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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/jacs.4c07878

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Authorship：Corresponding author   Publisher：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

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

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

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DOI： 10.1101/2024.05.10.593077

Language：English   Publishing type：Research paper (conference, symposium, etc.)   Publisher：Journal of Molecular Recognition  

DOI： 10.1002/jmr.3077

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

IgG molecules that bind antigen on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centered on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

DOI： 10.1093/intimm/dxae017

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Japanese Journal of Applied Physics  

High-speed atomic force microscopy (HS-AFM) is a technique that enables real-time imaging of nanoscale phenomena in solution. It was originally developed to visualize biomolecules, whose dynamics in solution significantly affect the manifestation of their functions, and has contributed to the understanding of molecular mechanisms based on the observation of single-molecule dynamics of proteins. In recent years, its application has broadened to include not only biomolecules, but also the structural dynamics of supramolecular assemblies that associate and dissociate in solution, as well as the evaluation of synthetic molecules such as polymer gels that swell in solution. In this paper, we review some of our recent studies on the application of HS-AFM to supramolecular polymers and hydrogel particles.

DOI： 10.35848/1347-4065/ad33f2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Journal of Applied Physics  

Compressed sensing (CS), a technique in signal processing that reconstructs sparse signals from a limited sampling number, has been valuable in topographic images obtained from atomic force microscopy (AFM). However, how CS is effective in reconstructing AFM mechanical images remains unclear. We investigated the reconstruction of topographic and mechanical images of living cells, such as developing embryos obtained from AFM mapping experiments using CS. The results showed that both topographic and mechanical images of embryonic cells in the different developmental stages were well reconstructed at a spatial resolution higher than the original AFM images. These results suggested that the CS approach enabled the cell mechanical properties, together with cell surface morphology, using AFM mapping measurements to be faster than the conventional AFM methods without reducing the spatial resolution.

DOI： 10.35848/1347-4065/ad34de

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The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368-372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.

DOI： 10.1038/s42003-024-06081-0

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

DOI： 10.1021/acs.nanolett.3c04877

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Archives of Biochemistry and Biophysics  

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 μM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 μM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 μM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

DOI： 10.1016/j.abb.2023.109854

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Pharmaceutical Society of Japan  

<p>This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding <i>via</i> the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked <i>N</i>-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.</p>

DOI： 10.1248/bpb.b23-00751

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Authorship：Corresponding author   Language：Japanese   Publisher：JAPANESE SOCIETY FOR BACTERIOLOGY  

<p>Proteins in the cells are born (synthesized), work, and die (decomposed). In the life of a protein, its birth is obviously important, but how it dies is equally important in living organisms. Proteases secreted into the outside of cells are used to decompose the external proteins and the degradation products are taken as the nutrients. On the other hand, there are also proteases that decompose unnecessary or harmful proteins which are generated in the cells. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for degradation of such proteins. Bacteria, which are prokaryotes, have a similar system as the proteasome. We would like to explain the bacterial degradation system of proteins or the death of proteins, which is performed by ATP-dependent protease Clp, with a particular focus on the ClpXP complex, and with an aspect as a target for antibiotics against bacteria.</p>

DOI： 10.3412/jsb.79.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

A polymer material shows phase separation-induced glass transition under critical miscible conditions, where the miscible/immiscible state is altered by temperature variations predicted by temperature-corrected Hansen solubility parameters.

DOI： 10.1039/d4ma00737a

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

Abstract

Kinesin‐driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo‐binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3‐APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two‐step manner, which suggests kinesin‐cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two‐step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

DOI： 10.15252/embr.202356864

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

DOI： 10.1093/jmicro/dfad051

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Angewandte Chemie - International Edition  

19F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19F MRI imaging. Importantly, our 19F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.

DOI： 10.1002/anie.202308565

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jmb.2023.168331

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

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

DOI： 10.1038/s42003-023-05373-1

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

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

DOI： 10.1038/s41467-023-41156-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

DOI： 10.3390/ijms241612865

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

The real-time visualization via high-speed atomic force microscopy revealed that single microgel exhibit heterogeneous degradation behavior.

DOI： 10.1039/d3sm00216k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41557-023-01216-y

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Other Link： https://www.nature.com/articles/s41557-023-01216-y

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/jacsau.3c00124

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

We propose a recycling strategy for tough polymers based on microparticles. The "microparticle-based" concept allows materials recycling without loss of their properties (‘closed-loop’ recycling).

DOI： 10.1039/d3gc00090g

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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

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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

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Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.63.104

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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

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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

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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

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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

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (conference, symposium, etc.)   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophysico.bppb-v20.s022

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.63.285

CiNii Research

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.langmuir.2c02742

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

DOI： 10.1126/sciadv.ade5155

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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

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Japanese Journal of Applied Physics  

DOI： 10.35848/1347-4065/ac7cc6

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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

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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

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

DOI： 10.1002/smll.202106401

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/smll.202106401

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

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

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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

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

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

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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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

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 80°C, 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

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[Figure: see text].

DOI： 10.1126/sciadv.abf2211

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Language：English   Publishing type：Research paper (international conference proceedings)  

DOI： 10.35848/1347-4065/ac0814

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

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

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

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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

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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

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DOI： 10.1039/d1ra02688g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

<p>Nanostructure and thermoresponsiveness of single and packed poly(<italic>N</italic>-isopropyl methacrylamide)-based microgels observed by temperature-controllable high speed atomic force microscopy.</p>

DOI： 10.1039/d1ra01650d

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FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. 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

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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

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Language：Japanese  

DOI： 10.14952/SEIKAGAKU.2021.930526

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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

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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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

DOI： 10.1039/d0cp02069a

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41428-020-0372-3

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Other Link： http://www.nature.com/articles/s41428-020-0372-3

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Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

DOI： 10.1073/pnas.2011366117

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High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.

DOI： 10.1039/d0cc03776a

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DOI： 10.35848/1347-4065/ab9f31

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The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

DOI： 10.1128/JB.00236-20

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Language：English   Publisher：(一社)日本生物物理学会  

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Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

DOI： 10.1021/acs.jpcb.0c03892

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Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

DOI： 10.1039/d0nr01121e

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The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

DOI： 10.1073/pnas.1916452117

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science ({AAAS})  

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

DOI： 10.1126/sciadv.aaz2441

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

Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

DOI： 10.1007/s12551-020-00670-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

DOI： 10.1074/jbc.RA119.012078

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Language：Japanese  

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

<p>A two-dimensional sheet assembly of a hexameric hemoprotein was constructed. A single cysteine residue was introduced onto each subunit surface in the hexameric hemoprotein and modified with a maleimide-tethering tripeptide FGG tag to provide a host–guest interaction between one cucurbit[8]uril molecule (CB8) and two FGG tags. The addition of CB8 into the engineered protein forms the assembly as a sheet-like precipitate. High-speed atomic force microscopic measurements directly reveal the detailed structure and the dynamic process of formation.</p>

DOI： 10.1246/cl.190855

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

Background: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.Methods: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.Results: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.Conclusions: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significance: We achieved an imaging rate of similar to 3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated similar to 39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

DOI： 10.1016/j.bbagen.2019.03.011

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

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

DOI： 10.1038/s41598-020-58371-2

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

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

DOI： 10.1021/jacs.9b10080

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DOI： 10.1016/j.jmb.2019.10.008

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

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

DOI： 10.1038/s41467-019-14025-0

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

Guillain-Barre syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the C(H)2 and C(H)3 domains. These data indicate that the IgG assembly is mediated through Fc-Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain-Barre syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

DOI： 10.3390/ijms21010147

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

The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.

DOI： 10.1038/s41467-019-13743-9

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

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

DOI： 10.1038/s41586-019-1604-6

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

Language：English  

The moment of protein uptake into hydrogel microspheres (microgels) was directly monitored at the nanoscale by high-speed atomic force microscopy. The dynamic morphological changes in the microgels during protein uptake are largely different depending on the type of protein, and the properties and structure of the microgels, which would affect the colloidal stability of protein-microgel hybrids both in vitro and in vivo.

DOI： 10.1039/c9cc05116c

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Language：English   Publisher：AMER CHEMICAL SOC  

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

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, Fc gamma RIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with Fc gamma RIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

DOI： 10.1038/s41598-019-48323-w

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Language：Japanese   Publisher：Japanese Journal of Applied Physics  

DOI： 10.7567/1347-4065/ab2864

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Language：Japanese   Publisher：2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings  

Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to ∼20 μm and ∼3 μm in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to ∼50 μm and ∼32 μm, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

DOI： 10.1109/3M-NANO46308.2019.8947429

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Language：Japanese   Publisher：2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings  

Atomic force microscopy (AFM) is a powerful tool to visualize biological molecules at nm resolution in liquid conditions and thus has been applied to a wide variety of biological molecules. One of the most coveted functions of AFM is 'fast recording' because it should allow us to directly visualize dynamic processes of biological molecules at work. We have been challenging to break this limitation over 15 years and established fast imaging of dynamic molecular processes. The most advanced high-speed AFM can now capture successive images at 30-60 ms/frame. Further, the interaction force between the tip and the sample, which significantly influences on soft biological molecules, is greatly reduced without deterioration of the scanning performance. In this short paper, typical applications of HS-AFM on biological and artificial molecules will be demonstrated.

DOI： 10.1109/3M-NANO46308.2019.8947375

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

DOI： 10.1038/s41467-019-11204-x

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Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ42 oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ42 species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ42 species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ42 species. The findings revealed that smaller Aβ42 species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to "permissive" binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.

DOI： 10.1016/j.jmb.2019.04.044

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

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

DOI： 10.1016/j.xphs.2019.02.002

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Language：Japanese   Publisher：Science Advances  

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

DOI： 10.1126/sciadv.aax0495

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Language：English  

Despite the tremendous efforts devoted to the structural analysis of hydrogel microspheres (microgels), many details of their structures remain unclear. Reported in this study is that thermoresponsive poly(N-isopropyl acrylamide) (pNIPAm)-based microgels exhibit not only the widely accepted core-shell structures, but also inhomogeneous decanano-sized non-thermoresponsive spherical domains within their dense cores, which was revealed by temperature-controlled high-speed atomic force microscopy (TC-HS-AFM). Based on a series of experiments, it is concluded that the non-thermoresponsive domains are characteristic for pNIPAm microgels synthesized by precipitation polymerization, and plausible structures for microgels prepared by other polymerization techniques are proposed.

DOI： 10.1002/anie.201903483

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ange.201903483

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Soft colloidal particles such as hydrogel microspheres assemble at air/water or oil/water interfaces, where the soft colloids are highly deformed and their surface polymer chains are highly entangled with each other. Herein, we report the formation of robust one-dimensional, string-like colloidal assemblies through self-organization of hydrogel microspheres with shape anisotropy at the air/water interface of sessile droplets. Shape-anisotropic hydrogel microspheres were synthesized via two-step polymerization, whereby a hydrogel shell was formed onto preformed rigid microellipsoids. The shape anisotropy of the hydrogel microspheres was confirmed by transmission electron microscopy and high-speed atomic force microscopy as well as by light-scattering measurements. The present findings are crucial for the understanding of natural self-organization phenomena, where "softness" influences microscopic assembled structures such as those of Nostoc bacteria.

DOI： 10.1002/anie.201901611

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

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

DOI： 10.3390/ijms20092308

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

Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

DOI： 10.1021/acssynbio.9b00019

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Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

DOI： 10.1038/s41467-019-09051-x

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

DOI： 10.1039/c8cc09314h

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Language：English   Publisher：Royal Society of Chemistry ({RSC})  

DOI： 10.1039/c8nr07392a

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

DOI： 10.1016/j.str.2018.09.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

V₁-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A₃B₃ complex and a central DF shaft. The nucleotide-free A₃B₃ complex of <italic>Enterococcus hirae</italic>, composed of three identical A₁B₁ heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A₃B₃. The nucleotide-free mutant A₃B₃ complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A₃B₃ forms a metastable (stressed) asymmetric structure composed of unstable A₁B₁ conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

DOI： 10.1126/sciadv.aau8149

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

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to similar to 20 mu m and similar to 3 mu m in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to similar to 50 mu m and similar to 32 mu m, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Science Publishing Group  

DOI： 10.11648/j.bs.20190501.12

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

Despite recent advances in mechanistic understanding and controlled-synthesis methodologies regarding synthetic supramolecular assemblies, it has remained challenging to capture the molecular-level phenomena in real time, thus hindering further progress in this research field. In this study, we applied high-speed atomic-force microscopy (AFM), which has extraordinary spatiotemporal resolution (1 nm and sub-100 ms), to capture dynamic events occurring during synthetic molecular self-assembly. High-speed AFM permitted the visualization of unique dynamic behavior, such as seeded growth and self-repair in real time. Furthermore, scanning-probe AFM permitted the site-specific manipulation and functionalization of a molecular self-assembly. This powerful combination of bottom-up and top-down approaches at the molecular level should enable targeted syntheses of unprecedented functional nanoarchitectures.

DOI： 10.1002/anie.201809165

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Real-time morphology/structure changes in individual hydrogel microspheres (microgels) were directly visualized at high spatiotemporal resolution using high-speed atomic force microscopy (HS-AFM) under temperature control ranging from room temperature to ∼40 °C. The recorded HS-AFM movies demonstrate that the size and morphology of thermoresponsive poly(N-isopropyl acrylamide)-based microgels change with increasing temperature at the individual microgel level. Specifically, the height of the microgels gradually decreases and domain structures appeared even below the volume phase transition temperature. Moreover, the domain structure is retained, even after the microgels have fully collapsed. The present study thus demonstrates that temperature-controlled HS-AFM is a useful tool for monitoring stimulus-responsiveness of microgels. In the near future, it should furthermore be possible to extend this temperature-controlled HS-AFM to other stimulus-responsive materials, including autonomously oscillating microgels.

DOI： 10.1021/acsomega.8b01770

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DOI： 10.1021/jacs.8b06690

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DOI： 10.1038/s41467-018-05438-4

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Language：Japanese   Publisher：Japanese Journal of Applied Physics  

DOI： 10.7567/JJAP.57.08N001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates. (C) Elsevier B.V. All rights reserved.

DOI： 10.1016/j.colsurfb.2018.04.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

A method for synthesizing coordination nanofibers by extracting the structural motifs of metal-organic frameworks (MOFs) is demonstrated. In these soluble nanofibers, multiple chromophores with largely different sizes and shapes can be arranged at desired compositions, and excited triplet energy migrates among the densely assembled chromophore arrays, showing an efficient photon upconversion even at very low concentration.

DOI： 10.1039/c8cc01594e

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

The ATP-dependent bacterial protein disaggregation machine, CIpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of CIpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize CIpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable CIpB to disentangle protein aggregates.

DOI： 10.1038/s41467-018-04587-w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO−], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m2, which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1–2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.

DOI： 10.1016/j.xphs.2018.01.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

DOI： 10.1038/s41598-018-26606-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc.  

Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.

DOI： 10.1074/jbc.RA118.001817

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

DOI： 10.1002/asia.201800252

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science  

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

DOI： 10.1126/sciadv.aao7054

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Publisher：Wiley  

DOI： 10.1002/asia.201800587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.

DOI： 10.1016/j.jmb.2017.12.020

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Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

DOI： 10.1039/c7cp04606e

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Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

DOI： 10.1039/c8cp90024h

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DOI： 10.1016/j.bpj.2017.11.421

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DOI： 10.1016/j.bbagen.2017.07.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications Ltd  

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallo-graphically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

DOI： 10.1093/protein/gzx066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society of Computer Chemistry, Japan  

<p>Recent progress of high-speed atomic force microscopy (HS-AFM) has enabled us to visualize dynamic conformational change and molecular interaction of proteins in realtime under a physiological condition. Molecular mechanisms through dynamic phenomena that have been deduced from the accumulation of experimental evidence by various analytical techniques are now able to be directly and intuitively understood from AFM movies. While a movie provides a great deal of information compared to a static image, extracting useful information based on objective criteria is essential for the next step. This review describes representative HS-AFM data for three typical dynamic biological phenomena; conformational dynamics of single proteins, linear movement of enzymes and velocity analysis and dynamic molecular interaction together with imaging processing techniques for interpreting AFM data and efficient analysis of successive images.</p>

DOI： 10.2477/jccj.2018-0001

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Language：English   Publisher：Methods in Molecular Biology  

Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

DOI： 10.1007/978-1-4939-8591-3_10

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Language：Japanese   Publisher：Optics InfoBase Conference Papers  

Scanning near-field optical microscopy (SNOM) has been recognized as a powerful technique for super-resolution optical imaging [1]. It has been even more unique compared with other super-resolution fluorescent imaging techniques because it utilizes near-field light at a metallic tip as a nano-light-source, which realizes super-resolution not only in fluorescence but also in any other optical signals such as Raman scattering, infrared absorption and photoluminescence. Such a strong advantage has been stimulating biological fields as a new class of analytical techniques. However, it has been yet highly challenging to apply SNOM for biological samples. Although there have been several issues for biological applications, one of the major problems is its low imaging speed. Because it requires to physically scan a metallic tip, the imaging rate was typically limited to several minutes per frame, which is too slow to capture dynamic biological samples.

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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The 20S proteasome is a core particle of the eukaryotic proteasome responsible for proteolysis and is composed of layered α and β hetero-heptameric rings. The α7 subunit, which is one of components of the α ring, is known to self-assemble into a double-ringed homo-tetradecamer composed of two layers of the α7 heptameric ring. The α7 tetradecamer is known to disassemble upon the addition of α6 subunit, producing a 1:7 hetero-octameric α6-α7 complex. However, the detailed disassembly mechanism remains unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to dissect the disassembly process of the α7 double ring caused by interaction with the α6. HS-AFM movies clearly demonstrated two different modes of interaction in which the α6 monomer initially cracks at the interface between the stacked two α7 single rings and the subsequent intercalation of the α6 monomer in the open pore of the α7 single ring blocks the re-association of the single rings into the double ring. This result provides a mechanistic insight about the disassembly process of non-native homo-oligomers formed by proteasome components which is crucial for the initial process for assembly of 20S proteasome.

DOI： 10.1038/s41598-017-15708-8

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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

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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

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Language：Japanese  

DOI： 10.1016/j.ajoms.2017.06.007

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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

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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

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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

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Language：Japanese   Publisher：Japanese Journal of Applied Physics  

DOI： 10.7567/JJAP.56.08L001

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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

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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

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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

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

<p>Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.</p>

DOI： 10.2142/biophysico.14.0_127

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Language：Japanese   Publisher：The Japan Society of Vacuum and Surface Science  

DOI： 10.14886/sssj2008.37.0_254

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Language：Japanese   Publisher：The Japan Society of Vacuum and Surface Science  

DOI： 10.14886/sssj2008.37.0_19

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DOI： 10.1038/ncomms13415

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DOI： 10.1016/j.ultramic.2015.10.017

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DOI： 10.1074/jbc.M114.627554

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DOI： 10.1063/1.4922381

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DOI： 10.1038/srep08724

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DOI： 10.1021/nl5045617

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

DOI： 10.1371/journal.pone.0116685

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

DOI： 10.1038/ncomms4975

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

DOI： 10.1074/jbc.M113.546085

PubMed

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

PubMed

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

DOI： 10.1021/ja4119994

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DOI： 10.1021/cr4003837

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DOI： 10.1016/j.jsb.2013.02.011

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DOI： 10.1016/j.bpj.2013.07.052

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DOI： 10.1063/1.4813280

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DOI： 10.1063/1.4803449

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DOI： 10.1146/annurev-biophys-083012-130324

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DOI： 10.1016/j.jmb.2012.05.018

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DOI： 10.1038/nprot.2012.047

PubMed

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Web of Science

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DOI： 10.1016/B978-0-12-415931-0.00009-4

PubMed

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DOI： 10.1126/science.1208386

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

DOI： 10.1126/science.1205510

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

DOI： 10.1002/anie.201007544

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DOI： 10.1007/978-1-61779-105-5_18

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DOI： 10.1038/NNANO.2010.7

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

DOI： 10.1063/1.2951594

Web of Science

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DOI： 10.1002/cphc.200800210

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

DOI： 10.1002/jmr.843

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

DOI： 10.1063/1.2766825

PubMed

Publishing type：Research paper (scientific journal)  

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

Web of Science

Responsible for pages：147-178   Language：English Book type：Scholarly book

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Responsible for pages：263-267   Language：English Book type：Dictionary, encyclopedia

Language：Japanese

Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

DOI： 10.1007/978-1-4939-8591-3_10

Scopus

PubMed

Language：Japanese Book type：Scholarly book

Responsible for pages：481-518   Language：English Book type：Scholarly book

Total pages：437   Responsible for pages：143-176  

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DOI： 10.4032/9789814364485

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DOI： 10.1007/978-1-4614-4921-8

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DOI： 10.1002/9783527649808

Responsible for pages：285-300   Language：English Book type：Scholarly book

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DOI： 10.4032/9789814267977

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Responsible for pages：289-308   Language：English Book type：Scholarly book

DOI： 10.1007/1-4020-3019-3

Responsible for pages：103-120   Language：English Book type：Scholarly book

DOI： 10.1007/978-94-010-0736-8

Responsible for pages：505-506   Language：English Book type：Scholarly book

DOI： 10.1007/978-94-011-0049-6

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER CHEMICAL SOC  

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Event date： 2020.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：オンライン   Country：Japan  

Event date： 2020.10

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Venue：オンライン   Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Poster presentation  

Event date： 2020.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2020.9

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2020.2

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：横浜市立大学 金沢八景キャンパス, 神奈川  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：ぎふ長良川 ホテルパーク, 岐阜  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Seoul National University, Korea  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Sommerhotel Julius-Raab-Heim, Linz, Austria  

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：Royton Sapporo, Sapporo  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：BVV Trade Fairs, Brno, Czech Republic  

Language：English   Presentation type：Oral presentation (invited, special)  

Language：English   Presentation type：Oral presentation (keynote)  

Venue：Nagoya University, Aichi  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Montanuniversität Leoben, Austria  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：the Arizona State University Tempe Campus, USA  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Catholic University of Leuven, Leuven, Belgium  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Okazaki Conference Center, Okazaki  

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Language：English   Presentation type：Oral presentation (general)  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Durham University, UK  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Yamate Campus, ExCELLS, Okazaki  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Telluride Intermediate School, Telluride, USA  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Academia Sinica, Taipei, Taiwan  

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Venue：Neve Ilan Hotel, Israel  

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Institute of Molecular Science, Okazaki  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：名古屋大学  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：京都大学吉田キャンパス, 京都  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：仙台国際センター, 宮城  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：横浜市立大学 金沢八景キャンパス, 神奈川  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：名古屋大学  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：神戸ポートピアホテル, 兵庫  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：呉羽ハイツ, 富山  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：産業技術総合研究所, つくば  

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：同志社大学, 京都  

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：仙台国際センター, 宮城  

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：自然科学研究機構、東京