研究者詳細 - 内橋　貴之

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

Open Access

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

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

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

Open Access

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

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

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