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A03 Proposed Research Projects (2016-2017)

Paper | Original Paper

2017

*Shinji Watanabe, Toshio Ando,
High-speed XYZ-nanopositioner for scanning ion conductance microscopy,
Applied Physics Letters, VOL. 111, PP. 113106-1/113106-4 111, 113606-1/113601-4 (2017).

[Summary] We describe a tip-scan-type high-speed XYZ-nanopositioner designed for scanning ion conductance microscopy (SICM), with a special care being devoted to the way of nanopipette holding. The nanopipette probe is mounted in the center of a hollow piezoactuator, both ends of which are attached to identical diaphragm flexures, for Z-positioning. This design minimizes the generation of undesirable mechanical vibrations. Mechanical amplification is used to increase the XY-travel range of the nanopositioner. The first resonance frequencies of the nanopositioner are measured as ~100kHz and ~2.3kHz for the Z- and XY-displacements, respectively. The travel ranges are ~6um and ~34lm for Z and XY, respectively. When this nanopositioner is used for hoppingmode imaging of SICM with a ~10-nm radius tip, the vertical tip velocity can be increased to 400nm/ms; hence, the one-pixel acquisition time can be minimized to ~1ms.

*Kei Fujiwara, Tsunehito Sawamura, Tatsuya Niwa, Tatsuki Deyama, Shin-ichiro M. Nomura, Hideki Taguchi, Nobuhide Doi,
In vitro transcription–translation using bacterial genome as a template to reconstitute intracellular profile,
Nucleic acids research gkx776, (2017).

[Summary] In vitro transcription–translation systems (TX–TL) can synthesize most of individual genes encoded in genomes by using strong promoters and translation initiation sequences. This fact raises a possibility that TX–TL using genome as a template can reconstitute the profile of RNA and proteins in living cells. By using cell extracts and genome prepared from different organisms, here we developed a system for in vitro genome transcription–translation (iGeTT) using bacterial genome and cell extracts, and surveyed de novo synthesis of RNA and proteins. Two-dimensional electrophoresis and nano LC–MS/MS showed that proteins were actually expressed by iGeTT. Quantitation of transcription levels of 50 genes for intracellular homeostasis revealed that the levels of RNA synthesis by iGeTT are highly correlated with those in growth phase cells. Furthermore, activity of iGeTT was influenced by transcription derived from genome structure and gene location in genome. These results suggest that intracellular profiles and characters of genome can be emulated by TX–TL using genome as a template.

*Natsuhiko Yoshinaga and Tanniemola B. Liverpool,
Hydrodynamic interactions in dense active suspensions: From polar order to dynamical clusters,
Physical Review E Rapid Communications 96, 020603(R) (2017).

[Summary] We study the role of hydrodynamic interactions in the collective behavior of collections of microscopic activeparticles suspended in a fluid. We introduce a calculational framework that allows us to separate the differentcontributions to their collective dynamics from hydrodynamic interactions on different length scales. Hence weare able to systematically show that lubrication forces when the particles are very close to each other play asimportant a role as long-range hydrodynamic interactions in determining their many-body behavior.We find thatmotility-induced phase separation is suppressed by near-field interactions, leading to open gel-like clusters ratherthan dense clusters. Interestingly, we find a globally polar ordered phase appears for neutral swimmers with noforce dipole that is enhanced by near-field lubrication forces in which the collision process rather than long-rangeinteraction dominates the alignment mechanism.

Yuki Sakamoto, and *Shoichi Toyabe,
Assembly of a functional and responsive microstructure by heat bonding of DNA-grafted colloidal brick,
Scientific Reports 7, 9104 (2017).

[Summary] A micromachine constructed to possess various chemical and mechanical functions is one of the ultimate targets of technology. Conventional lithographic processes can be used to form complicated structures. However, they are basically limited to rigid and static structures with poor surface properties. Here, we demonstrate a novel method for assembling responsive and functional microstructures from diverse particles modified with DNA strands. The DNA strands are designed to form hairpins at room temperature and denature when heated. Structures are assembled through the simultaneous manipulation and heating of particles with “hot” optical tweezers, which incorporates the particles one by one. The flexible connection formed by DNA strands allows the responsive deformation of the structures with local controllability of the structural flexibility. We assembled a microscopic robot arm actuated by an external magnet, a hinge structure with a locally controlled connection flexibility and a three-dimensional double helix structure. The method is simple and can also be applied to build complex biological tissues from cells.

Marcel Hörning, Masaki Nakahata, Philip Linke, Akihisa Yamamoto, Mariam Veschgini, Stefan Kaufmann, Yoshinori Takashima, Akira Harada & *Motomu Tanaka,
Dynamic mechano-regulation of myoblast cells on supramolecular hydrogels cross-linked by reversible host-guest interactions,
Scientific Reports 7, 7660 (2017).

[Summary] A new class of supramolecular hydrogels, cross-linked by host-guest interactions between β-cyclodextrin (βCD) and adamantane, were designed for the dynamic regulation of cell-substrate interactions. The initial substrate elasticity can be optimized by selecting the molar fraction of host-
and guest monomers for the target cells. Moreover, owing to the reversible nature of host-guest interactions, the magnitude of softening and sti ening of the substrate can be modulated by varying the concentrations of free, competing host molecules (βCD) in solutions. By changing the substrate elasticity at a desired time point, it is possible to switch the micromechanical environments of cells.
We demonstrated that the Young’s modulus of our “host-guest gels”, 4–11 kPa, lies in an optimal range not only for static (ex situ) but also for dynamic (in situ) regulation of cell morphology and cytoskeletal ordering of myoblasts. Compared to other stimulus-responsive materials that can either change the elasticity only in one direction or rely on less biocompatible stimuli such as UV light and temperature change, our supramolecular hydrogel enables to reversibly apply mechanical cues to various cell types in vitro without interfering cell viability.

Kazusa Beppu, Ziane, Izri, Jun Gohya, Kanta Eto, Masatoshi Ichikawa, and *Yusuke T. Maeda,
Geometry-driven collective ordering of bacterial vortices,
Soft Matter 13, 5038 (2017).

[Summary] Controlling the phases of matter is a challenge that spans from condensed materials to biological systems. Here, by imposing a geometric boundary condition, we study the controlled collective motion of Escherichia coli bacteria. A circular microwell isolates a rectified vortex from disordered vortices masked in the bulk. For a doublet of microwells, two vortices emerge but their spinning directions show transition from parallel to anti-parallel. A Vicsek-like model for confined self-propelled particles gives the point where the two spinning patterns occur in equal probability and one geometric quantity governs the transition as seen in experiments. This mechanism shapes rich patterns including chiral configurations in a quadruplet of microwells, thus revealing a design principle of active vortices.

Chikako Kurokawa, Kei Fujiwara, Masamune Morita, Ibuki Kawamata, Yui Kawagishi, Atsushi Sakai, Yoshihiro Murayama, Shin-ichiro M. Nomura, Satoshi Murata, *Masahiro Takinoue, and *Miho Yanagisawa,
DNA cytoskeleton for stabilizing artificial cells,
Proceedings of the National Academy of Sciences of the United States of America 114, 7228-7233 (2017).

[Summary] Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

Takahisa Matsuzaki, Hiroaki Ito, Veronika Chevyreva, Ali Makky, Stefan Kaufmann, Kazuki Okano, Naritaka Kobayashi, Masami Suganuma, Seiichiro Nakabayashi, Hiroshi Y. Yoshikawa and *Motomu Tanaka,
Adsorption of galloyl catechin aggregates significantly modulates membrane mechanics in the absence of biochemical cues,
Physical Chemistry Chemical Physics 19, 9937-19947 (2017).

[Summary] Physical interactions of four major green tea catechin derivatives with cell membrane models were systemically investigated. Catechins with the galloyl moiety caused the aggregation of small unilamellar vesicles and an increase in the surface pressure of lipid monolayers, while those without did not. Differential scanning calorimetry revealed that, in a low concentration regime (r10 mM), catechin molecules are not significantly incorporated into the hydrophobic core of lipid membranes as substitutional impurities. Partition coefficient measurements revealed that the galloyl moiety of catechin and the cationic quaternary amine of lipids dominate the catechin–membrane interaction, which can be attributed to the combination of electrostatic and cation–p interactions. Finally, we shed light on the mechanical consequence of catechin–membrane interactions using the Fourier-transformation of the membrane fluctuation. Surprisingly, the incubation of cell-sized vesicles with 1 mM galloyl catechins, which is comparable to the level in human blood plasma after green tea consumption, significantly increased the bending stiffness of the membranes by a factor of more than 60, while those without the galloyl moiety had no detectable influence. Atomic force microscopy and circular dichroism spectroscopy suggest that the membrane stiffening is mainly attributed to the adsorption of galloyl catechin aggregates to the membrane surfaces. These results contribute to our understanding of the physical and thus the generic functions of green tea catechins in therapeutics, such as cancer prevention.

Daisuke Nakane and Takayuki Nishizaka*,
Asymmetric distribution of type IV pili triggered by directional light in unicellular cyanobacteria,
Proceedings of the National Academy of Sciences 114, 6593-6598 (2017).

[Summary] The type IV pili (T4P) system is a supermolecular machine observed in prokaryotes. Cells repeat the cycle of T4P extension, surface attachment, and retraction to drive twitching motility. Although the properties of T4P as a motor have been scrutinized with biophysics techniques, the mechanism of regulation remains unclear. Here we provided the framework of the T4P dynamics at the single-cell level in Synechocystis sp. PCC6803, which can recognize light direction. We demonstrated that the dynamics was detected by fluorescent beads under an optical microscope and controlled by blue light that induces negative phototaxis; extension and retraction of T4P was activated at the forward side of lateral illumination to move away from the light source. Additionally, we directly visualized each pilus by fluorescent labeling, allowing us to quantify their asymmetric distribution. Finally, quantitative analyses of cell tracking indicated that T4P was generated uniformly within 0.2 min after blue-light exposure, and within the next 1 min the activation became asymmetric along the light axis to achieve directional cell motility; this process was mediated by the photo-sensing protein, PixD. This sequential process provides clues toward a general regulation mechanism of T4P system, which might be essentially common between archaella and other secretion apparatuses.

*Sho Fujii, Ryuta Fukano, Yoshihito Hayami, Hiroaki Ozawa, Eiro Muneyuki, Noboru Kitamura, and *Masa-aki Haga,
Simultaneous Formation and Spatial Patterning of ZnO on ITO Surfaces by Local Laser-Induced Generation of Microbubbles in Aqueous Solutions of [Zn(NH3)4]2+,
ACS Applied Materials & Interfaces 9, 8413-8419 (2017).

[Summary] We demonstrate the simultaneous formation and spatial patterning of ZnO nanocrystals on an indium−tin oxide (ITO) surface upon local heating using a laser (1064 nm) and subsequent formation of microbubbles. Laser irradiation of an ITO surface in aqueous [Zn(NH3)4]2+solution (1.0×10-2 M at pH 12.0) under an optical microscope produced ZnO nanocrystals, the presence of which was confirmed by X-ray diffraction analysis and Raman microspectroscopy. Scanning the focused laser beam over the ITO surface generated a spatial ZnO pattern (height:∼60 nm,width:∼1μm) in the absence of a template or mask. The Marangoni convection generated in the vicinity of the micro-bubbles resulted in a rapid concentration/accumulation of [Zn(NH3)4]2+ around the microbubbles, which led to the formation of ZnO at the solid−bubble−solution three-phase contact line around the bubbles and thus afforded ZnO nanocrystals on the ITO surface upon local heating with a laser.

Yuji Higaki,Benjamin Fröhlich, Akihisa Yamamoto, Ryo Murakami,Makoto Kaneko, *Atsushi Takahara, and *Motomu Tanaka,
Ion-specific modulation of interfacial interaction potentials between solid substrates and cell-sized particles mediated via zwitterionic, super-hydrophilic poly(sulfobetaine) brushes,
The Journal of Physical Chemistry B 121, 1396−1404 (2017).

[Summary] Zwitterionic polymer brushes draw increasing
attention not only because of their superhydrophilic, self-
cleaning capability but also due to their excellent antifouling
capacity. We investigated the ion-specific modulation of the
interfacial interaction potential via densely packed, uniform
poly(sulfobetaine) brushes. The vertical Brownian motion of a
cell-sized latex particle was monitored by microinterferometry,
yielding the effective interfacial interaction potentials V(Δh)
and the autocorrelation function of height fluctuation. The
potential curvature V′′(Δh) exhibited a monotonic increase
according to the increase in monovalent salt concentrations,
implying the sharpening of the potential confinement. An
opposite tendency was observed in CaCl2 solutions, suggesting that the ion specific modulation cannot be explained by the classical Hofmeister series. When the particle fluctuation was monitored in the presence of free sulfobetaine molecules, the increase in [sulfobetaine] resulted in a distinct increase in hydrodynamic friction. This was never observed in all the other salt solutions, suggesting the interference of zwitterionic pairing of sulfobetaine side chains by the intercalation of sulfobetaine molecules into the brush layer. Furthermore, poly(sulfobetaine) brushes exhibited a very low V′′(Δh) and hydrodynamic friction to human erythrocytes, which seems to explain the excellent blood repellency of zwitterionic polymer materials.

2016

Kano Suzuki, Kenji Mizutani, Shintaro Maruyama, Kazumi Shimono, Fabiana L. Imai, Eiro Muneyuki, Yoshimi Kakinuma, Yoshiko Ishizuka-Katsura, Mikako Shirouzu, Shigeyuki Yokoyama, Ichiro Yamato and *Takeshi Murata,
Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor.,
Nature Communications 7, 13235 (2016).

[Summary] V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.

Shunsuke Yabunaka, Natsuhiko Yoshinaga,
Collision between chemically-driven self-propelled drops,
Journal of Fluid Mechanics 809, 205-233 (2016).

[Summary] We use analytical and numerical approaches to investigate head-on collisions between two self-propelled drops described as a phase separated binary mixture. Each drop is driven by chemical reactions that isotropically produce or consume the concentration of a third chemical component, which affects the surface tension of the drop. The isotropic distribution of the concentration field is destabilized by motion of the drop, which is created by the Marangoni flow from the concentration-dependent surface tension. This symmetry-breaking self-propulsion is distinct from other self-propulsion mechanisms due to its intrinsic polarity of squirmers and self-phoretic motion; there is a bifurcation point below which the drop is stationary and above which it moves spontaneously. When two drops are moving in the opposite direction along the same axis, their interactions arise from hydrodynamics and concentration overlap. We found that two drops exhibit either an elastic collision or fusion, depending on the distance from their bifurcation point, which may be controlled, for example, by viscosity. An elastic collision occurs when there is a balance between dissipation and the injection of energy by chemical reactions. We derive the reduced equations for the collision between two drops and analyse the contributions from the two interactions. The concentration-mediated interaction is found to dominate the hydrodynamic interaction for a head-on collision.

Yoshiaki Kinosita, *Nariya Uchida, Daisuke Nakane and *Takayuki Nishizaka,
Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum,
Nature Microbiology 1, 16148/1-9 (2016).

[Summary] Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6–10%.

Yoshiaki Kinosita, *Nariya Uchida, Daisuke Nakane, and *Takayuki Nishizaka,
Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum,
Nature Microbiology 1, 16148/1-9 (2016).

*Yuko Sato, Tomoya Kujirai, Ritsuko Arai, Haruhiko Asakawa, Chizuru Ohtsuki, Naoki Horikoshi, Kazuo Yamagata, Jun Ueda, Takahiro Nagase, Tokuko Haraguchi, Yasushi Hiraoka, Akatsuki Kimura, Hitoshi Kurumizaka, and *Hiroshi Kimura,
A genetically encoded probe for live-cell imaging of H4K20 monomethylation.,
Journal of Molecular Biology 428, 3885-2902 (2016).

[Summary] Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

Ritsuya Niwayama, Hiromichi Nagao, Tomoya Kitajima, Lars Hufnagel, Kyosuke Shinohara, Tomoyuki Higuchi, Takuji Ishikawa, and *Akatsuki Kimura,
Bayesian Inference of Forces Causing Cytoplasmic Streaming in Caenorhabditis elegans Embryos and Mouse Oocytes.,
PLoS ONE 11, e0159917 (2016).

[Summary] Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.

Akihiro Tanaka, Daisuke Nakane, Masaki Mizutani, Takayuki Nishizaka, *Makoto Miyata,
Directed binding of gliding bacterium, Mycoplasma mobile, shown by detachment force and bond lifetime,
mBio 7, e00455-16 (2016).

*Shigeru Matsumura, Tomoko Kojidani, Yuji Kamioka, Seiichi Uchida, Tokuko Haraguchi, Akatsuki Kimura, and Fumiko Toyoshima,
Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.,
Nature Communications 7, 11858 (2016).

[Summary] Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry.

*Mitsuhiro Sugawa, Kei-ichi Okazaki, Masaru Kobayashi, Takashi Matsui, Gerhard Hummer, Tomoko Masaike, and *Takayuki Nishizaka,
F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements,
Proceedings of the National Academy of Sciences of the United States of America 113, E2916-2924 (2016).

Yi-Teng Hsiao, Kuan-Ting Wu, Nariya Uchida, and *Wei-Yen Woon,
Impurity-tuned non-equilibrium phase transition in a bacterial carpet,
Applied Physics Letters 108, 183701/1-5 (2016).

[Summary] The effects of impurity on the non-equilibrium phase transition in Vibrio alginolyticus bacterialcarpets are investigated through a position-sensitive-diode implemented optical tweezers-microsphere assay. The collective flow increases abruptly as we increase the rotation rate of flagellavia Na þ concentration. The effects of impurities on the transition behavior are examined by mixingcells of a wild type strain (VIO5) with cells of a mutant strain (NMB136) in different swimmingpatterns. For dilute impurities, the transition point is shifted toward higher Na þ concentration.Increasing the impurities’ ratio to over 0.25 leads to a significant drop in the collective force, sug-gesting a partial orientational order with a smaller correlation length.

Viktoria Frank, Stefan Kaufmann, Rebecca Wright, Patrick Horn, Hiroshi Yoshikawa, Patrick Wuchter, Jeppe Madsen, Andrew Lewis, Steven P. Armes, Anthony D. Ho, and *Motomu Tanaka,
Frequent mechanical stress suppresses proliferation of mesenchymal stem cells from human bone marrow without loss of multipotency,
Scientific Reports 6, 24264 (2016).

[Summary] Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not only to biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such “dynamic in vitro niche” can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage

Mariam Veschgini, F. Gebert, Nyamdorj Khangai, H. Ito, Ryo Suzuki, Thomas W. Holstein, Yasushi Mae, Takero Arai, and *Motomu Tanaka,
Tracking mechanical and morphological dynamics of regenerating Hydra tissue fragments using a two fingered micro-robotic hand,
Applied Physics Letters 108, 103702 (2016).

[Summary] Regeneration of a tissue fragment of freshwater polyp Hydra is accompanied by significant morphological fluctuations, suggesting the generation of active forces. In this study, we utilized a two fingered micro-robotic hand to gain insights into the mechanics of regenerating tissues. Taking advantage of a high force sensitivity (~1 nN) of our micro-hand, we non-invasively acquired the bulk elastic modulus of tissues by keeping the strain levels low (ε < 0.15). Moreover, by keeping the strain at a constant level, we monitored the stress relaxation of the Hydra tissue and determined both viscous modulus and elastic modulus simultaneously, following a simple Maxwell model. We further investigated the correlation between the frequency of force fluctuation and that of morpho- logical fluctuation by monitoring one “tweezed” tissue and the other “intact” tissue at the same time. The obtained results clearly indicated that the magnitude and periodicity of the changes in force and shape are directly correlated, confirming that our two fingered micro-hand can precisely quantify the mechanics of soft, dynamic tissue during the regeneration and development in a non- invasive manner.

*Jun Tamogami, Keitaro Sato, Sukuna Kurokawa, Takumi Yamada, Toshifumi Nara, Makoto Demura, Seiji Miyauchi, Takashi Kikukawa, Eiro Muneyuki, Naoki Kamo,
Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≧~8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH,
Biochemistry 55(7), 1036-48 (2016).

[Summary] Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H+-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (~9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. Based on these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.