaao7054 2018年4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：Science Advances

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

DOI： 10.1126/sciadv.aao7054

Front Cover: Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555 (Chem. Asian J. 10/2018) 査読有り

Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

Chemistry - An Asian Journal 13 巻 ( 10 ) 頁： 1229 2018年4月

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui, S; Uchihashi, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui, S; Uchihashi, T; Suzuki, D

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 255 巻頁： . 2018年3月

Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II 査読有り

Haruyama, T; Uchihashi, T; Yamada, Y; Kodera, N; Ando, T; Konno, H

JOURNAL OF MOLECULAR BIOLOGY 430 巻 ( 5 ) 頁： 602 - 610 2018年3月

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis 国際誌

Nakamura, A; Tasaki, T; Okuni, Y; Song, CH; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

PHYSICAL CHEMISTRY CHEMICAL PHYSICS 20 巻 ( 5 ) 頁： 3010 - 3018 2018年2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：Physical Chemistry Chemical Physics

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, kfastoff (3.2 s-1, 78%) and kslowoff (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, kfastoff, kslowoff and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

DOI： 10.1039/c7cp04606e

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis (vol 20, pg 3010, 2018) 国際誌

Nakamura, A; Tasaki, T; Okuni, Y; Song, C; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

PHYSICAL CHEMISTRY CHEMICAL PHYSICS 20 巻 ( 5 ) 頁： 3844 - 3844 2018年2月

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

Sugiyama, S; Mori, T; Byrne, M; Uchihashi, T; Johnson, CH; Ando, T

BIOPHYSICAL JOURNAL 114 巻 ( 3 ) 頁： 68A - 68A 2018年2月

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H, Ando Toshio

BIOPHYSICAL JOURNAL 114 巻 ( 3 ) 頁： 68A-68A 2018年2月

Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes

Uchihashi, T; Scheuring, S

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1862 巻 ( 2 ) 頁： 229 - 240 2018年2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：Biochimica et Biophysica Acta - General Subjects

Background Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work. Scope of review The scope of this review is overviewing recent progresses of HS-AFM for studying dynamic processes of biomolecular systems. In the technical descriptions, key developments enabling fast and non-invasive imaging of biological samples are briefly mentioned. Then recent successful applications of HS-AFM are overviewed to showcase the power of HS-AFM in biological research. Major conclusions We discuss examples where HS-AFM movies captured important dynamic biological processes, including conformational dynamics of membrane proteins, processive movements of enzymes, assembly and disassembly processes of protein supramolecular structures, and dynamics in a two-dimensional protein crystal. These examples demonstrate the usability of HS-AFM to reveal biomolecular processes at high spatiotemporal (nanometer and subsecond) resolution. General significance Real-time movies of unlabeled proteins at work captured by HS-AFM allowed us to directly gain insights into mechanisms of molecular actions. Together with further functional extensions, HS-AFM will enable researchers to investigate more complex biological systems involving multiple proteins and will become an indispensable technique for life science. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” Guest Editor: Dr., Professor Koichi Kato.

DOI： 10.1016/j.bbagen.2017.07.010

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis 査読有り

Takeda, T; Kozai, T; Yang, H; Ishikuro, D; Seyama, K; Kumagai, Y; Abe, T; Yamada, H; Uchihashi, T; Ando, T; Takei, K

ELIFE 7 巻 2018年1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：eLife

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation 査読有り国際誌

Yagi-Utsumi, M; Sikdar, A; Kozai, T; Inoue, R; Sugiyama, M; Uchihashi, T; Yagi, H; Satoh, T; Kato, K

PROTEIN ENGINEERING DESIGN & SELECTION 31 巻 ( 1 ) 頁： 29 - 36 2018年1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：Protein Engineering, Design and Selection

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

DOI： 10.1093/protein/gzx066

Visualization of Protein Dynamics using High-Speed Atomic Force Microscopy and Image Analysis

Uchihashi Takayuki

JOURNAL OF COMPUTER CHEMISTRY-JAPAN 17 巻 ( 1 ) 頁： 20-30 - 30 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：日本コンピュータ化学会

<p>最近の高速原子間力顕微鏡(Atomic Force Microscopy: AFM)の進展によって, 生理溶液中でのタンパク質の構造とその時間発展，分子の離散集合などダイナミクスをリアルタイムで可視化できるようになってきた. これまで様々な計測手法による実験事実の積み重ねから推定されてきた事実を動画として提示することで, タンパク質の動的振る舞いを介した機能発現の分子機構を直接的かつ直感的に把握できるようになった. 動画は静止画に比べて極めて多くの情報を与えるが, 一方で, 単に動画鑑賞に終わることなく, そこから有益な情報を客観的な指標を基に抽出すること, すなわち動画解析が次のステップには重要である. 本稿では, これまで高速AFMで観察されてきた代表的なタンパク質の動的現象をi)一分子の構造変化, ii)分子の直進運動と速度解析, iii)二分子間の結合と解離, の3つに大別し, それぞれについて筆者らの実験結果をデータの解釈と解析の効率化のために開発した画像解析法とともに解説する.</p>

DOI： 10.2477/jccj.2018-0001

Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy

Uchihashi, T; Watanabe, H; Kodera, N

NANOSCALE IMAGING: METHODS AND PROTOCOLS 1814 巻頁： 159 - 179 2018年

記述言語：英語出版者・発行元：Methods in Molecular Biology

Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

DOI： 10.1007/978-1-4939-8591-3_10

High-speed scanning near-field optical microscopy

Umakoshi T., Fukuda S., Uchihashi T., Verma P., Ando T.

Optics InfoBase Conference Papers Part F125-JSAP 2018 巻 2018年

記述言語：日本語出版者・発行元：Optics InfoBase Conference Papers

Scanning near-field optical microscopy (SNOM) has been recognized as a powerful technique for super-resolution optical imaging [1]. It has been even more unique compared with other super-resolution fluorescent imaging techniques because it utilizes near-field light at a metallic tip as a nano-light-source, which realizes super-resolution not only in fluorescence but also in any other optical signals such as Raman scattering, infrared absorption and photoluminescence. Such a strong advantage has been stimulating biological fields as a new class of analytical techniques. However, it has been yet highly challenging to apply SNOM for biological samples. Although there have been several issues for biological applications, one of the major problems is its low imaging speed. Because it requires to physically scan a metallic tip, the imaging rate was typically limited to several minutes per frame, which is too slow to capture dynamic biological samples.

Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy 査読有り

Takayuki Uchihashi, Hiroki Watanabe, Noriyuki Kodera

Methods in Molecular Biology 1814 巻頁： 159 - 179 2018年

エンドサイトーシス生物学の新展開ダイナミンによる膜切断過程の動態イメージング

竹田哲也, 小財稔矢, 楊恵然, 石黒大輝, 背山佳穂, 熊谷祐介, 阿部匡史, 山田浩司, 内橋貴之, 安藤敏夫, 竹居孝二

生命科学系学会合同年次大会 2017年度巻頁： [4AW17 - 2] 2017年12月

Two-step process for disassembly mechanism of proteasome α7 homo-tetradecamer by α6 revealed by high-speed atomic force microscopy 国際誌

Kozai, T; Sekiguchi, T; Satoh, T; Yagi, H; Kato, K; Uchihashi, T

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 15373 - 15373 2017年11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

The 20S proteasome is a core particle of the eukaryotic proteasome responsible for proteolysis and is composed of layered α and β hetero-heptameric rings. The α7 subunit, which is one of components of the α ring, is known to self-assemble into a double-ringed homo-tetradecamer composed of two layers of the α7 heptameric ring. The α7 tetradecamer is known to disassemble upon the addition of α6 subunit, producing a 1:7 hetero-octameric α6-α7 complex. However, the detailed disassembly mechanism remains unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to dissect the disassembly process of the α7 double ring caused by interaction with the α6. HS-AFM movies clearly demonstrated two different modes of interaction in which the α6 monomer initially cracks at the interface between the stacked two α7 single rings and the subsequent intercalation of the α6 monomer in the open pore of the α7 single ring blocks the re-association of the single rings into the double ring. This result provides a mechanistic insight about the disassembly process of non-native homo-oligomers formed by proteasome components which is crucial for the initial process for assembly of 20S proteasome.

DOI： 10.1038/s41598-017-15708-8

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

Shibata, M; Nishimasu, H; Kodera, N; Hirano, S; Ando, T; Uchihashi, T; Nureki, O

NATURE COMMUNICATIONS 8 巻 ( 1 ) 頁： 1430 2017年11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 CRISPR-Associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-Time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-Time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

DOI： 10.1038/s41467-017-01466-8

Na<SUP>+</SUP>-induced structural transition of MotPS for stator assembly of the <i>Bacillus</i> flagellar motor

Terahara, N; Kodera, N; Uchihashi, T; Ando, T; Namba, K; Minamino, T

SCIENCE ADVANCES 3 巻 ( 11 ) 頁： eaao4119 2017年11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：Science Advances

The bacterial flagellar motor consists of a rotor and a dozen stator units and regulates the number of active stator units around the rotor in response to changes in the environment. The MotPS complex is a Na+-type stator unit in the Bacillus subtilis flagellar motor and binds to the peptidoglycan layer through the peptidoglycan-binding (PGB) domain of MotS to act as the stator. The MotPS complex is activated in response to an increase in the Na+ concentration in the environment, but the mechanism of this activation has remained unknown. We report that activation occurs by a Na+-induced folding and dimer formation of the PGB domain of MotS, as revealed in real-time imaging by high-speed atomic force microscopy. The MotPS complex showed two distinct ellipsoid domains connected by a flexible linker. A smaller domain, corresponding to the PGB domain, became structured and unstructured in the presence and absence of 150 mM NaCl, respectively. When the amino-terminal portion of the PGB domain adopted a partially stretched conformation in the presence of NaCl, the center-to-center distance between these two domains increased by up to 5 nm, allowing the PGB domain to reach and bind to the peptidoglycan layer. We propose that assembly of the MotPS complex into a motor proceeds by means of Na+-induced structural transitions of its PGB domain.

DOI： 10.1126/sciadv.aao4119

Dedifferentiated liposarcoma in the maxillary gingiva: A clinical report and review of the literature

Enomoto Akifumi, Matsunaga Kazuhide, Shimoide Takeshi, Mukai Takao, Uchihashi Takayuki, Hamada Suguru

JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY MEDICINE AND PATHOLOGY 29 巻 ( 6 ) 頁： 542 - 545 2017年11月

Fast Adsorption of Soft Hydrogel Microspheres on Solid Surfaces in Aqueous Solution

Matsui, S; Kureha, T; Hiroshige, S; Shibata, M; Uchihashi, T; Suzuki, D

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 56 巻 ( 40 ) 頁： 12146 - 12149 2017年9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

The real-time adsorption behavior of polymeric colloidal microspheres onto solid surfaces in aqueous solution was visualized for the first time using high-speed atomic force microscopy (HS-AFM) to reveal how the softness of the microspheres affects their dynamic adsorption. Studies that focus on the deformability of microspheres upon dynamic adsorption have not yet been reported, most likely on account of a lack of techniques that appropriately depict the dynamic adsorption and deformation behavior of individual microspheres at the nanoscale in real time. In this study, the deformability of microspheres plays a crucial role on the adsorption kinetics, that is, soft hydrogel microspheres adsorb faster than harder elastomeric or rigid microspheres. These results should provide insight towards development of new colloidal nanomaterials that exhibit effective adsorption on specific sites in aqueous solution.

DOI： 10.1002/anie.201705808

Visualisation of a flexible modular structure of the ER folding-sensor enzyme UGGT

Satoh, T; Song, C; Zhu, T; Toshimori, T; Murata, K; Hayashi, Y; Kamikubo, H; Uchihashi, T; Kato, K

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 12142 2017年9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

In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates displayed on the protein surface serve as quality tags. The ER folding-sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a gatekeeper in the ER quality control system by specifically catalysing monoglucosylation onto incompletely folded glycoproteins, thereby enabling them to interact with lectin-chaperone complexes. Here we characterise the dynamic structure of this enzyme. Our crystallographic data demonstrate that the sensor region is composed of four thioredoxin-like domains followed by a β-rich domain, which are arranged into a C-shaped structure with a large central cavity, while the C-terminal catalytic domain undergoes a ligand-dependent conformational alteration. Furthermore, small-angle X-ray scattering, cryo-electron microscopy and high-speed atomic force microscopy have demonstrated that UGGT has a flexible modular structure in which the smaller catalytic domain is tethered to the larger folding-sensor region with variable spatial arrangements. These findings provide structural insights into the working mechanism whereby UGGT operates as a folding-sensor against a variety of glycoprotein substrates through its flexible modular structure possessing extended hydrophobic surfaces for the recognition of unfolded substrates.

DOI： 10.1038/s41598-017-12283-w

Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis

Harada, H; Onoda, A; Uchihashi, T; Watanabe, H; Sunagawa, N; Samejima, M; Igarashi, K; Hayashi, T

CHEMICAL SCIENCE 8 巻 ( 9 ) 頁： 6561 - 6565 2017年9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：Chemical Science

Cellobiose dehydrogenase (CDH) is a dual domain flavocytochrome, which consists of a dehydrogenase (DH) domain containing a flavin adenine dinucleotide and a cytochrome (CYT) domain containing b-type heme. To directly visualize the dynamic domain motion of class-I CDH from Phanerochaete chrysosporium (PcCDH) during catalysis using high-speed atomic force microscopy, the apo-form of PcCDH was anchored to a heme-immobilized flat gold surface that can specifically fix the orientation of the CYT domain. The two domains of CDH are found to be immobile in the absence of cellobiose, whereas the addition of cellobiose triggers an interdomain flip-flop motion involving domain-domain association and dissociation. Our results indicate that dynamic motion of a dual domain enzyme during catalysis induces efficient electron transfer to an external electron acceptor.

DOI： 10.1039/c7sc01672g

Scanning Probe Microscopy FOREWORD

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

JAPANESE JOURNAL OF APPLIED PHYSICS 56 巻 ( 8 ) 2017年8月

High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM

Keya, JJ; Inoue, D; Suzuki, Y; Kozai, T; Ishikuro, D; Kodera, N; Uchihashi, T; Kabir, AMR; Endo, M; Sada, K; Kakugo, A

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 6166 2017年7月

High-Speed Atomic Force Microscopy Reveals Loss of Nuclear Pore Resilience as a Dying Code in Colorectal Cancer Cells

Mohamed, MS; Kobayashi, A; Taoka, A; Watanabe-Nakayama, T; Kikuchi, Y; Hazawa, M; Minamoto, T; Fukumori, Y; Kodera, N; Uchihashi, T; Ando, T; Wong, RW

ACS NANO 11 巻 ( 6 ) 頁： 5567 - 5578 2017年6月

High-speed AFM revealed single-molecule blocking dynamics of a scorpion toxin on the KcsA potassium channel 査読有り

Ayumi Sumino, Takayuki Uchihashi, Takashi Sumikama, Shigetoshi Oiki

JOURNAL OF PHYSIOLOGICAL SCIENCES 67 巻頁： S117 2017年3月

Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging 査読有り

Sumino, A; Uchihashi, T; Oiki, S

JOURNAL OF PHYSICAL CHEMISTRY LETTERS 8 巻 ( 4 ) 頁： 785 - 793 2017年2月

Nuclear Pore Selective Barrier Dynamics as Revealed by High-Speed Atomic Force Microscopy in Colorectal Cancer Cells.

Mohamed, MS; Kobayashi, A; Taoka, A; Watanabe-Nakayama, T; Kikuchi, Y; Hazawa, M; Minamoto, T; Fukumori, Y; Kodera, N; Uchihashi, T; Ando, T; Wong, R

MOLECULAR BIOLOGY OF THE CELL 28 巻 2017年

High-speed atomic force microscopy imaging of live mammalian cells.

Shibata M, Watanabe H, Uchihashi T, Ando T, Yasuda R

Biophysics and physicobiology 14 巻 ( 0 ) 頁： 127-135 - 135 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人日本生物物理学会

<p>Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.</p>

DOI： 10.2142/biophysico.14.0_127

"Clusterase" model of dynamin-mediated membrane fission.

Takeda, T; Kozai, T; Yang, H; Kaho, S; Yusuke, K; Tadashi, A; Hiroshi, Y; Uchihashi, T; Ando, T; Takei, K

MOLECULAR BIOLOGY OF THE CELL 28 巻頁： . 2017年

"Clusterase" model of dynamin-mediated membrane fission.

Takeda T, Kozai T, Yang H, Kaho S, Yusuke K, Tadashi A, Hiroshi Y, Uchihashi T, Ando T, Takei K

MOLECULAR BIOLOGY OF THE CELL 28 巻頁： . 2017年

高速原子間力顕微鏡で可視化する生体・人工高分子の動態

内橋貴之

表面科学学術講演会要旨集 37 巻 ( 0 ) 頁： 254 2017年

記述言語：日本語出版者・発行元：公益社団法人日本表面真空学会

高速原子間力顕微鏡(HS-AFM)は1frame/s以上のフレームレートで溶液環境にある分子の様々なダイナミクスをリアルタイムで可視化でき、これまで多くのタンパク質の機能動態観察に成功している。本講演では、HS-AFMの要素技術について概説するとともに、最近の生体分子および人工超分子に応用した例について紹介する。

DOI： 10.14886/sssj2008.37.0_254

高速AFMを用いた生体分子のその場観察

内橋貴之

表面科学学術講演会要旨集 37 巻 ( 0 ) 頁： 19 2017年

A natural light-driven inward proton pump. 査読有り

Inoue K, Ito S, Kato Y, Nomura Y, Shibata M, Uchihashi T, Tsunoda SP, Kandori H

Nature communications 7 巻頁： 13415 2016年11月

Functional extension of high-speed AFM for wider biological applications. 査読有り

Uchihashi T, Watanabe H, Fukuda S, Shibata M, Ando T

Ultramicroscopy 160 巻頁： 182-96 2016年1月

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS. 査読有り

Sriwimol W, Aroonkesorn A, Sakdee S, Kanchanawarin C, Uchihashi T, Ando T, Angsuthanasombat C

The Journal of biological chemistry 290 巻 ( 34 ) 頁： 20793-803 2015年8月

Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope. 査読有り

Fukuda S, Uchihashi T, Ando T

The Review of scientific instruments 86 巻 ( 6 ) 頁： 063703 2015年6月

Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells. 査読有り

Shibata M, Uchihashi T, Ando T, Yasuda R

Scientific reports 5 巻頁： 8724 2015年3月

Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy. 査読有り

Imamura M, Uchihashi T, Ando T, Leifert A, Simon U, Malay AD, Heddle JG

Nano letters 15 巻 ( 2 ) 頁： 1331-5 2015年2月

Real-time dynamic adsorption processes of cytochrome c on an electrode observed through electrochemical high-speed atomic force microscopy. 査読有り

Takeda K, Uchihashi T, Watanabe H, Ishida T, Igarashi K, Nakamura N, Ohno H

PloS one 10 巻 ( 2 ) 頁： e0116685 2015年

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin. 査読有り

Igarashi K, Uchihashi T, Uchiyama T, Sugimoto H, Wada M, Suzuki K, Sakuda S, Ando T, Watanabe T, Samejima M

Nature communications 5 巻頁： 3975 2014年6月

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

289 巻 ( 20 ) 頁： 14056-65 2014年5月

[Direct observation of rotary catalysis of rotorless F1-ATPase with high-speed AFM].

Uchihashi T, Iino R, Ando T, Noji H

Seikagaku. The Journal of Japanese Biochemical Society 86 巻 ( 2 ) 頁： 127-36 2014年4月

Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.

Nakamura A, Watanabe H, Ishida T, Uchihashi T, Wada M, Ando T, Igarashi K, Samejima M

Journal of the American Chemical Society 136 巻 ( 12 ) 頁： 4584-92 2014年3月

Filming biomolecular processes by high-speed atomic force microscopy.

Ando T, Uchihashi T, Scheuring S

Chemical reviews 114 巻 ( 6 ) 頁： 3120-88 2014年3月

Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy.

Yamashita H, Inoue K, Shibata M, Uchihashi T, Sasaki J, Kandori H, Ando T

Journal of structural biology 184 巻 ( 1 ) 頁： 2-11 2013年10月

Real-time visualization of assembling of a sphingomyelin-specific toxin on planar lipid membranes.

Yilmaz N, Yamada T, Greimel P, Uchihashi T, Ando T, Kobayashi T

Biophysical journal 105 巻 ( 6 ) 頁： 1397-405 2013年9月

High-speed atomic force microscope combined with single-molecule fluorescence microscope.

Fukuda S, Uchihashi T, Iino R, Okazaki Y, Yoshida M, Igarashi K, Ando T

The Review of scientific instruments 84 巻 ( 7 ) 頁： 073706 2013年7月

Wide-area scanner for high-speed atomic force microscopy.

Watanabe H, Uchihashi T, Kobashi T, Shibata M, Nishiyama J, Yasuda R, Ando T

The Review of scientific instruments 84 巻 ( 5 ) 頁： 053702 2013年5月

High-speed AFM and applications to biomolecular systems.

Ando T, Uchihashi T, Kodera N

Annual review of biophysics 42 巻頁： 393-414 2013年

Single-molecule imaging on living bacterial cell surface by high-speed AFM.

Yamashita H, Taoka A, Uchihashi T, Asano T, Ando T, Fukumori Y

Journal of molecular biology 422 巻 ( 2 ) 頁： 300-9 2012年9月

Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy.

Uchihashi T, Kodera N, Ando T

Nature protocols 7 巻 ( 6 ) 頁： 1193-206 2012年5月

Direct Visualization of Cellobiohydrolase on Crystalline Cellulose using High-Speed Atomic Force Microscopy 査読有り

Uchihashi Takayuki, Igarashi Kiyohiko, Koivula Anu, Wada Masahisa, Penttil Merja, Samejima Masahiro, Ando Toshio

BIOPHYSICAL JOURNAL 102 巻 ( 3 ) 頁： 585A-586A 2012年1月

Visualization of cellobiohydrolase I from Trichoderma reesei moving on crystalline cellulose using high-speed atomic force microscopy.

Methods in enzymology 510 巻頁： 169-82 2012年

Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.

Science (New York, N.Y.) 333 巻 ( 6047 ) 頁： 1279-82 2011年9月

High-speed atomic force microscopy reveals rotary catalysis of rotorless F₁-ATPase.

333 巻 ( 6043 ) 頁： 755-8 2011年8月

Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy.

Shibata M, Uchihashi T, Yamashita H, Kandori H, Ando T

Angewandte Chemie (International ed. in English) 50 巻 ( 19 ) 頁： 4410-3 2011年5月

High-speed atomic force microscopy and biomolecular processes.

Uchihashi T, Ando T

Methods in molecular biology (Clifton, N.J.) 736 巻頁： 285-300 2011年

High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin 査読有り

Shibata Mikihiro, Yamashita Hayato, Uchihashi Takayuki, Kandori Hideki, Ando Toshio

NATURE NANOTECHNOLOGY 5 巻 ( 3 ) 頁： 208-212 2010年3月

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

内橋　貴之

JOURNAL OF STRUCTURAL BIOLOGY 167 巻 ( 153-158 ) 頁： . 2009年

High resonance frequency force microscope scanner using inertia balance support 査読有り

Fukuma Takeshi, Okazaki Yasutaka, Kodera Noriyuki, Uchihashi Takayuki, Ando Toshio

APPLIED PHYSICS LETTERS 92 巻 ( 24 ) 2008年6月

Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy

MIYAGI Atsushi, TSUNAKA Yasuo, TSUNAKA Yasuo, UCHIHASHI Takayuki, UCHIHASHI Takayuki, MAYANAGI Kouta, MAYANAGI Kouta, MAYANAGI Kouta, HIROSE Susumu, MORIKAWA Kosuke, MORIKAWA Kosuke, ANDO Toshio, ANDO Toshio

CHEMPHYSCHEM 9 巻 ( 13 ) 頁： 1859-1866 2008年

High-speed AFM and nano-visualization of biomolecular processes

内橋　貴之

Pflugers Archiv -Eur. J. Physiol. 456 巻頁： . 2008年

High resonance frequency force microscope scanner using inertia balance support

内橋　貴之

APPLIED PHYSICS LETTERS 92 巻 ( 243119 ) 頁： . 2008年

High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes

内橋　貴之

PROGRESS IN SURFACE SCIENCE 83 巻 ( 337-437 ) 頁： . 2008年

High-speed atomic force microscopy for observing dynamic biomolecular processes.

Ando T, Uchihashi T, Kodera N, Yamamoto D, Taniguchi M, Miyagi A, Yamashita H

Journal of molecular recognition : JMR 20 巻 ( 6 ) 頁： 448-58 2007年11月

Tip-sample distance control using photothermal actuation of a small cantilever for high-speed atomic force microscopy.

Yamashita H, Kodera N, Miyagi A, Uchihashi T, Yamamoto D, Ando T

The Review of scientific instruments 78 巻 ( 8 ) 頁： 083702 2007年8月

High-speed atomic force microscopy for observing dynamic biomolecular processes

内橋　貴之

JOURNAL OF MOLECULAR RECOGNITION 20 巻 ( 448-458 ) 頁： . 2007年

Capacitance XAFS method: A new site-selective and microscopic X-ray absorption Spectroscopy 査読有り

Ishii Masashi, Nakao Aiko, Uchihashi Takayuki

PHYSICA SCRIPTA T115 巻頁： 97-101 2005年

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