Faculty Profiles - UCHIHASHI Takayuki

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

Nishiguchi, S; Kasai, RS; Uchihashi, T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 120 ( 18 ) page： e2302047120 2023.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Toshinobu Nakajo, Shinpei Kusaka, Haruka Hiraoka, Kohei Nomura, Noriaki Matsubara, Rintaro Baba, Yuki Yoshida, Kosuke Nakamoto, Masakazu Honma, Hiroaki Iguchi, Takayuki Uchihashi, Hiroshi Abe, Ryotaro Matsuda

Chemical communications (Cambridge, England) Vol. 59 ( 33 ) page： 4974 - 4977 2023.4

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

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

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

Dimerization Processes of Cell-cell Adhesion Molecule Cadherin Revealed by High-speed Atomic Force Microscopy Invited Reviewed

Shigetaka Nishiguchi, Tadaomi Furuta, Takayuki Uchihashi

Seibutu Butsuri Vol. 63 ( 2 ) page： 104 - 106 2023.4

Mst. Rubaya Rashid, Christian Ganser, Mousumi Akter, Syeda Rubaiya Nasrin, Arif Md. Rashedul Kabir, Kazuki Sada, Takayuki Uchihashi, Akira Kakugo

Chemistry Letters Vol. 52 ( 2 ) page： 100 - 104 2023.2

Language：English Publishing type：Research paper (scientific journal) Publisher：The Chemical Society of Japan

<p>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 nanometer-level 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.</p>

DOI： 10.1246/cl.220491

Disulfide Bond-mediated Oligomerization of a Green Fluorescent Protein in Solution Reviewed

CiNii Research

Julian Wong Soon, Koji Oohora, Takayuki Uchihashi, Takashi Hayashi

Chemistry Letters Vol. 52 ( 2 ) page： 105 - 109 2023.2

Language：English Publishing type：Research paper (scientific journal) Publisher：The Chemical Society of Japan

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 Reviewed

CiNii Research

Yue Y., Yokota Y., Uchihashi T.

iScience Vol. 26 ( 1 ) page： 105864 2023.1

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

Yoshitaka Matsuo, Takayuki Uchihashi, Toshifumi Inada

Nature communications Vol. 14 ( 1 ) page： 79 - 79 2023.1

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

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

Activity of Regional Branch

Introduction of Session 2, “Advanced methods for retinal proteins” Invited

Uchihashi Takayuki, Kandori Hideki

Biophysics and Physicobiology Vol. 20 ( Supplemental ) page： n/a 2023

Seibutsu Butsuri Vol. 63 ( 5 ) page： 285 - 286 2023

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

Kanji Takahashi, Tatsuro Nishikino, Hiroki Kajino, Seiji Kojima, Takayuki Uchihashi, Michio Homma

BIOPHYSICS AND PHYSICOBIOLOGY Vol. 20 ( 2 ) page： n/a 2023

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

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 twotransmembrane 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 Reviewed

CiNii Research

Yuichiro Nishizawa, Takumi Inui, Ryuji Namioka, Takayuki Uchihashi, Takumi Watanabe, Daisuke Suzuki

Langmuir Vol. 38 ( 51 ) page： 16084 - 16093 2022.12

Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface Reviewed International coauthorship

Taku Uchiyama, Takayuki Uchihashi, Takuya Ishida, Akihiko Nakamura, Josh V. Vermaas, Michael F. Crowley, Masahiro Samejima, Gregg T. Beckham, Kiyohiko Igarashi

Science Advances Vol. 8 ( 51 ) page： eade5155 2022.12

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

Feng-Yueh Chan, Ryo Kurosaki, Christian Ganser, Tetsuya Takeda, Takayuki Uchihashi

Review of Scientific Instruments Vol. 93 ( 11 ) page： 113703 - 113703 2022.11

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

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

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

Scanning Probe Microscopy Invited

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

Japanese Journal of Applied Physics Vol. 61 ( SL ) 2022.9

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

BIOCHEMISTRY Vol. 61 ( 18 ) page： 1936 - 1944 2022.8

Language：English Publisher：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. Reviewed International journal

Shigetaka Nishiguchi, Tadaomi Furuta, Takayuki Uchihashi

Proceedings of the National Academy of Sciences of the United States of America Vol. 119 ( 30 ) page： e2208067119 2022.7

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

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