(

A03 Proposed Research Projects (2016-2017)

Paper | Original Paper

2017

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.

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.