研究者詳細 - 内橋　貴之

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 Salmonella 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

In Situ 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

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。

DOI： 10.3412/jsb.79.1

CiNii Research

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

Open Access

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

<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

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19F 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