研究者詳細 - 内橋　貴之

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

Shibata, M; Nishimasu, H; Kodera, N; Hirano, S; Ando, T; Uchihashi, T; Nureki, O

NATURE COMMUNICATIONS 8 巻 ( 1 ) 頁： 1430 2017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：Nature Communications

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

Terahara, N; Kodera, N; Uchihashi, T; Ando, T; Namba, K; Minamino, T

SCIENCE ADVANCES 3 巻 ( 11 ) 頁： eaao4119 2017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：Science Advances

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 29 巻 ( 6 ) 頁： 542 - 545 2017年11月

Matsui, S; Kureha, T; Hiroshige, S; Shibata, M; Uchihashi, T; Suzuki, D

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 56 巻 ( 40 ) 頁： 12146 - 12149 2017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：Angewandte Chemie - International Edition

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

Satoh, T; Song, C; Zhu, T; Toshimori, T; Murata, K; Hayashi, Y; Kamikubo, H; Uchihashi, T; Kato, K

SCIENTIFIC REPORTS 7 巻 ( 1 ) 頁： 12142 2017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：Scientific Reports

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 β-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

Harada, H; Onoda, A; Uchihashi, T; Watanabe, H; Sunagawa, N; Samejima, M; Igarashi, K; Hayashi, T

CHEMICAL SCIENCE 8 巻 ( 9 ) 頁： 6561 - 6565 2017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：Chemical Science

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