研究者詳細 - 内橋　貴之

論文 - 内橋　貴之

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Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of C<sub>H</sub>1 Domain Deletion 査読有り国際誌

Yanaka, S; Nishiguchi, S; Yogo, R; Watanabe, H; Shen, JA; Yagi, H; Uchihashi, T; Kato, K

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 23 巻 ( 4 ) 2022年2月

担当区分：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：International Journal of Molecular Sciences

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH 1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH 1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

DOI： 10.3390/ijms23042090

Shape-selective one-step synthesis of branched gold nanoparticles on the crystal surface of redox-active Pd<SUP>II</SUP>-macrocycles 査読有り

Yamashita, Y; Tashiro, S; Ishii, Y; Uchihashi, T; Matsushita, N; Kubota, R; Shionoya, M

DALTON TRANSACTIONS 51 巻 ( 4 ) 頁： 1318 - 1324 2022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：Dalton Transactions

The synthesis of branched gold nanoparticles (AuNPs) with shape- and size-specific optical properties requires effective control of the particle formation mechanism using appropriate reducing agents and protective agents that prevent particle aggregation in solution. In this context, the heterogeneous synthesis of AuNPs using solid surfaces of graphene oxides and metal–organic frameworks has attracted much attention. These materials are characterized by their ability to immobilize and stabilize the particles grown on the surface without the need for additional protective agents. However, the shape- and size-selective synthesis of AuNPs using solid surfaces remains challenging. Herein, we report the shape-selective one-step synthesis of monodisperse branched AuNPs using a metal–macrocycle framework (MMF), a porous molecular crystal of PdII3-tris(phenylenediamine) macrocycle. Konpeito-Shaped branched AuNPs with uniform size were obtained on the surface of MMF by mixing HAuCl4·4H2O, l-ascorbic acid and MMF microcrystals. Spectroscopic and microscopic observations confirmed that MMF promoted the reduction of gold by its reductive activity as well as acted as a solid support to electrostatically immobilize the pseudo-seed particles for further growth on the crystal surface. In addition, the MMF also served as a substrate for in situ high-speed AFM imaging due to the effective immobilization of AuNPs on the surface, allowing direct visualization of the particle growth. Since the chemical structural features of MMF allow the growth of branched AuNPs via pseudo-seeding, this approach would provide new synthetic methods for obtaining a variety of gold nanostructures.

DOI： 10.1039/d1dt03973c

The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes 査読有り国際誌

Hamasaki E., Wakita N., Yasuoka H., Nagaoka H., Morita M., Takashima E., Uchihashi T., Takeda T., Abe T., Lee J.W., Iimura T., Saleem M.A., Ogo N., Asai A., Narita A., Takei K., Yamada H.

Frontiers in Cell and Developmental Biology 10 巻頁： 884509 - 884509 2022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：Frontiers in Cell and Developmental Biology

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

DOI： 10.3389/fcell.2022.884509

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion 査読有り

Saeko Yanaka, Shigetaka Nishiguchi, Rina Yogo, Hiroki Watanabe, Jiana Shen, Hirokazu Yagi, Takayuki Uchihashi, and Koichi Kato

International Journal of Molecular Sciences 23 巻 ( 4 ) 頁： 2090 2021年12月

担当区分：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：MDPI AG

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

DOI： 10.3390/ijms23042090.

Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin 査読有り

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

JOURNAL OF PHYSICAL CHEMISTRY LETTERS 12 巻 ( 51 ) 頁： 12225 - 12229 2021年12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 Physical Chemistry Letters

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax ∼ 490 nm) and green-absorbing PR (GPR; λmax ∼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.

DOI： 10.1021/acs.jpclett.1c03355

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade 査読有り国際誌

Yagi-Utsumi, M; Aoki, K; Watanabe, H; Song, CH; Nishimura, S; Satoh, T; Yanaka, S; Ganser, C; Tanaka, S; Schnapka, V; Goh, EW; Furutani, Y; Murata, K; Uchihashi, T; Arakawa, K; Kato, K

SCIENTIFIC REPORTS 11 巻 ( 1 ) 頁： 21328 - 21328 2021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：Scientific Reports

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades’ response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol–gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

DOI： 10.1038/s41598-021-00724-6

Construction of ferritin hydrogels utilizing subunit-subunit interactions 査読有り

Yamanaka, M; Mashima, T; Ogihara, M; Okamoto, M; Uchihashi, T; Hirota, S

PLOS ONE 16 巻 ( 11 ) 頁： e0259052 - e0259052 2021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：PLoS ONE

Various proteins form nanostructures exhibiting unique functions, making them attractive as next-generation materials. Ferritin is a hollow spherical protein that incorporates iron ions. Here, we found that hydrogels are simply formed from concentrated apoferritin solutions by acid denaturation and subsequent neutralization. The water content of the hydrogel was approximately 80%. The apoferritin hydrogel did not decompose in the presence of 1 M HCl, 2-mercaptoethanol, or methanol but was dissolved in the presence of 1 M NaOH, by heating at 80C, or by treatment with trypsin or 6 M guanidine hydrochloride. The Young’s modulus of the hydrogel was 20.4 ± 12.1 kPa according to local indentation experimentes using atomic force microscopy, indicating that the hydrogel was relatively stiff. Transition electron microscopy measurements revealed that a fibrous network was constructed in the hydrogel. The color of the hydrogel became yellow-brown upon incubation in the presence of Fe3+ ions, indicating that the hydrogel adsorbed the Fe3+ ions. The yellow-brown color of the Fe3 +-adsorbed hydrogel did not change upon incubation in pure water, whereas it became pale by incubating it in the presence of 100 mM ethylenediaminetetraacetic acid (EDTA). The apoferritin hydrogel also adsorbed Co2+ and Cu2+ ions and released them in the presence of EDTA, while it adsorbed less Ni2+ ions; more Fe3+ ions adsorbed to the apoferritin hydrogel than other metal ions, indicating that the hydrogel keeps the iron storage characteristic of ferritin. These results demonstrate a new property of ferritin: the ability to form a hydrogel that can adsorb/desorb metal ions, which may be useful in designing future biomaterials.

DOI： 10.1371/journal.pone.0259052

Deformation of microtubules regulates translocation dynamics of kinesin 査読有り国際共著国際誌

Nasrin, SR; Ganser, C; Nishikawa, S; Kabir, AMR; Sada, K; Yamashita, T; Ikeguchi, M; Uchihashi, T; Hess, H; Kakugo, A

SCIENCE ADVANCES 7 巻 ( 42 ) 頁： eabf2211 2021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：Science Advances

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

DOI： 10.1126/sciadv.abf2211

Scanning Probe Microscopy FOREWORD 招待有り

Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

JAPANESE JOURNAL OF APPLIED PHYSICS 60 巻 ( SE ) 2021年9月

高速原子間力顕微鏡による一分子動態計測：最近の応用研究招待有り

内橋貴之

生化学 93 巻頁： 526 - 561 2021年8月

Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible β-Barrel Structure in Solution and Keeps This Structure in Dehydration 査読有り国際誌

Miyazawa, K; Itoh, SG; Watanabe, H; Uchihashi, T; Yanaka, S; Yagi-Utsumi, M; Kato, K; Arakawa, K; Okumura, H

JOURNAL OF PHYSICAL CHEMISTRY B 125 巻 ( 32 ) 頁： 9145 - 9154 2021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：Journal of Physical Chemistry B

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

DOI： 10.1021/acs.jpcb.1c04850

Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images 査読有り国際誌

Dasgupta, B; Miyashita, O; Uchihashi, T; Tama, F

FRONTIERS IN MOLECULAR BIOSCIENCES 8 巻頁： 704274 - 704274 2021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：Frontiers in Molecular Biosciences

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

DOI： 10.3389/fmolb.2021.704274

JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes 査読有り国際誌

Sakane, A; Yano, T; Uchihashi, T; Horikawa, K; Hara, Y; Imoto, I; Kurisu, S; Yamada, H; Takei, K; Sasaki, T

COMMUNICATIONS BIOLOGY 4 巻 ( 1 ) 頁： 551 - 551 2021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：Communications Biology

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

DOI： 10.1038/s42003-021-02080-7

Non-close-packed arrangement of soft elastomer microspheres on solid substrates 査読有り

Sasaki, Y; Hiroshige, S; Takizawa, M; Nishizawa, Y; Uchihashi, T; Minato, H; Suzuki, D

RSC ADVANCES 11 巻 ( 24 ) 頁： 14562 - 14567 2021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：RSC Advances

Unlike rigid microparticles, soft and deformable elastomer (rubber) microspheres were found to exhibit a non-close-packed arrangement on solid substrates after the evaporation of water from their dispersions. The microscopic observation revealed that individual microspheres are ordered in regular intervals at the air/water interface of a sessile droplet and remain fixed on the substrate without being affected by the capillary forces during evaporation due to their deformability. Moreover, using the Langmuir-Blodgett method, thin films of non-close-packed structures could be successfully generated over large areas. Our findings may potentially help to control the arranged structures of elastomer microspheres, which can be expected to improve the nano-science and technology for the precise control for e.g. surface patterning. This journal is

DOI： 10.1039/d1ra02688g

Nanostructure and thermoresponsiveness of poly(<i>N</i>-isopropyl methacrylamide)-based hydrogel microspheres prepared <i>via</i> aqueous free radical precipitation polymerization 査読有り

Nishizawa, Y; Minato, H; Inui, T; Saito, I; Kureha, T; Shibayama, M; Uchihashi, T; Suzuki, D

RSC ADVANCES 11 巻 ( 22 ) 頁： 13130 - 13137 2021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：RSC Advances

Thermoresponsive hydrogel microspheres (microgels) are smart materials that quickly respond to external stimuli, and their thermoresponsiveness can be tuned by varying the constituent chemical species. Although uniformly sized microgels can be preparedviaaqueous free radical precipitation polymerization, the nanostructure of the obtained microgels is complex and remains unclear so far. In the present study, the nanostructure and thermoresponsiveness of poly(N-isopropyl methacrylamide) (pNIPMAm)-based microgels, which have a volume-transition temperature of ∼43 °C, were evaluated mainly using temperature-controllable high-speed atomic force microscopy. These observations, which are characterized by high spatio-temporal resolution, revealed that the pNIPMAm microgels have a peculiar heterogeneous structure, for example a core-shell and non-thermoresponsive nanostructure in the core region, that originates from the precipitation polymerization process. Furthermore, it was found that the adsorption concentration of the microgels on the substrate is one of the keys for controlling their thermoresponsiveness. These findings can be expected to advance the design of new materials such as thermoresponsive nanosheets and stimuli-responsive coatings.

DOI： 10.1039/d1ra01650d

Dynamic Assembly/Disassembly of <i>Staphylococcus aureus</i> FtsZ Visualized by High-Speed Atomic Force Microscopy 査読有り国際誌

Fujita, J; Sugiyama, S; Terakado, H; Miyazaki, M; Ozawa, M; Ueda, N; Kuroda, N; Tanaka, S; Yoshizawa, T; Uchihashi, T; Matsumura, H

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22 巻 ( 4 ) 頁： 1 - 10 2021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：International Journal of Molecular Sciences

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architec-tures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.

DOI： 10.3390/ijms22041697

Nanostructures, Thermoresponsiveness, and Assembly Mechanism of Hydrogel Microspheres during Aqueous Free-Radical Precipitation Polymerization 査読有り国際誌

Nishizawa, Y; Minato, H; Inui, T; Uchihashi, T; Suzuki, D

LANGMUIR 37 巻 ( 1 ) 頁： 151 - 159 2021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：Langmuir

Although techniques to produce uniformly sized hydrogel microspheres (microgels) by aqueous free-radical precipitation polymerization are well established, the details of the polymerization process remain mysterious. In the present study, the structural evolution and thermoresponsiveness of the developing microgels during the polymerization were evaluated by temperature-controlled high-speed atomic force microscopy. This analysis clarified that the swelling properties of the precursor microgels formed in the early stages of the polymerization are quite low due to the high incorporation of cross-linkers and that non-thermoresponsive deca-nanosized spherical domains are already present in the precursor microgels. Furthermore, we succeeded in tracking the formation of nuclei and their growth process, which has never been fully understood, in aqueous solution by real-time observations. These findings will help us to design functional microgels with the desired nanostructures via precipitation polymerization.

DOI： 10.1021/acs.langmuir.0c02654

Single-molecule dynamics observed by high-speed atomic force microscopy: Recent applications

Uchihashi T.

Seikagaku 93 巻 ( 4 ) 頁： 526 - 531 2021年

Structural insights into the mechanism of rhodopsin phosphodiesterase 査読有り国際誌

Ikuta, T; Shihoya, W; Sugiura, M; Yoshida, K; Watari, M; Tokano, T; Yamashita, K; Katayama, K; Tsunoda, SP; Uchihashi, T; Kandori, H; Nureki, O

NATURE COMMUNICATIONS 11 巻 ( 1 ) 頁： 5605 - 5605 2020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

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

DOI： 10.1038/s41467-020-19376-7

Novel <i>Babesia bovis</i> exported proteins that modify properties of infected red blood cells 査読有り国際誌

Hakimi, H; Templeton, TJ; Sakaguchi, M; Yamagishi, J; Miyazaki, S; Yahata, K; Uchihashi, T; Kawazu, S; Kaneko, O; Asada, M

PLOS PATHOGENS 16 巻 ( 10 ) 頁： e1008917 2020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：PLoS Pathogens

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV-III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

DOI： 10.1371/journal.ppat.1008917

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