Faculty Profiles - UCHIHASHI Takayuki

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

Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, Osamu Nureki

NATURE COMMUNICATIONS Vol. 8 ( 1 ) page： 1430 2017.11

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

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+-induced structural transition of MotPS for stator assembly of the Bacillus flagellar motor

Naoya Terahara, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Keiichi Namba, Tohru Minamino

SCIENCE ADVANCES Vol. 3 ( 11 ) page： eaao4119 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC ADVANCEMENT SCIENCE

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

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

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 Vol. 29 ( 6 ) page： 542 - 545 2017.11

Shusuke Matsui, Takuma Kureha, Seina Hiroshige, Mikihiro Shibata, Takayuki Uchihashi, Daisuke Suzuki

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Vol. 56 ( 40 ) page： 12146 - 12149 2017.9

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

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

Tadashi Satoh, Chihong Song, Tong Zhu, Takayasu Toshimori, Kazuyoshi Murata, Yugo Hayashi, Hironari Kamikubo, Takayuki Uchihashi, Koichi Kato

SCIENTIFIC REPORTS Vol. 7 ( 1 ) page： 12142 2017.9

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

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 beta-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

Hirofumi Harada, Akira Onoda, Takayuki Uchihashi, Hiroki Watanabe, Naoki Sunagawa, Masahiro Samejima, Kiyohiko Igarashi, Takashi Hayashi

CHEMICAL SCIENCE Vol. 8 ( 9 ) page： 6561 - 6565 2017.9

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

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 Vol. 56 ( 8 ) 2017.8

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

Jakia Jannat Keya, Daisuke Inoue, Yuki Suzuki, Toshiya Kozai, Daiki Ishikuro, Noriyuki Kodera, Takayuki Uchihashi, Arif Md. Rashedul Kabir, Masayuki Endo, Kazuki Sada, Akira Kakugo

SCIENTIFIC REPORTS Vol. 7 ( 1 ) page： 6166 2017.7

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

In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.

DOI： 10.1038/s41598-017-06249-1

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

Mahmoud Shaaban Mohamed, Akiko Kobayashi, Azuma Taoka, Takahiro Watanabe-Nakayama, Yosuke Kikuchi, Masaharu Hazawa, Toshinari Minamoto, Yoshihiro Fukumori, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Richard W. Wong

ACS NANO Vol. 11 ( 6 ) page： 5567 - 5578 2017.6

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

Nuclear pore complexes (NPCs) are the sole turnstile implanted in the nuclear envelope (NE), acting as a central nanoregulator of transport between the cytosol and the nucleus. NPCs consist of similar to 30 proteins, termed nucleoporins. About one-third of nucleoporins harbor natively unstructured, intrinsically disordered phenylalanine-glycine strings (FG-Nups), which engage in transport selectivity. Because the barriers insert deeply in the NPC, they are nearly inaccessible. Several in vitro barrier models have been proposed; however, the dynamic FG-Nups protein molecules themselves are imperceptible in vivo. We show here that high-speed atomic force microscopy (HS-AFM) can be used to directly visualize nanotopographical changes of the nuclear pore inner channel in colorectal cancer (CRC) cells. Furthermore, using MLN8237/alisertib, an apoptotic and autophagic inducer currently being tested in relapsed cancer clinical trials, we unveiled the functional loss of nucleoporins, particularly the deformation of the FG-Nups barrier, in dying cancer cells. We propose that the loss of this nanoscopic resilience is an irreversible dying code in cells. These findings not only illuminate the potential application of HS-AFM as an intracellular nanoendoscopy but also might aid in the design of future nuclear targeted nanodrug delivery tailored to the individual patient.

DOI： 10.1021/acsnano.7b00906

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

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

Ayumi Sumino, Takayuki Uchihashi, Takashi Sumikama, Shigetoshi Oiki

JOURNAL OF PHYSIOLOGICAL SCIENCES Vol. 67 page： S117 2017.3

Ayumi Sumino, Takayuki Uchihashi, Shigetoshi Oiki

JOURNAL OF PHYSICAL CHEMISTRY LETTERS Vol. 8 ( 4 ) page： 785 - 793 2017.2

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

Here, we have developed a method of oriented reconstitution of the KcsA potassium channel amenable to high-resolution AFM imaging. The solubilized liposome full-length KcsA channels with histidine-tagged (His-tag) C-terminal ends were attached to a Ni2+-coated mica surface, and then detergent-destabilized liposomes were added to fill the interchannel space. AFM revealed that the membrane embedded KcsA channels were oriented with their extracellular faces upward, seen as a tetrameric square shape. This orientation was corroborated,,by the visible binding of a peptide scorpion toxin, agitoxin-2. To observe the cytoplasmic side of the channel, a His-tag was inserted into the extracellular loop, and the oppositely oriented channels provided wholly different images. In either orientation, the channels were individually dispersed at acidic pH, whereas they were self-assembled at neutral pH, indicating that the oriented channels are allowed to diffuse in the membrane. This method is readily applicable to membrane proteins' in general for AFM imaging.

DOI： 10.1021/acs.jpclett.6b03058