A03 Proposed Research Projects (2014-2015)

Paper | Original Paper


Chika Okimura, Yuichi Sakumura, Katsuya Shimabukuro, and *Yoshiaki Iwadate,
Sensing of substratum rigidity and directional migration by fast-crawling cells,
Physical Review E, in press.

[Summary] Living cells sense the mechanical properties of their surrounding environment and respond accordingly. Crawling cells detect the rigidity of their substratum and migrate in certain directions. They can be classified into two categories: slow-moving and fast-moving cell types. Slow-moving cell types, such as fibroblasts, smooth muscle cells, mesenchymal stem cells, etc., move toward rigid areas on the substratum in response to a rigidity gradient. However, rigidity sensing has hitherto not been recorded in fast-moving cell types whose size is ~10 µm and migration velocity is ~10 µm/min. In this study, we used both isotropic substrata with different rigidities and an anisotropic substratum that is rigid on the x-axis but soft on the y-axis to demonstrate rigidity sensing by fast-moving Dictyostelium cells and neutrophil-like differentiated HL-60 cells. Dictyostelium cells exerted larger traction forces on more rigid isotropic substratum. Dictyostelium cells and HL-60 cells migrated in the “soft” direction on the anisotropic substratum, although myosin II-null Dictyostelium cells migrated in random directions, indicating that rigidity sensing of fast-moving cell types differs from that of slow types and is induced by a myosin II-related process.


Chika Okimura. and Yoshiaki Iwadate,
Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes,
Cell Adhesion & Migration 10, 406-418 (2016).

[Summary] Crawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration.

Ayane Sonoda, Chika Okimura, and *Yoshiaki Iwadate,
Shape and area of keratocytes are related to the distribution and magnitude of their traction forces,
Cell Structure and Function 41(1), 33-43 (2016).

[Summary] Fish epidermal keratocytes maintain an overall fan shape during their crawling migration. The shape-determination mechanism has been described theoretically and experimentally on the basis of graded radial extension of the leading edge, but the relationship between shape and traction forces has not been clarified. Migrating keratocytes can be divided into fragments by treatment with the protein kinase inhibitor staurosporine. Fragments containing a nucleus and cytoplasm behave as mini-keratocytes and maintain the same fan shape as the original cells. We measured the shape of the leading edge, together with the areas of the ventral region and traction forces, of keratocytes and mini-keratocytes. The shapes of keratocytes and mini-keratocytes were similar. Mini-keratocytes exerted traction forces at the rear left and right ends, just like keratocytes. The magnitude of the traction forces was proportional to the area of the keratocytes and mini-keratocytes. The myosin II ATPase inhibitor blebbistatin decreased the forces at the rear left and right ends of the keratocytes and expanded their shape laterally. These results suggest that keratocyte shape depends on the distribution of the traction forces, and that the magnitude of the traction forces depends on the area of the cells.

Chika Okimura, Kazuki Ueda, Yuichi Sakumura, *Yoshiaki Iwadate,
Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching,
Cell Adhesion & Migration 10, 331-341 (2016).

[Summary] To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.

Takako Nakata, Chika Okimura, Takafumi Mizuno, and *Yoshiaki Iwadate,
The role of stress fibers in the shape determination mechanism of fish keratocytes,
Biophysical Journal 110, 481-492 (2016).

[Summary] Crawling cells have characteristic shapes that are a function of their cell types. How their different shapes are determined is an interesting question. Fish epithelial keratocytes are an ideal material for investigating cell shape determination, since they maintain a nearly constant fan-shape during their crawling locomotion. We compared the shape and related molecular mechanisms in keratocytes from different fish species to elucidate the key mechanisms that determine cell shape. Wide keratocytes from cichlids applied large traction forces at the rear due to large focal adhesions, and showed a spatially loose gradient associated with actin retrograde flow rate, whereas round keratocytes from black tetra applied low traction forces at the rear small focal adhesions and showed a spatially steep gradient of actin retrograde flow rate. Laser ablation of stress fibers (contractile fibers connected to rear focal adhesions) in wide keratocytes from cichlids increased actin retrograde flow rate and led to slowed leading edge extension near the ablated region. Stress fibers thus might play an important role in the mechanism of maintaining cell shape by regulating actin retrograde flow rate.


Hiroki Himeno, Hiroaki Ito, Yuji Higuchi, Tsutomu Hamada, *Naofumi Shimokawa, Masahiro Takagi,
Coupling between pore formation and phase separation in charged lipid membranes,
Physical Review E 92, 62713 (2015).

[Summary] We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids.

Naoki Narematsu, Raymond Quek, *Keng‐Hwee Chiam, and *Yoshiaki Iwadate,
Ciliary metachronal wave propagation on the compliant surface of Paramecium cells,
Cytoskeleton 72, 633-646 (2015).

[Summary] Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves.

Wonju Lee, Yoshiaki Kinosita, Youngjin Oh, Nagisa Mikami, Heejin Yang, Makoto Miyata, Takayuki Nishizaka, and *Donghyun Kim,
Three-Dimensional Superlocalization Imaging of Gliding Mycoplasma mobile by Extraordinary Light Transmission through Arrayed Nanoholes,
ACS Nano 9, 10896-10908 (2015).

[Summary] In this paper, we describe super-resolved sampling of live bacteria based on extraordinary optical transmission (EOT) of light. EOT is produced by surface plasmon confinement and coupling with nanostructures. Bacterial fluorescence is excited by the localized fields for subdiffraction-limited sampling. The concept was applied to elucidating bacterial dynamics of gliding Mycoplasma mobile (M. mobile). The results analyzed with multiple M. mobile bacteria show individual characters and reveal that M. mobile undergoes a significant axial variation at 94 nm. The sampling error of the method is estimated to be much smaller than 1/10 of the diffraction limit both in the lateral and depth axis. The method provides a powerful tool for investigation of biomolecular dynamics at subwavelength precision.

Thapakorn Tree-udom, Jiraporn Seemork, Kazuki Shigyou, Tsutomu Hamada, Naunpun Sangphech, Tanapat Palaga, Numpon Insin, Porntip Pan-In, and *Supason Pattanaargson Wanichwecharungruang ,
Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake and Cytotoxicity,
ACS Applied Materials & Interfaces 7, 23993–24000 (2015).

[Summary] We study the effect of particle shape on the particle–membrane interaction by monitoring the association between particles of various shapes and lipid bilayer membrane of artificial cell-sized liposomes.

Hitomi Nakashima, Chika Okimura, and *Yoshiaki Iwadate,
The molecular dynamics of crawling migration in microtubule-disrupted keratocytes,
Biophysics and Physicobiology 12, 21-29 (2015).

[Summary] Cell-crawling migration plays an essential role in complex biological phenomena. It is now generally believed that many processes essential to such migration are regulated by microtubules in many cells, including fibroblasts and neurons. However, keratocytes treated with nocodazole, which is an inhibitor of microtubule polymerization – and even keratocyte fragments that contain no microtubules – migrate at the same velocity and with the same directionality as normal keratocytes. In this study, we discovered that not only these migration properties, but also the molecular dynamics that regulate such properties, such as the retrograde flow rate of actin filaments, distributions of vinculin and myosin II, and traction forces, are also the same in nocodazole-treated keratocytes as those in untreated keratocytes. These results suggest that microtubules are not in fact required for crawling migration of keratocytes, either in terms of migrating properties or of intracellular molecular dynamics.

*Yutetsu Kuruma and Takuya Ueda,
The PURE system for the cell-free synthesis of membrane proteins,
Nature Protocols 10, 1328-1344 (2015).

[Summary] Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large molecular weight complexes, such as SecYEG translocon or ATP synthase. Here, we describe a protocol for the cell-free synthesis of membrane proteins using the PURE system, and subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane, and complex formation in the membrane. Additionally, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within one day. This protocol can also avoid the risks in membrane protein purification and in reconstruction to lipid membrane, compared to alternative methods using living cells.

*Aya Tanaka, Ryosuke Tanaka, Nahoko Kasai, Shingo Tsukada, Takaharu Okajima, Koji Sumitomo,
Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy,
Journal of Structural Biology 191, 32–38 (2015).

Yuki Koyano, Natsuhiko Yoshinaga, and *Hiroyuki Kitahata,
General criteria for determining rotation or oscillation in a two-dimensional axisymmetric system,
Journal of Chemical Physics 143, 014117/1-6 (2015).

[Summary] A self-propelled particle in a two-dimensional axisymmetric system, such as a particle in a central force field or confined in a circular region, may show rotational or oscillatory motion. These motions do not require asymmetry of the particle or the boundary, but arise through spontaneous symmetry breaking.We propose a generic model for a self-propelled particle in a two-dimensional axisymmetric system. A weakly nonlinear analysis establishes criteria for determining rotational or oscillatory motion.

Ryosuke Takahashi, *Takaharu Okajima,
Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy,
Applied Physics Letters 107, 173702 (2015).

*Tsutomu Hamada, Rie Fujimoto, Shunsuke F. Shimobayashi, Masatoshi Ichikawa, *Masahiro Takagi,
Molecular behavior of DNA in a cell-sized compartment coated by lipids,
Physical Review E 91, 62717 (2015).

[Summary] The behavior of long DNA molecules in a cell-sized confined space was investigated. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space.

Hiroaki Ito, Navina Kuss, Bastian E. Rapp, Masatoshi Ichikawa, Thomas Gutsmann, Klaus Brandenburg, Johannes M. B. Pöschl, and *Motomu Tanaka,
Quantification of the Influence of Endotoxins on the Mechanics of Adult and Neonatal Red Blood Cells,
Journal of Physical Chemistry B 119, 7837−7845 (2015).

[Summary] In this study, we physically modeled the influence of endotoxin-induced sepsis symptoms on human red blood cells (RBCs) by quantifying the impact of endotoxins on the cell mechanics by the analysis of Fourier-transformed mean square amplitude of shape fluctuation, called flicker spectroscopy. With the aid of a microfluidic diffusion chamber, we noninvasively determined principal mechanical parameters of human RBCs in the absence and presence of endotoxins for individual RBCs for the first time. Because of the elongation of saccharide chain length of endotoxins, we found an increase in the morphological transition from discocytes to echinocytes, and monotonic changes in the mechanical parameters. Since septic shocks often cause lethal risks of neonates, we measured the mechanical parameters of neonatal RBCs, and compared them to those of adult RBCs. The quantitative comparison reveals that neonatal RBCs are more susceptible to the effect of endotoxins than adult RBCs. Furthermore, coincubation with the antiseptic peptide P19-2.5 (Aspidasept) with endotoxin results in a slight suppression of the impact of the endotoxin. The strategy proposed in our study can potentially be applied for the quantitative diagnosis of RBCs based on mechanical readouts.

Sunatda Arayachukeat, Jiraporn Seemork, Porntip Pan-In, Kittima Amornwachirabodee, Naunpun Sangphech, Titiporn Sansureerungsikul, Kamonluck Sathornsantikun, Chotima Vilaivan, Kazuki Shigyou, Prompong Pienpinijtham, Tirayut Vilaivan, Tanapat Palaga, Wijit Banlunara, Tsutomu Hamada, and *Supason Pattanaargson Wanichwecharungruang,
Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles,
Nano Letters 15(5), 3370-3376 (2015).

[Summary] A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Alexandra S. Burk, Cornelia Monzel, Hiroshi Y. Yoshikawa, Patrick Wuchter, Rainer Saffrich, Volker Eckstein, *Motomu Tanaka and *Anthony D. Ho,
Quantifying Adhesion Mechanisms and Dynamics of Human Hematopoietic Stem and Progenitor Cells.,
Scientific Reports 5, 9370 (2015).

[Summary] Using planar lipid membranes with precisely defined concentrations of specific ligands, we have determined the binding strength between human hematopoietic stem cells (HSC) and the bone marrow niche. The relative significance of HSC adhesion to the surrogate niche models via SDF1a-CXCR4 or N-cadherin axes was quantified by (a) the fraction of adherent cells, (b) the area of tight adhesion, and (c) the critical pressure for cell detachment. We have demonstrated that the binding of HSC to the niche model is a cooperative process, and the adhesion mediated by the CXCR4- SDF1a axis is stronger than that by homophilic N-cadherin binding. The statistical image analysis of stochastic morphological dynamics unraveled that HSC dissipated energy by undergoing oscillatory deformation. The combination of an in vitro niche model and novel physical tools has enabled us to quantitatively determine the relative significance of binding mechanisms between normal HSC versus leukemia blasts to the bone marrow niche.

Shin Yamaguchi, Kei Saito, Miki Sutoh, Takayuki Nishizaka, Yoko Y Toyoshima, Junichiro Yajima,
Torque generation by axonemal outer-arm dynein,
Biophysical Journal 108, 872-879 (2015).

[Summary] Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (alphabetagamma) as well as proteolytically generated two-headed (betagamma) and one-headed (alpha) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed alphabetagamma and one-headed alpha exhibited significantly longer pitch. In contrast, the pitch driven by two-headed betagamma did not display this sensitivity. In the assays on lawns containing mixtures of alpha and betagamma at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of alpha in the mixture increased. Even small proportions of alpha-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (alphabetagamma) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

PingGen Cai and Takaharu Okajima,
Precision of cell-to-cell variation in power-law rheology characterized by atomic force microscopy,
Japanese Journal of Applied Physics 54, 037001 (2015).

Jun Kurushima, Daisuke Nakane, Takayuki Nishizaka, and *Haruyoshi Tomita,
Bacteriocin protein BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes L-Ala2-crossbridged peptidoglycan,
Journal of Bacteriology 197, 286-295 (2015).

[Summary] Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis, and consists of two extracellular proteins BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids; 64.5 kDa) was a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase inducing cell lysis of E. faecalis when an accessory factor BacA is co-present. However, the target of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell division associated site including the equatorial ring, division septum, and nascent cell wall on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the 329-590 amino acid region of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and the targeting of BacL1 to the cell division associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with l-Ala-l-Ala crossbridging peptidoglycan including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae but not among bacteria with alternate peptidoglycan structure such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the l-Ala-l-Ala-crossbridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.

Mari Takahashi, Priyank Mohan, Akiko Nakade, Koichi Higashimine, Derek Mott, Tsutomu Hamada, Kazuaki Matsumura, Tomohiko Taguchi, and *Shinya Maenosono,
Ag/FeCo/Ag Core/shell/shell Magnetic Nanoparticles with Plasmonic Imaging Capability,
Langmuir 31, 2228–2236 (2015).

[Summary] Magnetic nanoparticles (NPs) have been used to separate various species such as bacteria, cells, and proteins. In this study, we synthesized Ag/FeCo/Ag core/shell/shell NPs designed for magnetic separation of subcellular components like intracellular vesicles. A benefit of these NPs is that their silver metal content allows plasmon scattering to be used as a tool to observe detection by the NPs easily and semipermanently. Therefore, these NPs are considered a potential alternative to existing fluorescent probes like dye molecules and colloidal quantum dots. In addition, the Ag core inside the NPs suppresses the oxidation of FeCo because of electron transfer from the Ag core to the FeCo shell, even though FeCo is typically susceptible to oxidation. The surfaces of the Ag/FeCo/Ag NPs were functionalized with ε-poly-l-lysine-based hydrophilic polymers to make them water-soluble and biocompatible. The imaging capability of the polymer-functionalized NPs induced by plasmon scattering from the Ag core was investigated. The response of the NPs to a magnetic field using liposomes as platforms and applying a magnetic field during observation by confocal laser scanning microscopy was assessed. The results of the magnetophoresis experiments of liposomes allowed us to calculate the magnetic force to which each liposome was subjected.

Shoichi Toyabe and Eiro Muneyuki,
Single molecule thermodynamics of ATP synthesis by F1-ATPase,
New Journal of Physics 17, 015008/1-7 (2015).

[Summary] FoF1-ATP synthase is a factory for synthesizing ATP in virtually all cells. Its core machinery is the subcomplex F1-motor (F1-ATPase) and performs the reversible mechanochemical coupling. The isolated F1-motor hydrolyzes ATP, which is accompanied by unidirectional rotation of its central γ−shaft. When a strong opposing torque is imposed, theγ-shaft rotates in the opposite direction and drives the F1 -motor to synthesize ATP. This mechanical-to-chemical free-energy transduction is the final and central step of the multistep cellular ATP-synthetic pathway. Here, we determined the amount of mechanical work exploited by the F1 -motor to synthesize an ATP molecule during forced rotations using a methodology combining a nonequilibrium theory and single molecule measurements of responses to external torque. We found that the internal dissipation of the motor is negligible even during rotations far from a quasistatic process.


Hideaki Matsubayashi, Yutetsu Kuruma, and Takuya Ueda,
Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery,
Origins of Life and Evolution of Biospheres 44, 331-334 (2014).

Nataliya Frenkel, Ali Makky, Ikhwan Resmala Sudji, *Michael Wink, and *Motomu Tanaka,
Mechanistic Investigation of Interactions between Steroidal Saponin Digitonin and Cell Membrane Models,
The Journal of Physical Chemistry B 118, 14632−14639 (2014).

Harden Rieger, Hiroshi Y. Yoshikawa, Katharina Quadt, Morten A. Nielsen, Cecilia P. Sanchez, Ali Salanti, *Motomu Tanaka and Michael Lanzer,
Cytoadhesion of Plasmodium falciparum–infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced,
Blood 125, 383-391 (2014).

Hiroshi Ueno, Yoshihiro Minagawa, Mayu Hara, Suhaila Rahman, Ichiro Yamato, Eiro Muneyuki, Hiroyuki Noji, Takeshi Murata, *Ryota Iino,
Torque Generation of Enterococcus hirae V-ATPase,
The Journal of Biology Chemistry 289, 31212-31223 (2014).

[Summary] V-ATPase (VoV1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in VoV1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae VoV1 (EhVoV1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhVoV1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhVoV1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhVoV1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhVoV1 showed faster rotation than EhV1, and the torque of EhVoV1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhVoV1. These results indicate that rotor-stator interactions of the Vo moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhVoV1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhVoV1.

Hiroki Himeno, Naofumi Shimokawa, Shigeyuki Komura, David Andelman, *Tsutomu Hamada, Masahiro Takagi,
Charge-induced phase separation in lipid membranes,
Soft Matter 10, 7959-7967 (2014).

[Summary] Phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG(−)), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG(−)), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in the stability of membrane phase separation. DPPG(−) enhances phase separation, while DOPG(−) suppresses it. Furthermore, the addition of DPPG(−) to a binary mixture of DPPC/cholesterol induces phase separation between DPPG(−)-rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG(−) was added to a neutral ternary system of DOPC/DPPC/cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach.

Yasuaki Komuro, Suyong Re, Chigusa Kobayashi, Eiro Muneyuki, and *Yuji Sugita,
CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca2+-ATPase,
Journal of Chemical Theory and Computation 10, 4133−4142 (2014).

[Summary] Adenosine triphosphate (ATP) is an indispensableenergy source in cells. In a wide variety of biologicalphenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATPhydrolysis is converted to mechanical forces to bring aboutlarge-scale conformational changes in proteins. Investigation of structure−function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca2+-ATPase and four soluble proteins. The ATP-bound structure of Ca2+-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using originalparameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified arameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.

Phuc Nghia Nguyen, Mariam Veschgini, Motomu Tanaka, Gilles Waton, Thierry Vandammec and *Marie Pierre Krafft,
Counteracting the inhibitory effect of proteins towards lung surfactant substitutes: a fluorocarbon gas helps displace albumin at the air/water interfac,
Chemical Communications 50, 11576-11579 (2014).

Mihoko Kajita, Kaoru Sugimura, Atsuko Ohoka, Jemima Burden, Hitomi Suganuma, Masaya Ikegawa, Takashi Shimada, Tetsuya Kitamura, Masanobu Shindoh, Susumu Ishikawa, Sayaka Yamamoto, Sayaka Saitoh, Yuta Yako, Ryosuke Takahashi, Takaharu Okajima, Junichi Kikuta, Yumiko Maijima, Masaru Ishii, Masazumi Tada, and *Yasuyuki Fujita,
Filamin acts as a key regulator in epithelial defence against transformed cells,
Nature Communications 5, 4428/1-13 (2014).

Shunchi Kawasaki, *Takahiro Muraoka, Haruki Obara, Takerou Ishii, Tsutomu Hamada, *Kazushi kinbara,
Thermodriven Micrometer-Scale Aqueous-Phase Separation of Amphiphilic Oligoethylene Glycol Analogues,
Chemistry-An Asian Journal 9, 2778-2788 (2014).

[Summary] Thermoresponsive materials with a lower critical solution temperature (LCST) are receiving growing attention, of which examples of non-polymeric small molecules are limited. Monodisperse oligoethylene glycol amphiphiles that contain aromatic units with a LCST in water have been developed and applied to peptide extraction. Concentration-dependent hysteretic transmittance changes were observed in response to temperature elevation and reduction. Dynamic light scattering measurements and phase contrast microscopy revealed the formation of micrometer-sized aggregates upon heating at a concentration above 5.0 mM; these aggregates self-assembled to form larger aggregates upon cooling before dissolution. The “interaggregate” interactions are likely to cause the hysteretic behavior. As an application of this thermodriven phase separation, selective extraction of peptide fragments containing high percentages of hydrophobic and aromatic amino acid residues was successfully demonstrated.

Hideaki Matsubayashi, Yutetsu Kuruma, and Takuya Ueda,
In vitro synthesis of the E. coli Sec Translocon from DNA,
Angewandte Chemie International Edition 53, 7535-7538 (2014).

Yoshiaki Kinosita, Daisuke Nakane, Mitsuhiro Sugawa, Tomoko Masaike, Kana Mizutani, Makoto Miyata, and *Takayuki Nishizaka,
Unitary step of gliding machinery in Mycoplasma mobile,
Proceedings of the National Academy of Sciences 111, 8601-8606 (2014).

[Summary] Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0-4.5 mums(-1) with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.

Tatsuya Shima, Takahiro Muraoka, Tsutomu Hamada, Masamune Morita, Masahiro Takagi, Hajime Fukuoka, Yuichi Inoue, Takashi Sagawa, Akihiko Ishijima, Yuki Omata, Takashi Yamashita, *Kazushi Kinbara,
Micrometer-Size Vesicle Formation Triggered by UV Light,
Langmuir 30, 7289-7295 (2014).

[Summary] Vesicle formation is a fundamental kinetic process related to the vesicle budding and endocytosis in a cell. In the vesicle formation by artificial means, transformation of lamellar lipid aggregates into spherical architectures is a key process and known to be prompted by e.g. heat, infrared irradiation, and alternating electric field induction. Here we report UV-light-driven formation of vesicles from particles consisting of crumpled phospholipid multilayer membranes involving a photoactive amphiphilic compound composed of 1,4-bis(4-phenylethynyl)benzene (BPEB) units. The particles can readily be prepared from a mixture of these components, which is casted on the glass surface followed by addition of water under ultrasonic radiation. Interestingly, upon irradiation with UV light, micrometer-size vesicles were generated from the particles. Neither infrared light irradiation nor heating prompted the vesicle formation. Taking advantage of the benefits of light, we successfully demonstrated micrometer-scale spatiotemporal control of single vesicle formation. It is also revealed that the BPEB units in the amphiphile are essential for this phenomenon.

Masamune Morita, *Tsutomu Hamada, Mun’delanji C. Vestergaard, Masahiro Takagi,
Endo- and Exocytic Budding Transformation of Slow-Diffusing Membrane Domains Induced by Alzheimer’s Amyloid Beta,
Physical Chemistry Chemical Physics 16, 8773-8777 (2014).

[Summary] Cell-sized liposomes are a powerful tool for clarifying physicochemical mechanisms that govern molecular interactions. Herein, budding transformation of membrane domains was induced by amyloid beta peptides. The peptides increased the membrane viscosity as demonstrated by the Brownian motion of membrane domains. These results could aid in understanding the physicochemical mechanism of Alzheimer's disease.

Nataliya Frenkel, Jens Wallys, Sara Lippert, Jörg Teubert, Stefan Kaufmann, Aparna Das, Eva Monroy, Martin Eickhoff and *Motomu Tanaka,
High Precision, Electrochemical Detection of Reversible Binding of Recombinant Proteins on Wide Bandgap GaN Electrodes Functionalized with Biomembrane Models,
Advanced Functional Materials 24, 4927–4934 (2014).

*Yoshihiro Shimizu, Yutetsu Kuruma, Takashi Kanamori, Takuya Ueda ,
The PURE system for protein production,
Methods in Molecular Biology 1118, 275-284 (2014).