Faculty Profiles - UCHIHASHI Takayuki

Application of high-speed atomic force microscopy in visualizing the dynamics of synthetic polymers Invited Reviewed

Uchihashi, T; Nishizawa, Y

JAPANESE JOURNAL OF APPLIED PHYSICS Vol. 63 ( 4 ) 2024.4

Authorship：Corresponding author Language：English Publishing type：Research paper (international conference proceedings) Publisher：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

Compressed sensing reconstruction of cell mechanical images obtained from atomic force microscopy Reviewed

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

JAPANESE JOURNAL OF APPLIED PHYSICS Vol. 63 ( 4 ) 2024.4

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

FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella. International journal

Miki Kinoshita, Tohru Minamino, Takayuki Uchihashi, Keiichi Namba

Communications biology Vol. 7 ( 1 ) page： 366 - 366 2024.3

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

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

In Situ Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope Reviewed

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

NANO LETTERS Vol. 24 ( 9 ) page： 2805 - 2811 2024.2

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

Tanaka, Y; Uchihashi, T; Nakamura, A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 752 page： 109854 2024.2

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

Yanaka Saeko, Watanabe Hiroki, Yogo Rina, Kongsema Mesayamas, Kondo Sachiko, Yagi Hirokazu, Uchihashi Takayuki, Kato Koichi

Biological and Pharmaceutical Bulletin Vol. 47 ( 1 ) page： 334 - 338 2024.1

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：The Pharmaceutical Society of Japan

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

DOI： 10.1248/bpb.b23-00751

CiNii Research

Protein degradation in bacteria: focus on the ClpP protease Reviewed

ISHIKAWA Fumihiro, HOMMA Michio, TANABE Genzoh, UCHIHASHI Takayuki

Nippon Saikingaku Zasshi Vol. 79 ( 1 ) page： 1 - 13 2024

Authorship：Corresponding author Language：Japanese Publisher：JAPANESE SOCIETY FOR BACTERIOLOGY

Proteins in the cells are born (synthesized), work, and die (decomposed). In the life of a protein, its birth is obviously important, but how it dies is equally important in living organisms. Proteases secreted into the outside of cells are used to decompose the external proteins and the degradation products are taken as the nutrients. On the other hand, there are also proteases that decompose unnecessary or harmful proteins which are generated in the cells. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for degradation of such proteins. Bacteria, which are prokaryotes, have a similar system as the proteasome. We would like to explain the bacterial degradation system of proteins or the death of proteins, which is performed by ATP-dependent protease Clp, with a particular focus on the ClpXP complex, and with an aspect as a target for antibiotics against bacteria.

DOI： 10.3412/jsb.79.1

CiNii Research

Phase separation-induced glass transition under critical miscible conditions Reviewed

Mayu Watanabe, Dong Shi, Ryuji Kiyama, Kagari Maruyama, Yuichiro Nishizawa, Takayuki Uchihashi, Jian Ping Gong, Takayuki Nonoyama

Materials Advances 2024

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

A polymer material shows phase separation-induced glass transition under critical miscible conditions, where the miscible/immiscible state is altered by temperature variations predicted by temperature-corrected Hansen solubility parameters.

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

Publisher：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

Open Access

The two‐step cargo recognition mechanism of heterotrimeric kinesin Reviewed

Xuguang Jiang, Tadayuki Ogawa, Kento Yonezawa, Nobutaka Shimizu, Sotaro Ichinose, Takayuki Uchihashi, Wataru Nagaike, Toshio Moriya, Naruhiko Adachi, Masato Kawasaki, Naoshi Dohmae, Toshiya Senda, Nobutaka Hirokawa

EMBO reports Vol. 24 ( 11 ) page： e56864 2023.11

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

Abstract

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

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19F MR Imaging Reviewed

Measuring mechanical properties with high-speed atomic force microscopy Invited Reviewed

Ganser, C; Uchihashi, T

MICROSCOPY Vol. 73 ( 1 ) page： 14 - 21 2023.10

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 Vol. 25 ( 4 ) page： 271 - 278 2023.10

Language：Japanese Publisher：分子シミュレーション学会

<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>

DOI： 10.11436/mssj.25.271

CiNii Research

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

Angewandte Chemie - International Edition Vol. 62 ( 40 ) page： e202308565 2023.10

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

Kentaro Mannen, Takashi Nagata, Andrey Rozenberg, Masae Konno, María del Carmen Marín, Reza Bagherzadeh, Oded Béjà, Takayuki Uchihashi, Keiichi Inoue

Journal of Molecular Biology page： 168331 - 168331 2023.10

Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication. Reviewed International coauthorship International journal

Carol Cho, Christian Ganser, Takayuki Uchihashi, Koichi Kato, Ji-Joon Song

Communications biology Vol. 6 ( 1 ) page： 993 - 993 2023.9

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

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. Reviewed International coauthorship International journal

Eric H-L Chen, Chun-Hsiung Wang, Yi-Ting Liao, Feng-Yueh Chan, Yui Kanaoka, Takayuki Uchihashi, Koichi Kato, Longsheng Lai, Yi-Wei Chang, Meng-Chiao Ho, Rita P-Y Chen

Nature communications Vol. 14 ( 1 ) page： 5464 - 5464 2023.9

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

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 Reviewed

Maho Yagi-Utsumi, Haruko Miura, Christian Ganser, Hiroki Watanabe, Methanee Hiranyakorn, Tadashi Satoh, Takayuki Uchihashi, Koichi Kato, Kei-ichi Okazaki, Kazuhiro Aoki

International Journal of Molecular Sciences Vol. 24 ( 16 ) page： 12865 - 12865 2023.8

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

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 Reviewed

Yuichiro Nishizawa, Hiroki Yokoi, Takayuki Uchihashi, Daisuke Suzuki

Soft Matter Vol. 19 ( 27 ) page： 5068 - 5075 2023.7

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

The real-time visualization via high-speed atomic force microscopy revealed that single microgel exhibit heterogeneous degradation behavior.

DOI： 10.1039/d3sm00216k