業 績  

  1. *Chikara Furusawa, Tomoyuki Yamaguchi,
    Robust and accurate discrimination of self/non-self antigen presentations by regulatory T cell suppression,
    PLoS One 11(9), e0163134 (2016).
    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.
  2. Shingo Suzuki, Takaaki Horinouchi, *Chikara Furusawa,
    Phenotypic changes associated with the fitness cost in antibiotic resistant Escherichia coli strains,
    Molecular Biosystems 12(2), 414-20 (2016).
    The acquisition of antibiotic resistance in bacterial cells is often accompanied with a reduction of fitness in the absence of antibiotics, known as the “fitness cost.” The magnitude of this fitness cost is an important biological parameter that influences the degree to which antibiotic resistant strains become widespread. However, the relationship between the fitness cost and comprehensive phenotypic and genotypic changes remains unclear. Here, we quantified the fitness cost of resistant strains obtained by experimental evolution in the presence of various antibiotics, and analyzed how the cost correlated to phenotypic and genotypic changes in the resistant strains.
  3. Shingo Suzuki, Takaaki Horinouchi, *Chikara Furusawa,
    Expression profiling of antibiotic resistant bacteria obtained by laboratory evolution,
    Methods in Molecular Biology 1520, 263-279 (2017).
    To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.
  4. *古澤力, 鈴木真吾, 堀之内貴明,
    大腸菌進化実験を用いた遺伝子発現量からの抗生物質耐性メカニズムの解析,
    バイオサイエンスとインダストリー 74(2), 138-140 (2016).
     抗生物質が効かない耐性菌、特に複数の抗生物質への耐性を持つ多剤耐性菌の出現が世界的な問題となっている。そうした薬剤耐性を持つ病原菌を出現を抑制するためには、その進化のプロセスを理解することが重要となる。一方で、耐性獲得の進化プロセスは、表現型と遺伝子型の変化が絡み合う複雑なダイナミクスを持ち、その全貌が理解されているとは言いがたい。そこで本研究では、様々な抗生物質を添加した環境下において、大腸菌の進化実験を行い、その耐性進化のプロセスを解析した1)。以下、そこでの表現型と遺伝子型の解析から見えてきた、薬剤耐性の進化プロセスが持つ性質を紹介する。
  5. Nen Saito and Kunihiko Kaneko,
    Collective motion switches motor direction along filament,
    Scientific reports, in press.
  6. Takahiro Kohsokabe, Kunihiko Kaneko,
    Boundary-Induced Pattern Formation from Temporal Oscillation: Spatial Map Analysis,
    Europhysics Letters 116, 48005/1-6 (2017).
  7. Kenji Shinoda and Kunihiko Kaneko,
    Chaotic Griffiths Phase with Anomalous Lyapunov Spectra in Coupled Map Networks,
    Physical Review Letters 117, 254101/1-6 (2016).
  8. Jumpei F Yamagishi, Nen Saito, Kunihiko Kaneko,
    Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates,
    PLOS Computational Biology 12(10), e1005042/1-17 (2016).
    As cells grow and divide under a given environment, they become crowded and resourcesare limited, as seen in bacterial biofilms and multicellular aggregates. These cells oftenshow strong interactions through exchanging chemicals, as evident in quorum sensing, toachieve mutualism and division of labor. Here, to achieve stable division of labor, threecharacteristics are required. First, isogenous cells differentiate into several types. Second,this aggregate of distinct cell types shows better growth than that of isolated cells withoutinteraction and differentiation, by achieving division of labor. Third, this cell aggregate isrobust with respect to the number distribution of differentiated cell types. Indeed, theoreticalstudies have thus far considered how such cooperation is achieved when the ability of celldifferentiation is presumed. Here, we address how cells acquire the ability of cell differentiationand division of labor simultaneously, which is also connected with the robustness of acell society. For this purpose, we developed a dynamical-systems model of cells consistingof chemical components with intracellular catalytic reaction dynamics. The reactions convertexternal nutrients into internal components for cellular growth, and the divided cellsinteract through chemical diffusion. We found that cells sharing an identical catalytic networkspontaneously differentiate via induction from cell-cell interactions, and then achievedivision of labor, enabling a higher growth rate than that in the unicellular case. This symbioticdifferentiation emerged for a class of reaction networks under the condition of nutrientlimitation and strong cell-cell interactions. Then, robustness in the cell type distribution wasachieved, while instability of collective growth could emerge even among the cooperativecells when the internal reserves of products were dominant. The present mechanism issimple and general as a natural consequence of interacting cells with limited resources,and is consistent with the observed behaviors and forms of several aggregates of unicellularorganisms.
  9. Tomoki Kurikawa, Kunihiko Kaneko,
    Dynamic Organization of Hierarchical Memories,
    PLoS ONE 11, e0162640/1-19 (2016).
    In the brain, external objects are categorized in a hierarchical way. Although it is widelyaccepted that objects are represented as static attractors in neural state space, this viewdoes not take account interaction between intrinsic neural dynamics and external input,which is essential to understand how neural system responds to inputs. Indeed, structuredspontaneous neural activity without external inputs is known to exist, and its relationshipwith evoked activities is discussed. Then, how categorical representation is embedded intothe spontaneous and evoked activities has to be uncovered. To address this question, westudied bifurcation process with increasing input after hierarchically clustered associativememories are learned. We found a “dynamic categorization”; neural activity without inputwanders globally over the state space including all memories. Then with the increase ofinput strength, diffuse representation of higher category exhibits transitions to focused onesspecific to each object. The hierarchy of memories is embedded in the transition probabilityfrom one memory to another during the spontaneous dynamics. With increased inputstrength, neural activity wanders over a narrower state space including a smaller set ofmemories, showing more specific category or memory corresponding to the applied input.Moreover, such coarse-to-fine transitions are also observed temporally during transient processunder constant input, which agrees with experimental findings in the temporal cortex.These results suggest the hierarchy emerging through interaction with an external inputunderlies hierarchy during transient process, as well as in the spontaneous activity.
  10. Nen Saito, Yuki Sughiyama, and Kunihiko Kaneko,
    Motif analysis for small-number effects in chemical reaction dynamics,
    The Journal of Chemical Physics 145, 094111/1-7 (2016).
    The number of molecules involved in a cell or subcellular structure is sometimes rather small.In this situation, ordinary macroscopic-level fluctuations can be overwhelmed by non-negligible largefluctuations, which results in drastic changes in chemical-reaction dynamics and statistics comparedto those observed under a macroscopic system (i.e., with a large number of molecules). In order tounderstand how salient changes emerge from fluctuations in molecular number, we here quantitativelydefine small-number effect by focusing on a “mesoscopic” level, in which the concentration distributionis distinguishable both from micro- and macroscopic ones and propose a criterion for determiningwhether or not such an effect can emerge in a given chemical reaction network. Using the proposedcriterion, we systematically derive a list of motifs of chemical reaction networks that can showsmall-number effects, which includes motifs showing emergence of the power law and the bimodaldistribution observable in a mesoscopic regime with respect to molecule number. The list of motifsprovided herein is helpful in the search for candidates of biochemical reactions with a small-numbereffect for possible biological functions, as well as for designing a reaction system whose behavior canchange drastically depending on molecule number, rather than concentration.
  11. Yohei Saito, Yuki Sughiyama, Kunihiko Kaneko, and Tetsuya J. Kobayashi,
    Discreteness-induced transitions in multibody reaction systems,
    Physical Review E 94, 022140/1-8 (2016).
  12. Atsushi Kamimura, Kunihiko Kaneko,
    Negative scaling relationship between molecular diversity and resource abundances,
    Physical Review E 93, 062419/1-13 (2016).
    Cell reproduction involves replication of diverse molecule species, in contrast to a simple replication system with fewer components. To address this question of diversity, we study theoretically a cell system with catalytic reaction dynamics that grows by uptake of environmental resources. It is shown that limited resources lead to increased diversity of components within the system, and the number of coexisting species increases with a negative power of the resource uptake. The relationship is explained from the optimum growth speed of the cell, determined by a tradeoff between the utility of diverse resources and the concentration onto fewer components to increase the reaction rate.
  13. Benjamin Pfeuty and Kunihiko Kaneko,
    Requirements for efficient cell-type proportioning: regulatory timescales, stochasticity and lateral inhibition,
    Physical Biology 13, 026007/1-9 (2016).
    The proper functioning of multicellular organisms requires the robust establishment of preciseproportions between distinct cell types. This developmental differentiation process typically involvesintracellular regulatory and stochastic mechanisms to generate cell-fate diversity as well as intercellularsignaling mechanisms to coordinate cell-fate decisions at tissue level. We thus surmise that keyinsights about the developmental regulation of cell-type proportion can be captured by the modelingstudy of clustering dynamics in population of inhibitory-coupled noisy bistable systems. This generalclass of dynamical system is shown to exhibit a very stable two-cluster state, but also metastability,collective oscillations or noise-induced state hopping, which can prevent from timely and reliablyreaching a robust and well-proportioned clustered state. To circumvent these obstacles or to avoidfine-tuning, we highlight a general strategy based on dual-time positive feedback loops, such asmediated through transcriptional versus epigenetic mechanisms, which improves proportionregulation by coordinating early and flexible lineage priming with late and firm commitment. Thisresult sheds new light on the respective and cooperative roles of multiple regulatory feedback,stochasticity and lateral inhibition in developmental dynamics.
  14. Nobuto Takeuchi, Kunihiko Kaneko, Paulien Hogeweg,
    Evolutionarily stable disequilibrium: endless dynamics of evolution in a stationary population,
    Proceedings of the Royal Society B 283, 20153109 (2016).
    Evolution is often conceived as changes in the properties of a population over generations. Does this notion exhaust the possible dynamics of evolution? Life is hierarchically organized, and evolution can operate at multiple levels with conflicting tendencies. Using a minimal model of such conflicting multilevel evolution, we demonstrate the possibility of a novel mode of evolution that challenges the above notion: individuals ceaselessly modify their genetically inherited phenotype and fitness along their lines of descent, without involving apparent changes in the properties of the population. The model assumes a population of primitive cells (protocells, for short), each containing a population of replicating catalytic molecules. Protocells are selected towards maximizing the catalytic activity of internal molecules, whereas molecules tend to evolve towards minimizing it in order to maximize their relative fitness within a protocell. These conflicting evolutionary tendencies at different levels and genetic drift drive the lineages of protocells to oscillate endlessly between high and low intracellular catalytic activity, i.e. high and low fitness, along their lines of descent. This oscillation, however, occurs independently in different lineages, so that the population as a whole appears stationary. Therefore, ongoing evolution can be hidden behind an apparently stationary population owing to conflicting multilevel evolution.
  15. Yusuke Himeoka and Kunihiko Kaneko,
    Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism:consequence of anti-phase coupling between reaction flux and affinity,
    Physical Biology 13, 026002/1-12 (2016).
    Cells generally convert nutrient resources to products via energy transduction. Accordingly, thethermodynamic efficiency of this conversion process is one of the most essential characteristics ofliving organisms. However, although these processes occur under conditions of dynamicmetabolism, most studies of cellular thermodynamic efficiency have been restricted to examiningsteady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, wedevelop a simple model of metabolic reactions with anabolism–catabolism coupling catalyzed byenzymes. Through application of external oscillation in the enzyme abundances, the thermodynamicefficiency of metabolism was found to be improved. This result is in strong contrast withthat observed in the oscillatory input, in which the efficiency always decreased with oscillation. Thisimprovement was effectively achieved by separating the anabolic and catabolic reactions, whichtend to disequilibrate each other, and taking advantage of the temporal oscillations so that each ofthe antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillationbetween the reaction flux and chemical affinity through oscillation of enzyme abundances isessential. This improvement was also confirmed in a model capable of generating autonomousoscillations in enzyme abundances. Finally, the possible relevance of the improvement inthermodynamic efficiency is discussed with respect to the potential for manipulation of metabolicoscillations in microorganisms
  16. 高井啓, 岡田康志,
    3色の高輝度発光タンパク質プローブの開発と応用,
    Journal of Japanese Biochemical Society 88(5), 669-673 (2016).
  17. 岡田康志,
    超解像蛍光顕微鏡法の現状と生体イメージングへの応用,
    レーザー研究 44(10), 643-647 (2016).
  18. 岡田康志,
    超解像蛍光顕微鏡,
    Clinical Neuroscience 34(6), 638-642 (2016).
  19. Takumi Washio, Seine A. Shintani, Hideo Higuchi and Toshiaki Hisada,
    Analysis of spontaneous oscillations for a three state power stroke model,
    Physical Review E 95, 022411 (2017).
  20. Morito Sakuma, Sayaka Kita, and Hideo Higuchi,
    Quantitative evaluation of malignant gliomas damage induced by photoactivation of IR700 dye,
    Science and Technology of Advanced Materials 22, 473-482 (2016).
  21. 樋口秀男,
    生物物理学と他の領域をゆらぐ,
    理学部ニュース 48, 08 (2017).
  22. Gaigai Yu, Hiroyuki Onodera, Yuki Aono, Fuun Kawano, Yoshibumi Ueda, Akihiro Furuya, Hideyuki Suzuki, and *Moritoshi Sato,
    Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems,
    Scientific Reports 6, 35777 (2016).
    Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Herewe report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More speci cally, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two di erent classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to di erent kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.
  23. Fuun Kawano, Risako Okazaki, Masayuki Yazawa, and *Moritoshi Sato,
    A photoactivatable Cre–loxP recombination system for optogenetic genome engineering,
    Nature Chemical Biology 12, 1059-1064 (2016).
    Genome engineering techniques represented by the Cre–loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engi- neering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illu- mination (~0.04 W m−2) or short periods of pulsed illumination (~30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The pres- ent PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.
  24. Keiji Fushimi, Takahiro Nakajima, Yuki Aono, Tatsuro Yamamoto, Ni-Ni-Win, Masahiko Ikeuchi, Moritoshi Sato and *Rei Narikawa,
    Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin,
    Frontiers in Microbiology 7, 588 (2016).
    Cyanobacteriochromes (CBCRs) are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB) and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV). The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2) from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near- infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.
  25. *佐藤守俊,
    CRISPR-Cas9システムの光操作技術,
    実験医学増刊号「All about ゲノム編集」 34, 47-52 (2016).
    光を使って生命現象を光で「操る」ことができるとしたら,ライフサイエンスや医療はどうなるだろう?例えば,細胞内シグナル伝達を光で操作できるようになれば,代謝,分泌,細胞増殖,細胞分化,細胞死等の生命機能を自由自在にコントロールできるようになるかもしれない.ゲノムの塩基配列を光で自由自在に書き換えたり,遺伝子のはたらきを自由自在に制御できるようになったらどうだろう?光が得意とする高い時間・空間制御能をもってすれば,狙ったtime windowのみで,狙った生体部位のみで,様々な生命機能や疾患をコントロールできるかもしれない.本稿では,このような未来の実現を目指した筆者らの研究の一端を紹介する.
  26. Takasho Nozoe, Edo Kussell, and *Yuichi Wakamoto,
    Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data,
    PLoS Genetics, in press.
    Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells' response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.
  27. Mikihiro Hashimoto, Takashi Nozoe, Hidenori Nakaoka, Reiko Okura, Sayo Akiyoshi, Kunihiko Kaneko, Edo Kussell, and *Yuichi Wakamoto,
    Noise-driven growth rate gain in clonal cellular populations,
    PNAS 113, 3251-3256 (2016).
    Cellular populations in both nature and the lab are comprised of phenotypically heterogeneous individuals that compete with each other resulting in complex population dynamics. Predicting population growth characteristics based on knowledge of heterogeneous single-cell dynamics remains challenging. By observing groups of cells for hundreds of generations at single-cell resolution, we reveal that growth noise causes clonal populations of Escherichia coli to double faster than the mean doubling time of their constituent single cells across a broad set of balanced-growth conditions. We show that the population-level growth rate gain as well as age structures of populations and of cell lineages in competition are predictable. Furthermore, we theoretically reveal that the growth rate gain can be linked with the relative entropy of lineage generation time distributions. Unexpectedly, we find an empirical linear relation between the means and the variances of generation times across conditions, which provides a general constraint on maximal growth rates. Together, these results demonstrate a fundamental benefit of noise for population growth, and identify a growth law that sets a ‘speed limit’ for proliferation.
  28. Maroš Pleška, Long Qian, Reiko Okura, Tobias Bergmiller, Yuichi Wakamoto, Edo Kussell, and *Călin C. Guet,
    Bacterial autoimmunity due to a restriction-modification system,
    Current Biology 26, 404-409 (2016).
    Restriction-modification (RM) systems represent a minimal and ubiquitous biological system of self/non-self discrimination in prokaryotes, which protects hosts from exogenous DNA. The mechanism is based on the balance between methyltransferase (M) and cognate restriction endonuclease (R). M tags endogenous DNA as self by methylating short specific DNA sequences called restriction sites, whereas R recognizes unmethylated restriction sites as non-self and introduces a double-stranded DNA break. Restriction sites are significantly underrepresented in prokaryotic genomes, suggesting that the discrimination mechanism is imperfect and occasionally leads to autoimmunity due to self-DNA cleavage (self-restriction). Furthermore, RM systems can promote DNA recombination and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution. However, cleavage of self-DNA by RM systems as elements shaping prokaryotic genomes has not been directly detected, and its cause, frequency, and outcome are unknown. We quantify self-restriction caused by two RM systems of Escherichia coli and find that, in agreement with levels of restriction site avoidance, EcoRI, but not EcoRV, cleaves self-DNA at a measurable rate. Self-restriction is a stochastic process, which temporarily induces the SOS response, and is followed by DNA repair, maintaining cell viability. We find that RM systems with higher restriction efficiency against bacteriophage infections exhibit a higher rate of self-restriction, and that this rate can be further increased by stochastic imbalance between R and M. Our results identify molecular noise in RM systems as a factor shaping prokaryotic genomes.
  29. 中岡秀憲, 梅谷実樹, *若本祐一,
    1細胞レベルの環境応答と選択の計測,
    化学と生物, in press.
    バクテリアは様々なストレス環境を生き抜くことができるが、その生存戦略を理解するためには、1細胞レベルで起こる応答や選択の詳細を知る必要がある。本稿では「パーシスタンス現象」を例に、1細胞計測によりもたらされた新たな知見を紹介する。
  30. Ayako Uno, Ryohei Nakamura, Tatsuya Tsukahara, Wei Qu, Sumio Sugano, Yutaka Suzuki, Shinichi Morishita, and Hiroyuki Takeda,
    Comparative analysis of genome and epigenome in the closely related medaka species identifies conserved sequence preferences for DNA hypomethylated domains,
    Zoological Science 33, 368-365 (2016).
  31. Ryohei Seki, Cai Li, Qi Fang, Shinichi Hayashi, Shiro Egawa, Jiang Hu, Luohao Xu, Hailin Pan, Mao Kondo, Tomohiko Sato, Haruka Matsubara, Namiko Kamiyama, Keiichi Kitajima, Daisuke Saito, Yang Liu, M. Thomas P. Gilbert, Qi Zhou, Xing Xu, Toshihiko Shiroishi, Naoki Irie*, Koji Tamura* & Guojie Zhang*,
    Functional roles of aves class-specific cis-regulatory elements on macroevolution of bird-specific features,
    Nature Communications 8, 14229 (2017).
    Unlike microevolutionary processes, little is known about the genetic basis of macroevolutionary processes. One of these magnificent examples is the transition from non-avian dinosaurs to birds that has created numerous evolutionary innovations such as self-powered flight and its associated wings with flight feathers. By analysing 48 bird genomes, we identified millions of avian-specific highly conserved elements (ASHCEs) that predominantly (>99%) reside in non-coding regions. Many ASHCEs show differential histone modifications that may participate in regulation of limb development. Comparative embryonic gene expression analyses across tetrapod species suggest ASHCE-associated genes have unique roles in developing avian limbs. In particular, we demonstrate how the ASHCE driven avian-specific expression of gene Sim1 driven by ASHCE may be associated with the evolution and development of flight feathers. Together, these findings demonstrate regulatory roles of ASHCEs in the creation of avian-specific traits, and further highlight the importance of cis-regulatory rewiring during macroevolutionary changes.
  32. Takane Ozawa, Tomoko Mizuhara, Masataka Arata, Masakazu Shimada, Teruyuki Niimi, Kensuke Okada, *Yasukazu Okada, and *Kunihiro Ohta,
    Histone deacetylases control module-specific phenotypic plasticity in beetle weapons,
    PNAS 113, 15042–15047 (2016).
    Nutritional conditions during early development influence the plastic expression of adult phenotypes. Among several body modules of animals, the development of sexually selected exaggerated traits exhibits striking nutrition sensitivity, resulting in positive allometry and hypervariability distinct from other traits. Using de novo RNA sequencing and comprehensive RNA interference (RNAi) for epigenetic modifying factors, we found that histone deacetylases (HDACs) and polycomb group (PcG) proteins preferentially influence the size of mandibles (exaggerated male weapon) and demonstrate nutrition-dependent hypervariability in the broad-horned flour beetle, Gnatocerus cornutus. RNAi-mediated HDAC1 knockdown (KD) in G. cornutus larvae caused specific curtailment of mandibles in adults, whereas HDAC3 KD led to hypertrophy. Notably, these KDs conferred opposite effects on wing size, but little effect on the size of the core body and genital modules. PcG RNAi also reduced adult mandible size. These results suggest that the plastic development of exaggerated traits is controlled in a module-specific manner by HDACs.
  33. Koji Hashimoto, Kohei Kurosawa, Akiho Murayama, *Hidetaka Seo, and Kunihiro Ohta,
    B Cell-Based Seamless Engineering of Antibody Fc Domains,
    PLoS One 11, e0167232 (2016).
    Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.
  34. Naomichi Takemata and *Kunihiro Ohta,
    Role of non-coding RNA transcription around gene regulatory elements in transcription factor recruitment,
    RNA Biology 14, 1-5 (2016).
    Eukaryotic cells produce a variety of non-coding RNAs (ncRNAs), many of which have been shown to play pivotal roles in biological processes such as differentiation, maintenance of pluripotency of stem cells, and cellular response to various stresses. Genome-wide analyses have revealed that many ncRNAs are transcribed around regulatory DNA elements located proximal or distal to gene promoters, but their biological functions are largely unknown. Recently, it has been demonstrated in yeast and mouse that ncRNA transcription around gene promoters and enhancers facilitates DNA binding of transcription factors to their target sites. These results suggest universal roles of promoter/enhancer-associated ncRNAs in the recruitment of transcription factors to their binding sites.
  35. *Yoshito Hirata, *Arisa Oda, Kunihiro Ohta, and Kazuyuki Aihara,
    Three-dimensional reconstruction of single-cell chromosome structure using recurrence plots,
    Scientific Reports 6, 34982 (2016).
    Single-cell analysis of the three-dimensional (3D) chromosome structure can reveal cell-to-cell variability in genome activities. Here, we propose to apply recurrence plots, a mathematical method of nonlinear time series analysis, to reconstruct the 3D chromosome structure of a single cell based on information of chromosomal contacts from genome-wide chromosome conformation capture (Hi-C) data. This recurrence plot-based reconstruction (RPR) method enables rapid reconstruction of a unique structure in single cells, even from incomplete Hi-C information.
  36. Atsuko Miki, Josephine Galipon, Satoshi Sawai, Toshifumi Inada, and *Kunihiro Ohta,
    RNA decay systems enhance reciprocal switching of sense and antisense transcripts in response to glucose starvation,
    Genes to Cells 21, 1276-1289 (2016).
    Antisense RNA has emerged as a crucial regulator of opposite-strand protein-coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1-as) are expressed in glucose-rich conditions and anticorrelated with transcription of metabolic stress-induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation. We show that fbp1-as shares many features with mRNAs, such as a 5′-cap and poly(A)-tail, and that its decay partially depends upon Rrp6, a cofactor of the nuclear exosome complex involved in 3′–5′ degradation of RNA. Fluorescence in situ hybridization and polysome fractionation show that the majority of remaining fbp1-as localizes to the cytoplasm and binds to polyribosomes in glucose-rich conditions. Furthermore, fbp1-as and antisense RNA at other stress-responsive loci are promptly degraded via the cotranslational nonsense-mediated decay (NMD) pathway. These results suggest NMD may potentiate the swift disappearance of antisense RNAs in response to cellular stress.
  37. Naoyoshi Kumakura, Hiroka Otsuki, Masaru Ito, Mika Nomoto, Yasuomi Tada, Kunihiro Ohta, and *Yuichiro Watanabe,
    Arabidopsis AtRRP44 has ribonuclease activity that is required for cell viability,
    Plant Biotechnology 33, 77–85 (2016).
    The RNA exosome is a multiprotein complex responsible for 3′ to 5′ degradation and processing of various classes of RNAs in eukaryotes. Rrp44/Dis3 is the catalytic center of the RNA exosome in yeast and human. Previously, we identified Arabidopsis thaliana RRP44 (AtRRP44) as a single functional homolog of Rrp44/Dis3. Although AtRRP44 is potentially a catalytic center of the plant RNA exosome, the ribonuclease activity of AtRRP44 has not been tested. Here, we show that AtRRP44 has ribonuclease activity using in vitro translated recombinant proteins. Mutation of the aspartic acid residue D489 of AtRRP44 to asparagine (D489N) resulted in loss of ribonuclease activity, indicating that aspartic acid is at the active site. The wild-type AtRRP44 protein rescued the growth defect of Saccharomyces cerevisiae rrp44 mutants, but the D489N mutated AtRRP44 did not. This finding suggests that the ribonuclease activity of wild-type AtRRP44 is required for yeast cell viability. We also showed that AtRRP44 was highly expressed in organs experiencing active cell turnover, such as shoot apical meristem, root apical meristem, and lateral root primordium. Along with previous studies showing that loss of RRP44 in Arabidopsis is lethal, our results suggest that AtRRP44 has ribonuclease activity that is related to plant development.
  38. Risa Mitsumori, Tomoe Ohashi, Kazuto Kugou, Ayako Ichino ,Kei Taniguchi, Kunihiro Ohta, Hiroyuki Uchida, and *Masaya Oki,
    Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae,
    The Journal of Biochemistry 160, 11-17 (2016).
    In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.
  39. Naomichi Takemata, Arisa Oda, Takatomi Yamada, Josephine Galipon, Tomoichiro Miyoshi, Yutaka Suzuki, Sumio Sugano, Charles S. Hoffman, Kouji Hirota,and *Kunihiro Ohta,
    Local potentiation of stress-responsive genes by upstream noncoding transcription,
    Nucleic Acids Research 44 , 5174-5189 (2016).
    It has been postulated that a myriad of long noncoding RNAs (lncRNAs) contribute to gene regulation. In fission yeast, glucose starvation triggers lncRNA transcription across promoter regions of stress-responsive genes including fbp1 (fructose-1,6-bisphosphatase1). At the fbp1 promoter, this transcription promotes chromatin remodeling and fbp1 mRNA expression. Here, we demonstrate that such upstream noncoding transcription facilitates promoter association of the stress-responsive transcriptional activator Atf1 at the sites of transcription, leading to activation of the downstream stress genes. Genome-wide analyses revealed that ∼50 Atf1-binding sites show marked decrease in Atf1 occupancy when cells are treated with a transcription inhibitor. Most of these transcription-enhanced Atf1-binding sites are associated with stress-dependent induction of the adjacent mRNAs or lncRNAs, as observed in fbp1 These Atf1-binding sites exhibit low Atf1 occupancy and high histone density in glucose-rich conditions, and undergo dramatic changes in chromatin status after glucose depletion: enhanced Atf1 binding, histone eviction, and histone H3 acetylation. We also found that upstream transcripts bind to the Groucho-Tup1 type transcriptional corepressors Tup11 and Tup12, and locally antagonize their repressive functions on Atf1 binding. These results reveal a new mechanism in which upstream noncoding transcription locally magnifies the specific activation of stress-inducible genes via counteraction of corepressors.
  40. Sanki Tashiro, Tetsuya Handa, Atsushi Matsuda, Takuto Ban, Toru Takigawa, Kazumi Miyasato, Kojiro Ishii, Kazuto Kugou, Kunihiro Ohta, Yasushi Hiraoka, Hisao Masukata and *Junko Kanoh,
    Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing,
    Nature Communications 7, 10393 (2016).
    A chromosome is composed of structurally and functionally distinct domains. However, the molecular mechanisms underlying the formation of chromatin structure and the function of subtelomeres, the telomere-adjacent regions, remain obscure. Here we report the roles of the conserved centromeric protein Shugoshin 2 (Sgo2) in defining chromatin structure and functions of the subtelomeres in the fission yeast Schizosaccharomyces pombe. We show that Sgo2 localizes at the subtelomeres preferentially during G2 phase and is essential for the formation of a highly condensed subtelomeric chromatin body ‘knob’. Furthermore, the absence of Sgo2 leads to the derepression of the subtelomeric genes and premature DNA replication at the subtelomeric late origins. Thus, the subtelomeric specialized chromatin domain organized by Sgo2 represses both transcription and replication to ensure proper gene expression and replication timing.
  41. Atsuko Miki, Josephine Galipon, Satoshi Sawai, Toshifumi Inada, and Kunihiro Ohta,
    RNA decay systems enhance reciprocal switching of sense and antisense transcripts in response to glucose starvation. Genes to Cells,
    Genes to Cells 21, 1276-1289 (2016).
    Antisense RNA has emerged as a crucial regulator of opposite-strand protein-coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1-as) are expressed in glucose-rich conditions and anticorrelated with transcription of metabolic stress-induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation. We show that fbp1-as shares many features with mRNAs, such as a 5'-cap and poly(A)-tail, and that its decay partially depends upon Rrp6, a cofactor of the nuclear exosome complex involved in 3'-5' degradation of RNA. Fluorescence in situ hybridization and polysome fractionation show that the majority of remaining fbp1-as localizes to the cytoplasm and binds to polyribosomes in glucose-rich conditions. Furthermore, fbp1-as and antisense RNA at other stress-responsive loci are promptly degraded via the cotranslational nonsense-mediated decay (NMD) pathway. These results suggest NMD may potentiate the swift disappearance of antisense RNAs in response to cellular stress.
  42. Akihiko Nakajima, Motohiko Ishida, Taihei Fujimori, Yuichi Wakamoto, and Satoshi Sawai,
    The Microfluidic lighthouse: an omnidirectional gradient generator,
    Lab on a Chip 16, 4382-4394 (2016).
    Studies of chemotactic cell migration rely heavily on various assay systems designed to evaluate the ability of cells to move in response to attractant molecules. In particular, the development of microfluidics-based devices in recent years has made it possible to spatially distribute attractant molecules in graded profiles that are sufficiently stable and precise to test theoretical predictions regarding the accuracy and efficiency of chemotaxis and the underlying mechanism of stimulus perception. However, because the gradient is fixed in a direction orthogonal to the laminar flow and thus the chamber geometry, conventional devices are limited for the study of cell re-orientation to gradients that move or change directions. Here, we describe the development of a simple radially symmetric microfluidics device that can deliver laminar flow in 360°. A stimulant introduced either from the central inlet or by photo uncaging is focused into the laminar flow in a direction determined by the relative rate of regulated flow from multiple side channels. Schemes for flow regulation and an extended duplexed device were designed to generate and move gradients in desired orientations and speed, and then tested to steer cell migration of Dictyostelium and neutrophil-like HL60 cells. The device provided a high degree of freedom in the positioning and orientation of attractant gradients, and thus may serve as a versatile platform for studying cell migration, re-orientation, and steering.
  43. Satoshi Kuwana, Hiroshi Senoo, Satoshi Sawai, and Masashi Fukuzawa,
    A novel, lineage-primed prestalk cell subtype involved in the morphogenesis of D. discoideum,
    Developmental Biology 416, 286-299 (2016).
    Dictyostelium morphogenesis requires the tip, which acts as an organizer and conducts orchestrated cell movement and cell differentiation. At the slug stage the tip region contains prestalk A (pstA) cells, which are usually recognized by their expression of reporter constructs that utilize a fragment of the promoter of the ecmA gene. Here, using the promoter region of the o-methyl transferase 12 gene (omt12) to drive reporter expression, we demonstrate the presence, also within the pstA region, of a novel prestalk cell subtype: the pstVA cells. Surprisingly, a sub-population of the vegetative cells express a pstVA: GFP marker and, sort out to the tip, both when developing alone and when co-developed with an excess of unmarked cells. The development of such a purified GFP-marked population is greatly accelerated: by precocious cell aggregation and tip formation with accompanying precocious elevation of developmental gene transcription. We therefore suggest that the tip contains at least two prestalk cell subtypes: the developmentally-specified pstA cells and the lineage-primed pstVA cells. It is presumably the pstVA cells that play the dominant role in morphogenesis during the earlier stages of development. The basis for the lineage priming is, however, unclear because we can find no correlation between pstVA differentiation and nutrient status during growth or cell cycle position at the time of starvation, the two known determinants of probable cell fate.
  44. Fumihito Fukujin, Akihiko Nakajima, Nao Shimada, and Satoshi Sawai,
    Self-organization of chemoattractant waves in Dictyostelium depends on F-actin and cell–substrate adhesion,
    Journal of The Royal Society Interface 13 (2016).
    In the social amoeba Dictyostelium discoideum, travelling waves of extracellular cyclic adenosine monophosphate (cAMP) self-organize in cell populations and direct aggregation of individual cells to form multicellular fruiting bodies. In contrast to the large body of studies that addressed how movement of cells is determined by spatial and temporal cues encoded in the dynamic cAMP gradients, how cell mechanics affect the formation of a self-generated chemoattractant field has received less attention. Here, we show, by live cell imaging analysis, that the periodicity of the synchronized cAMP waves increases in cells treated with the actin inhibitor latrunculin. Detail analysis of the extracellular cAMP-induced transients of cytosolic cAMP (cAMP relay response) in well-isolated cells demonstrated that their amplitude and duration were markedly reduced in latrunculin-treated cells. Similarly, in cells strongly adhered to a poly-l-lysine-coated surface, the response was suppressed, and the periodicity of the population-level oscillations was markedly lengthened. Our results suggest that cortical F-actin is dispensable for the basic low amplitude relay response but essential for its full amplification and that this enhanced response is necessary to establish high-frequency signalling centres. The observed F-actin dependence may prevent aggregation centres from establishing in microenvironments that are incompatible with cell migration.
  45. Akihiko Nakajima, Satoshi Sawai,
    Dissecting Spatial and Temporal Sensing in Dictyostelium Chemotaxis Using a Wave Gradient Generator,
    Methods in Molecular Biology 1407, 107-122 (2016).
    External cues that dictate the direction of cell migration are likely dynamic during many biological processes such as embryonic development and wound healing. Until recently, how cells integrate spatial and temporal information to determine the direction of migration has remained elusive. In Dictyostelium discoideum, the chemoattractant cAMP that directs cell aggregation propagates as periodic waves. In light of the fact that any temporally evolving complex signals, in principle, can be expressed as a sum of sinusoidal functions with various frequencies, the Dictyostelium system serves as a minimal example, where the dynamic signal is in the simplest form of near sinusoidal wave with one dominant frequency. Here, we describe a method to emulate the traveling waves in a fluidics device. The text provides step-by-step instructions on the device setup and describes ways to analyze the acquired data. These include quantification of membrane translocation of fluorescently labeled proteins in individual Dictyostelium cells and estimation of exogenous cAMP profiles. The described approach has already helped decipher spatial and temporal aspects of chemotactic sensing in Dictyostelium. More specifically, it allowed one to discriminate the temporal and the spatial sensing aspects of directional sensing. With some modifications, one should be able to implement similar analysis in other cell types.
  46. Taro Ozaki, Kona Yamashita, Yuki Goto, Morito Shimomura, Shohei Hayashi, Shumpei Asamizu, Yoshinori Sugai, Haruo Ikeda, Hiroaki Suga, Hiroyasu Onaka,
    Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analoges expressed in vivo,
    Nature Communications 8, 14207 (2017).
    Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the 'flexible' in vitro translation system, referred to as the FIT-GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.
  47. S. A. K. Jongkees, S. Umemoto, Hiroaki Suga,
    Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides,
    Chemical Science 8, 1474-1481 (2017).
  48. Takayuki Katoh, Kenya Tajima, Hiroaki Suga,
    Consecutive Elongation of D-Amino Acids in Translation,
    Cell Chemical Biology 24, 46-54 (2017).
    Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond
  49. Naohiro Terasaka, Kazuki Futai, Takayuki Katoh, Hiroaki Suga,
    A human microRNA precursor binding to folic acid discovered by small RNA transcriptomic SELEX,
    RNA 22(12), 1918-1928 (2016).
    RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions.
  50. Yukiko Matsunaga, Nasir K. Bashiruddin, Yu Kitago, Junichi Takagi, Hiroaki Suga ,
    Allosteric inhibition of a semaphorin 4D receptor plexin B1 by a high affinity macrocyclic peptide,
    Cell Chemical Biology 23, 1341-1350 (2016).
  51. Yuki Goto, Hiroaki Suga,
    A post-translational cyclodehydratase, PatD, tolerates sequence variation in the C-terminal region of the substrate peptides,
    Chemistry Letters 45, 1247-1249 (2016).
  52. Kazuki Futai, Naohiro Terasaka, Takayuki Katoh, Hiroaki Suga ,
    tRid, an enabling method to isolate previously inaccessible small RNA fractions,
    Methods 106, 105-111 (2016).
    Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200 nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coll.
  53. Takayuki Katoh, Ingo Wohlgemuth, Masanobu Nagano, Marina V. Rodnina, and Hiroaki Suga,
    Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling,
    Nature Communications 7, 11657 (2016).
    The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA(Pro) is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA(Pro) isoacceptors and tRNA(fMet) in Escherichia coli, and the D-arm of tRNA(fMet) is essential for EF-P-induced acceleration of fMet-puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm
  54. Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, and Hiroaki Suga,
    Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes,
    Nature Chemistry 8(4), 317-325 (2016).
  55. *Taisuke Banno, Yuki Tanaka, Kouichi Asakura, *Taro Toyota,
    Self-propelled oil droplets and their morphological change to giant vesicles induced by a surfactant solution at low pH,
    Langmuir 32, 9591-9597 (2016).
  56. Taisuke Banno, Arisa Asami, Naoko Ueno, Hiroyuki Kitahata, Yuki Koyano, Kouichi Asakura, *Taro Toyota,
    Deformable self-propelled micro-object comprising underwater oil droplets,
    Scientific Reports 6, 31292 (2016).
  57. *Yuno Natsume, Taro Toyota,
    Appearance of crystalline pattern for colloidal particles encapsulated in giant vesicles,
    Transactions of the Materials Research Society of Japan 41, 147-149 (2016).
  58. Masahito Hayashi, Masayoshi Nishiyama, Yuki Kazayama, Taro Toyota, Yoshie Harada, *Kingo Takiguchi,
    Reversible morphological control of tubulin-encapsulating giant liposomes by hydrostatic pressure,
    Langmuir 32, 3794-3802 (2016).
  59. Yuki Kazayama, Tetsuhiko Teshima, Toshihisa Osaki, *Shoji Takeuchi, *Taro Toyota,
    Integrated microfluidic system for size-based selection and trapping of giant vesicles,
    Analytical Chemistry 88, 1111-1116 (2016).
  60. Kazuma Gotoh, Tomonori Nomoto, Taro Toyota, *Masanori Fujinami,
    Effects of halide ions on the acceptor phase in spontaneous chemical oscillations in donor/membrane/acceptor systems,
    Journal of Colloid and Interface Science 462, 351-358 (2016).
  61. Yuno Natsume, *Taro Toyota,
    Asymmetrical polyhedral configuration of giant vesicles induced by orderly array of encapsulated colloidal particles,
    PLOS ONE 11, e0146683 (2016).
  62. *豊田太郎, 風山祐輝, 大崎寿久, 竹内昌治,
    ジャイアントベシクルのダイナミクスと人工細胞型センサーへの展開,
    分析化学 65, 715-727 (2017).
  63. *豊田太郎, 本多智,
    合成両親媒性分子で創る細胞様システム,
    生物物理 56, 165-167 (2016).
  64. Tomoyuki Mano, *Jean-Baptiste Delfau, Junichiro Iwasawa, and Masaki Sano,
    Optimal run-and-tumble based transportation of a Janus particle with active steering,
    Proceedings of the National Academy of Sciences, in press (2017).
    Commanding the swimming of micrometric objects subjected to thermal agitation is always challenging both for artificial and living systems. Now, artificial swimmers can be designed whose self-propelling force can be tuned at will. However, orienting such small particles to an arbitrary direction requires counterbalancing the random rotational diffusion. Here, we introduce a simple concept to reorient artificial swimmers, granting them a motion similar to the run-and-tumbling behavior of Escherichia coli. We demonstrate it using Janus particles with asymmetric surface coating and moving under an AC electric field. Moreover, we determine the optimal strategy and compare it with biological swimmers. Our results encourage further investigation into dynamical behavior of colloidal particles, as well as application to microscopic devices.
  65. *Daiki Nishiguchi, Ken H. Nagai, Hugues Chate, and Masaki Sano,
    Long-range nematic order and anomalous fluctuations in suspensions of swimming filamentous bacteria,
    Physical Review E 95, 020601(R) /1-6 (2017).
    We study the collective dynamics of elongated swimmers in a very thin fluid layer by devising long, filamentous, non-tumbling bacteria. The strong confinement induces weak nematic alignment upon collision, which, for large enough density of cells, gives rise to global nematic order. This homogeneous but fluctuating phase, observed on the largest experimentally-accessible scale of millimeters,  exhibits the properties predicted by standard models for flocking such as the Vicsek-style model of polar particles  with nematic alignment: true long-range nematic order and non-trivial giant number fluctuations.
  66. *J.-B. Delfau, John J. Molina and M. Sano,
    Collective behavior of strongly confined suspensions of squirmers,
    Europhysics Letters 114, 24001/1-5 (2016).
    We run numerical simulations of strongly confined suspensions of model spherical swimmers called “squirmers”. Because of the confinement, the Stokeslet dipoles generated by the particles are quickly screened and the far-field flow is dominated by the source dipole for all the different kinds of squirmers. However, we show that the collective behavior of the suspension still depends on the self-propelling mechanism of the swimmers as polar states can only be observed for neutral squirmers. We demonstrate that the near-field hydrodynamic interactions play a crucial role in the alignment of the orientation vectors of spherical particles. Moreover, we point out thatthe enstrophy and the fluid fluctuations of an active suspension also depend on the nature of the squirmers.
  67. Tetsuya Hiraiwa, and Guillaume Salbreux,
    Role of Turnover in Active Stress Generation in a Filament Network,
    Physical Review Letters 116, 188101 (2016).
    We study the effect of turnover of cross-linkers, motors, and filaments on the generation of a contractile stress in a network of filaments connected by passive cross-linkers and subjected to the forces exerted by molecular motors. We perform numerical simulations where filaments are treated as rigid rods and molecular motors move fast compared to the time scale of an exchange of cross-linkers. We show that molecular motors create a contractile stress above a critical number of cross-linkers. When passive cross-linkers are allowed to turn over, the stress exerted by the network vanishes due to the formation of clusters. When both filaments and passive cross-linkers turn over, clustering is prevented and the network reaches a dynamic contractile steady state. A maximum stress is reached for an optimum ratio of the filament and cross-linker turnover rates. Taken together, our work reveals conditions for stress generation by molecular motors in a fluid isotropic network of rearranging filaments.
  68. *Takao Ohta, Mitsusuke Tarama and Masaki Sano,
    Simple model of cell crawling,
    Physica D 318-319, 3-11 (2016).
    Based on symmetry consideration of migration and shape deformations, we formulate phenomenologically the dynamics of cell crawling in two dimensions. Forces are introduced to change the cell shape. The shape deformations induce migration of the cell on a substrate. For time-independent forces we show that not only a stationary motion but also a limit cycle oscillation of the migration velocity and the shape occurs as a result of nonlinear coupling between different deformation modes. Time-dependent forces are generated in a stochastic manner by utilizing the so-called coherence resonance of an excitable system. The present coarse-grained model has a flexibility that it can be applied, e.g., both to keratocyte cells and to View the MathML source cells, which exhibit quite different dynamics from each other. The key factors for the motile behavior inherent in each cell type are identified in our model.
  69. *John J. Molina, Kotaro Otomura, Hayato Shiba, Hideki Kobayashi, Masaki Sano, and Ryoichi Yamamoto,
    Rheological evaluation of colloidal dispersions using the smoothed profile method: formulation and applications,
    Journal of Fluid Mechanics 792, 590-619 (2016).
    The smoothed profile method is extended to study the rheological behaviour of colloidal dispersions under shear flow by using the Lees–Edwards boundary conditions. We start with a reformulation of the smoothed profile method, a direct numerical simulation method for colloidal dispersions, so that it can be used with the Lees–Edwards boundary condition, under steady or oscillatory-shear flow. By this reformulation, all the resultant physical quantities, including local and total shear stresses, become available through direct calculation. Three simple rheological simulations are then performed for (1) a spherical particle, (2) a rigid bead chain and (3) a collision of two spherical particles under shear flow. Quantitative validity of these simulations is examined by comparing the viscosity with that obtained from theory and Stokesian dynamics calculations. Finally, we consider the shear-thinning behaviour of concentrated colloidal dispersions.
  70. *Masaki Sano and Keiichi Tamai,
    A Universal Transition to Turbulence in Channel Flow,
    Nature Physics 12, 249-253 (2016).
    Transition from laminar to turbulent flow drastically changes the mixing, transport, and drag properties of fluids, yet when and how turbulence emerges is elusive even for simple flow within pipes and rectangular channels1,2. Unlike the onset of temporal disorder, which is identified as the universal route to chaos in confined flows3,4, characterization of the onset of spatio-temporal disorder has been an outstanding challenge because turbulent domains irregularly decay or spread as they propagate downstream. Here, through extensive experimental investigation of channel flow, we identify a distinctive transition with critical behavior. Turbulent domains continuously injected from an inlet ultimately decayed, or in contrast, spread depending on flow rates. Near a transition point, critical behavior was observed. We investigate both spatial and temporal dynamics of turbulent clusters, measuring four critical exponents, a universal scaling function and a scaling relation, all in agreement with the (2+1) dimensional directed percolation universality class.
  71. *佐野雅己,
    現象のモデル化と数理モデルの普遍性,
    数理科学 631, 1-4 (2016).
    「数理モデルと普遍性」- モデル化で捉える自然現象の本質 -と題する特集を企画し、統計物理のモデルを中心に単純な数理モデルが自然現象の普遍性をみごとに表す場合があることを延べ、各記事の概要を紹介した。
  72. Masashi Fujii, Kaoru Ohashi, Yasuaki Karasawa, Minori Hikichi, *Shinya Kuroda,
    Small-volume effect enables robust, sensitive, and efficient information transfer in the spine,
    Biophysical Journal 112, 813-826 (2017).
    Why is the spine of a neuron so small that it can contain only small numbers of molecules and reactions inevitably become stochastic? We previously showed that, despite such noisy conditions, the spine exhibits robust, sensitive, and efficient features of information transfer using the probability of Ca2+ increase; however, the mechanisms are unknown. In this study, we show that the small volume effect enables robust, sensitive, and efficient information transfer in the spine volume, but not in the cell volume. In the spine volume, the intrinsic noise in reactions becomes larger than the extrinsic noise of input, resulting in robust information transfer despite input fluctuation. In the spine volume, stochasticity makes the Ca2+ increase occur with a lower intensity of input, causing higher sensitivity to lower intensity of input. The volume-dependency of information transfer increases its efficiency in the spine volume. Thus, we propose that the small-volume effect is the functional reason why the spine has to be so small.
  73. Hideki Terajima, *Hikari Yoshitane, Haruka Ozaki, Yutaka Suzuki,Shigeki Shimba, Shinya Kuroda, Wataru Iwasaki and *Yoshitaka Fukada,
    ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm,
    Nature Genetics 49, 146-151 (2017).
    It has been proposed that the CLOCK-ARNTL (BMAL1) complex drives circadian transcription of thousands of genes, including Per and Cry family genes that encode suppressors of CLOCK-ARNTL-dependent transcription. However, recent studies demonstrated that 70-80% of circadian-oscillating mRNAs have no obvious rhythms in their de novo transcription, indicating the potential importance of post-transcriptional regulation. Our CLOCK-ChIP-seq analysis identified rhythmic expression of adenosine deaminase, RNA-specific, B1 (Adarb1, also known as Adar2), an adenosine-to-inosine (A-to-I) RNA-editing enzyme. RNA-seq showed circadian rhythms of ADARB1-mediated A-to-I editing in a variety of transcripts. In Adarb1-knockout mice, rhythms of large populations of mRNA were attenuated, indicating a profound impact of ADARB1-mediated A-to-I editing on RNA rhythms. Furthermore, Adarb1-knockout mice exhibited short-period rhythms in locomotor activity and gene expression. These phenotypes were associated with abnormal accumulation of CRY2. The present study identifies A-to-I RNA editing as a key mechanism of post-transcriptional regulation in the circadian clockwork.
  74. *Katsuyuki Yugi,*Shinya Kuroda ,
    Metabolism-Centric Trans-Omics,
    Cell Systems 4, 19–20 (2017).
    Two recent studies in Cell and Science demonstrate the reconstruction of global mechanistic networks and identification of regulatory principles from multi-omics data.
  75. Takanori Sano, Kentaro Kawata, Satoshi Ohono, Katsuyuki Yugi, Hiroaki Kakuda,Hiroyuki Kubota, Shinsuke Uda, Masashi Fujii, Katsuyuki Kunida, Daisuke Hoshino,Atsushi Hatano, Yuki Ito, Miharu Sato, Yutaka Suzuki, Shinya Kuroda,
    Selective control of up-regulated and down-regulated genes by temporal patterns and doses of insulin,
    Science Signaling 9, ra112 (2016).
    Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the down-regulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii)transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness.
  76. Takamasa Kudo, *Shinsuke Uda, Takaho Tsuchiya, Takumi Wada, Yasuaki Karasawa, Masashi Fujii, Takeshi H. Saito, *Shinya Kuroda,
    Laguerre filter analysis with partial least square regression reveals a priming effect of ERK and CREB on c-FOS induction,
    PLoS ONE 11, e0160548 (2016).
    Signaling networks are made up of limited numbers of molecules and yet can code information that controls different cellular states through temporal patterns and a combination of signaling molecules. In this study, we used a data-driven modeling approach, the Laguerre filter with partial least square regression, to describe how temporal and combinatorial patterns of signaling molecules are decoded by their downstream targets. The Laguerre filter is a time series model used to represent a nonlinear system based on Volterra series expansion. Furthermore, with this approach, each component of the Volterra series expansion is expanded by Laguerre basis functions. We combined two approaches, application of a Laguerre filter and partial least squares (PLS) regression, and applied the combined approach to analysis of a signal transduction network. We applied the Laguerre filter with PLS regression to identify input and output (IO) relationships between MAP kinases and the products of immediate early genes (IEGs). We found that Laguerre filter with PLS regression performs better than Laguerre filter with ordinary regression for the reproduction of a time series of IEGs. Analysis of the nonlinear characteristics extracted using the Laguerre filter revealed a priming effect of ERK and CREB on c-FOS induction. Specifically, we found that the effects of a first pulse of ERK enhance the subsequent effects on c-FOS induction of treatment with a second pulse of ERK, a finding consistent with prior molecular biological knowledge. The variable importance of projections and output loadings in PLS regression predicted the upstream dependency of each IEG. Thus, a Laguerre filter with partial least square regression approach appears to be a powerful method to find the processing mechanism of temporal patterns and combination of signaling molecules by their downstream gene expression.
  77. Sarah Filippi, Chris P. Barnes, Paul D.W. Kirk, Takamasa Kudo, Katsuyuki Kunida, Siobhan S. McMahon, Takaho Tsuchiya, Takumi Wada, Shinya Kuroda, and *Michael P.H. Stumpf ,
    Robustness of MEK-ERK dynamics and origins of Cell-to-Cell variability in MAPK signaling,
    Cell Reports 15, 2524-2535 (2016).
    Cellular signaling processes can exhibit pronounced cell-to-cell variability in genetically identical cells. This affects how individual cells respond differentially to the same environmental stimulus. However, the origins of cell-to-cell variability in cellular signaling systems remain poorly understood. Here, we measure the dynamics of phosphorylated MEK and ERK across cell populations and quantify the levels of population heterogeneity over time using high-throughput image cytometry. We use a statistical modeling framework to show that extrinsic noise, particularly that from upstream MEK, is the dominant factor causing cell-to-cell variability in ERK phosphorylation, rather than stochasticity in the phosphorylation/dephosphorylation of ERK. We furthermore show that without extrinsic noise in the core module, variable (including noisy) signals would be faithfully reproduced downstream, but the within-module extrinsic variability distorts these signals and leads to a drastic reduction in the mutual information between incoming signal and ERK activity.
  78. Teruyuki Matsunaga, Hiroshi Kohsaka, and *Akinao Nose,
    Gap junction-mediated signaling from motor neurons regulates motor generation in the central circuits of larval Drosophila,
    The Jounal of Neuroscience 37(8), 2045-2060 (2017).
    In this study, we used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost in shaking-B2 (shakB2) or ogre2, gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggested shakB and ogre function in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input–output relationship among the component neurons in this system.SIGNIFICANCE STATEMENT Motor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the central pattern-generating circuits in larval Drosophila, providing novel insights into motor circuit control. The experimental system introduced in this study also presents a new approach for studying intersegmentally coordinated locomotion. Unlike traditional electrophysiology methods, this system enables the simultaneous recording and manipulation of populations of neurons that are genetically specified and span multiple segments.
  79. Eri Hasegawa, James W. Truman, and *Akinao Nose,
    Identification of excitatory premotor interneurons which regulate local muscle contraction during Drosophila larval locomotion,
    Scientific Reports 6, 30806 (2016).
    We use Drosophila larval locomotion as a model to elucidate the working principles of motor circuits. Larval locomotion is generated by rhythmic and sequential contractions of body-wall muscles from the posterior to anterior segments, which in turn are regulated by motor neurons present in the corresponding neuromeres. Motor neurons are known to receive both excitatory and inhibitory inputs, combined action of which likely regulates patterned motor activity during locomotion. Although recent studies identified candidate inhibitory premotor interneurons, the identity of premotor interneurons that provide excitatory drive to motor neurons during locomotion remains unknown. In this study, we searched for and identified two putative excitatory premotor interneurons in this system, termed CLI1 and CLI2 (cholinergic lateral interneuron 1 and 2). These neurons were segmentally arrayed and activated sequentially from the posterior to anterior segments during peristalsis. Consistent with their being excitatory premotor interneurons, the CLIs formed GRASP- and ChAT-positive putative synapses with motoneurons and were active just prior to motoneuronal firing in each segment. Moreover, local activation of CLI1s induced contraction of muscles in the corresponding body segments. Taken together, our results suggest that the CLIs directly activate motoneurons sequentially along the segments during larval locomotion.
  80. Lana Sinapayen, Atsushi Masumori, Takashi Ikegami,
    Learning by Stimulation Avoidance: A Principle to Control Spiking Neural Networks Dynamics,
    PLoS One 12(2), e0170388 (2017).
  81. Aleksandr Drozd, Olaf Witkowski, Satoshi Matsuoka, Takashi Ikegami,
    Critical Mass in the Emergence of Collective Intelligence: a Parallelized Simulation of Swarms in Noisy Environments,
    Artificial Life and Robotics 21, 317-323 (2016).
  82. Tim Taylor, Mark Bedau, Alastair Channon, David Ackley, Wolfgang Banzhaf, Guillaume Beslon, Emily Dolson, Tom Froese, Simon Hickinbotham, Takashi Ikegami, Barry McMullin, Norman Packard, Steen Rasmussen, Nathaniel Virgo, Eran Agmon, Edward Clark, Simon McGregor,Charles Ofria, Glen Ropella, Lee Spector, Kenneth O. Stanley, Adam Stanton, Christopher Timperley, Anya Vostinar, and Michael Wiser,
    Open-Ended Evolution: Perspectives from the OEE Workshop in York,
    Artificial Life 22, 408-423 (2016).
  83. Olaf Witkowski, Takashi Ikegami,
    Emergence of Swarming Behavior: Foraging Agents Evolve Collective Motion Based on Signaling,
    PLoS ONE 11(4), e0152756 (2016).
  84. Julien Hubert and Takashi Ikegami,
    How long did it last? Memorizing interval timings in a simple robotic task,
    Proceedings of the Artificial Life Conference 2016, 406-407 (2016).
  85. Martin Biehl, Takashi Ikegami, Daniel Polani,
    Towards information based spatiotemporal patterns as a foundation for agent representation in dynamical systems,
    Proceedings of the Artificial Life Conference 2016, 722-729 (2016).
  86. *Yoichi Kosodo, Taeko Suetsugu, Tetsuya J. Kobayashi, Fumio Matsuzaki,
    Systematic time-dependent visualization and quantitation of the neurogenic rate in brain organoids,
    Biochemical and Biophysical Research Communications 483, 94-100 (2017).
  87. *Yuki Sughiyama, Tetsuya J. Kobayaashi,
    Steady-state thermodynamics for population growth in fluctuating environments,
    Physical Review E 95, 012131 (2017).
  88. *Yohei Saito, Yuki Sughiyama, Kunihiko Kaneko, Tetsuya J. Kobayashi,
    Discreteness-induced transitions in multibody reaction systems,
    Physical Review E 94, 22140 (2016).
  89. *Yukinobu Arata, Michio Hiroshima, Chan-Gi Pack, Ravikrishna Ramanujam, Fumio Motegi, Kenichi Nakazato, Hitoshi Sawa, Tetsuya J. Kobayashi, Tatsuo Shibata, and Yasushi Sako,
    Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary,
    Cell Reports 16, 2156-2168 (2016).
  90. *Tetsuya J. Kobayashi, Ryo Yokota, Kazuyuki Aihara,
    Feedback Regulation and its Efficiency in Biochemical Networks,
    Journal of Statistical Physics 162, 1425-1449 (2016).
  91. *秋山泰身, 小林徹也,
    システム生物学と免疫系の自己-非自己識別,
    医学の歩み 259, 839-842 (2016).