Kazuki Horikawa : How Low Can You Go? The Numbers of Cells That Make Up Bodies: Large Numbers and Small Numbers, Minorities and Small Numbers from Molecules to Organisms in Biology-Toward a New Understanding of Biological Phenomena, pp.127-133,, Springer, Apr. 2018.
Kazuki Horikawa : 少数性生物学, Nippon-Hyoron-Sha Co.,Ltd., Tokyo, Mar. 2017.
Academic Paper (Judged Full Paper):
1.
Masaki Morishima, Pu Wang, Kosuke Horii, Kazuki Horikawa and Katsushige Ono : Channel Decline Caused by Saturated Fatty Acids via Both Free Fatty Acid Receptor 4-Dependent and -Independent Pathways in Cardiomyocytes., International Journal of Molecular Sciences, Vol.25, No.14, 2024.
(Summary)
channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.
Yusuke Hara, Aya Ichiraku, Tomoki Matsuda, Ayuko Sakane, Takuya Sasaki, Takeharu Nagai and Kazuki Horikawa : High-affinity tuning of single fluorescent protein-type indicators by flexible linker length optimization in topology mutant., Communications Biology, Vol.7, No.1, 2024.
(Summary)
dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.
Maiko Sakai, Kohta Ohnishi, Masashi Masuda, Erika Harumoto, Teppei Fukuda, Aika Ohnishi, Shunsuke Ishii, Hirokazu Ohminami, Hisami Yamanaka-Okumura, Kazuto Ohashi, Eisuke Itakura, Kazuki Horikawa, Shigenobu Yonemura, Taichi Hara and Yutaka Taketani : Modulations of the mTORC2-GATA3 axis by an isorhamnetin activated endosomal-lysosomal system of the J774.1 macrophage-like cell line., Journal of Clinical Biochemistry and Nutrition, Vol.75, No.1, 24-32, 2024.
(Summary)
The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.
(Keyword)
protein degradation / endocytosis / GATA3 / mTORC2 / isorhamnetin
Kazuki Horikawa and Tatsuya Takemoto : Analysis of the singularity cells controlling the pattern formation in multi-cellular systems, Biophysics and Physicobiology, Vol.21, e211001, 2024.
Udoy S. Basak, Sulimon Sattari, Md Motaleb Hossain, Kazuki Horikawa, Mikito Toda and Tamiki Komatsuzaki : Comparison of particle image velocimetry and the underlying agents dynamics in collectively moving self propelled particles., Scientific Reports, Vol.13, No.1, 12566, 2023.
(Summary)
Collective migration of cells is a fundamental behavior in biology. For the quantitative understanding of collective cell migration, live-cell imaging techniques have been used using e.g., phase contrast or fluorescence images. Particle tracking velocimetry (PTV) is a common recipe to quantify cell motility with those image data. However, the precise tracking of cells is not always feasible. Particle image velocimetry (PIV) is an alternative to PTV, corresponding to Eulerian picture of fluid dynamics, which derives the average velocity vector of an aggregate of cells. However, the accuracy of PIV in capturing the underlying cell motility and what values of the parameters should be chosen is not necessarily well characterized, especially for cells that do not adhere to a viscous flow. Here, we investigate the accuracy of PIV by generating images of simulated cells by the Vicsek model using trajectory data of agents at different noise levels. It was found, using an alignment score, that the direction of the PIV vectors coincides with the direction of nearby agents with appropriate choices of PIV parameters. PIV is found to accurately measure the underlying motion of individual agents for a wide range of noise level, and its condition is addressed.
(Keyword)
Rheology / Cell Movement / Hydrodynamics / Blood Flow Velocity
Kazuki Horikawa and Takeharu Nagai : Live Imaging of cAMP Signaling in D. discoideum Based on a Bioluminescent Indicator, Nano-lantern (cAMP)., Methods in Molecular Biology, Vol.2483, 231-240, 2022.
(Summary)
Bioluminescence imaging of cellular function is a promising strategy. It has advantages over fluorescence imaging such as high sensitivity, no phototoxicity or no autofluorescence, and compatibility to deep-tissue imaging or optogenetics. However, functional imaging of cellular signaling by bioluminescence is not so easy due to the limited availability of bright bioluminescent indicators.Here we describe a detailed strategy to detect cellular cAMP dynamics by using Nano-lantern (cAMP1.6), one of the brightest bioluminescent indicator for cAMP . Both induced and spontaneous cAMP signaling in social amoeba, with a large and small signal change, respectively, were imaged by this method.
Keisho Hirota, Tsuyoshi Hirashima, Kazuki Horikawa, Akihiro Yasoda and Michiyuki Matsuda : C-type Natriuretic Peptide-induced PKA Activation Promotes Endochondral Bone Formation in Hypertrophic Chondrocytes., Endocrinology, Vol.163, No.3, 2022.
(Summary)
Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.
(Keyword)
Animals / cell proliferation and differentiation / Cell Line / Chondrocytes / Cyclic AMP / Cyclic AMP-Dependent Protein Kinases / Cyclic GMP / Enzyme Activation / Fluorescence Resonance Energy Transfer / Growth Plate / knockout mice / Mice, Transgenic / Natriuretic Peptide, C-Type / Osteogenesis / signal transduction
Kana Beppu, Rie Tsutsumi, Satoshi Ansai, Nana Ochiai, Mai Terakawa, Marie Mori, Masashi Kuroda, Kazuki Horikawa, Takumi Tomoi, Joe Sakamoto, Yasuhiro Kamei, Kiyoshi Naruse and Hiroshi Sakaue : Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka., Biochemical and Biophysical Research Communications, Vol.601, 65-72, 2022.
(Summary)
Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.
(Keyword)
Animals / CRISPR-Cas Systems / Dysgeusia / gene expression / Green Fluorescent Proteins / Oryzias / quality of life / Taste
Atsushi Mitsuhashi, Kensuke Kondoh, Kazuki Horikawa, Kazuya Koyama, Thi Na Nguyen, Tania Afroj, Hiroto Yoneda, Kenji Otsuka, Hirokazu Ogino, Hiroshi Nokihara, Tsutomu Shinohara and Yasuhiko Nishioka : Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker., Cancer Science, Vol.112, No.12, 4853-4866, 2021.
(Summary)
Immune checkpoint inhibitor (ICI) programmed death (PD)-1/PD-ligand 1 (PD-L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti-PD-L1/PD-1 Ab in tumor angiogenesis. In syngeneic mouse models, anti-PD-L1 Ab inhibited tumor angiogenesis and induces net-like hypoxia only in ICI-sensitive cell lines. In tumor tissue and serum of ICI-sensitive cell line-bearing mice, interferon-γ (IFN-γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD-L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN-γ stimulation in tumor cell lines correlated with the sensitivity of PD-L1 blockade. The CXCL10/11 receptor CXCR3-neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD-L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti-PD-1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD-1/PD-L1 blockade and identify tumor-derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.
T Ichimura, T Kakizuka, Kazuki Horikawa, K Seiriki, A Kasai, H Hashimoto, K Fujita, M T Watanabe and T Nagai : Exploring rare cellular activity in more than one million cells by a transscale scope., Scientific Reports, Vol.11, No.1, 2021.
(Summary)
In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
Akinobu Z. Suzuki, Taichi Sakano, Hirona Sasaki, Rei Watahiki, Masaki Sone, Kazuki Horikawa and Toshiaki Furuta : Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity, Chemical Communications, Vol.57, No.46, 5630-5633, 2021.
(Summary)
We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.
Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.
Basak S. Udoy, Sattari Sulimon, Hossain Motaleb, Kazuki Horikawa and Komatsuzaki Tamiki : Transfer entropy dependent on distance among agents in quantifying leader-follower relationships, Biophysics and Physicobiology, Vol.18, 131-144, 2021.
(Summary)
Synchronized movement of (both unicellular and multicellular) systems can be observed almost everywhere. Understanding of how organisms are regulated to synchronized behavior is one of the challenging issues in the field of collective motion. It is hypothesized that one or a few agents in a group regulate(s) the dynamics of the whole collective, known as leader(s). The identification of the leader (influential) agent(s) is very crucial. This article reviews different mathematical models that represent different types of leadership. We focus on the improvement of the leader-follower classification problem. It was found using a simulation model that the use of interaction domain information significantly improves the leader-follower classification ability using both linear schemes and information-theoretic schemes for quantifying influence. This article also reviews different schemes that can be used to identify the interaction domain using the motion data of agents.
Anamaria D. Sarca, Luca Sardo, Hirofumi Fukuda, Hiroyuki Matsui, Kotaro Shirakawa, Kazuki Horikawa, Akifumi Takaori-Kondo and Taisuke Izumi : FRET-Based Detection and Quantification of HIV-1 Virion Maturation, Frontiers in Microbiology, Vol.12, 647452, 2021.
(Summary)
HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation , we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.
Basak S. Udoy, Sattari Sulimon, Hossain Motaleb Md., Kazuki Horikawa and Komatsuzaki Tamiki : An information-theoretic approach to infer the underlying interaction domain among elements from finite length trajectories in a noisy environment, The Journal of Chemical Physics, Vol.154, No.3, 034901, 2021.
(Summary)
Transfer entropy in information theory was recently demonstrated [Basak et al., Phys. Rev. E 102, 012404 (2020)] to enable us to elucidate the interaction domain among interacting elements solely from an ensemble of trajectories. Therefore, only pairs of elements whose distances are shorter than some distance variable, termed cutoff distance, are taken into account in the computation of transfer entropies. The prediction performance in capturing the underlying interaction domain is subject to the noise level exerted on the elements and the sufficiency of statistics of the interaction events. In this paper, the dependence of the prediction performance is scrutinized systematically on noise level and the length of trajectories by using a modified Vicsek model. The larger the noise level and the shorter the time length of trajectories, the more the derivative of average transfer entropy fluctuates, which makes the identification of the interaction domain in terms of the position of global minimum of the derivative of average transfer entropy difficult. A measure to quantify the degree of strong convexity at the coarse-grained level is proposed. It is shown that the convexity score scheme can identify the interaction distance fairly well even while the position of the global minimum of the derivative of average transfer entropy does not. We also derive an analytical model to explain the relationship between the interaction domain and the change in transfer entropy that supports our cutoff distance technique to elucidate the underlying interaction domain from trajectories.
Udoy S. Basak, Sulimon Sattari, Kazuki Horikawa and Tamiki Komatsuzaki : Inferring domain of interactions among particles from ensemble of trajectories, Physical Review E, Vol.102, No.1-1, 012404, 2020.
(Summary)
An information-theoretic scheme is proposed to estimate the underlying domain of interactions and the timescale of the interactions for many-particle systems. The crux is the application of transfer entropy which measures the amount of information transferred from one variable to another, and the introduction of a "cutoff distance variable" which specifies the distance within which pairs of particles are taken into account in the estimation of transfer entropy. The Vicsek model often studied as a metaphor of collectively moving animals is employed with introducing asymmetric interactions and an interaction timescale. Based on ensemble data of trajectories of the model system, it is shown that using the interaction domain significantly improves the performance of classification of leaders and followers compared to the approach without utilizing knowledge of the domain. Given an interaction timescale estimated from an ensemble of trajectories, the first derivative of transfer entropy averaged over the ensemble with respect to the cutoff distance is presented to serve as an indicator to infer the interaction domain. It is shown that transfer entropy is superior for inferring the interaction radius compared to cross correlation, hence resulting in a higher performance for inferring the leader-follower relationship. The effects of noise size exerted from environment and the ratio of the numbers of leader and follower on the classification performance are also discussed.
Yusaku Ohta, Toshiaki Furuta, Takeharu Nagai and Kazuki Horikawa : Red fluorescent cAMP indicator with increased affinity and expanded dynamic range., Scientific Reports, Vol.8, No.1, 1866, 2018.
(Summary)
cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit I and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (K = 0.3 M) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub- M concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.
Ayuko Sakane, Shin Yoshizawa, Masaomi Nishimura, Yuko Tsuchiya, Natsuki Matsushita, Kazuhisa Miyake, Kazuki Horikawa, Issei Imoto, Chiharu Mizuguchi, Hiroyuki Saito, Takato Ueno, Sachi Matsushita, Hisashi Haga, Shinji Deguchi, Kenji Mizuguchi, Hideo Yokota and Takuya Sasaki : Conformational plasticity of JRAB/MICAL-L2 provides "law and order" in collective cell migration., Molecular Biology of the Cell, Vol.27, No.20, 3095-3108, 2016.
(Summary)
In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration.
Yusaku Ohta, Takanori Kamagata, Asuka Mukai, Shinji Takada, Takeharu Nagai and Kazuki Horikawa : Nontrivial Effect of the Color-Exchange of a Donor/Acceptor Pair in the Engineering of Förster Resonance Energy Transfer (FRET)-Based Indicators., ACS Chemical Biology, Vol.11, No.7, 1816-1822, 2016.
(Summary)
Genetically encoded indicators driven by the Förster resonance energy transfer (FRET) mechanism are reliable tools for live imaging. While the properties of FRET-based indicators have been improved over the years, they often suffer from a poor dynamic range due to the lack of comprehensive understanding about how to apply an appropriate strategy to optimize the FRET parameters. One of the most successful optimizations is the incorporation of circularly permuted fluorescent proteins (cpFPs). To better understand the effects of this strategy, we systematically investigated the properties of the indicators by utilizing a set of FRET backbones consisting of native or one of the most effective cp variants (cp173FPs) with considerations of their order. As a result, the ordering of donor and acceptor FPs, which has been ignored in previous studies, was found to significantly affect the dynamic range of indicators. By utilizing these backbones, we succeeded in improving a cGMP indicator with 3.6-fold increased dynamic range and in generating an ultrasensitive cAMP indicator capable of environmental imaging, demonstrating the practical importance of the ordering of donors and acceptors in the engineering of FRET-based indicators.
Asuka Mukai, Aya Ichiraku and Kazuki Horikawa : Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum., BMC Biotechnology, Vol.16, 37, 2016.
(Summary)
Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation. Its haploid genome facilitates functional analysis of genes by a single round of mutagenesis including targeted disruption. Although the efficient generation of knockout strains based on an intrinsically high homologous recombination rate has been demonstrated, successful reports for knockin strains have been limited. As social amoeba has an exceptionally high adenine and thymine (A/T)-content, conventional plasmid-based vector construction has been constrained due to deleterious deletion in E. coli. We describe here a simple and efficient strategy to construct GFP-knockin cassettes by using a linear DNA cloning vector derived from N15 bacteriophage. This allows reliable handling of DNA fragments whose A/T-content may be as high as 85 %, and which cannot be cloned into a circular plasmid. By optimizing the length of recombination arms, we successfully generate GFP-knockin strains for five genes involved in cAMP signalling, including a triple-colour knockin strain. This robust strategy would be useful in handling DNA fragments with biased A/T-contents such as the genome of lower organisms and the promoter/terminator regions of higher organisms.
James Derek Hanson, Shingen Nakamura, Ryota Amachi, Masahiro Hiasa, Asuka Oda, Daisuke Tsuji, Kohji Itoh, Takeshi Harada, Kazuki Horikawa, Jumpei Teramachi, Hirokazu Miki, Toshio Matsumoto and Masahiro Abe : Effective impairment of myeloma cells and their progenitors by blockade of monocarboxylate transportation., Oncotarget, Vol.6, No.32, 33568-33586, 2015.
(Summary)
Cancer cells robustly expel lactate produced through enhanced glycolysis via monocarboxylate transporters (MCTs) and maintain alkaline intracellular pH. To develop a novel therapeutic strategy against multiple myeloma (MM), which still remains incurable, we explored the impact of perturbing a metabolism via inhibiting MCTs. All MM cells tested constitutively expressed MCT1 and MCT4, and most expressed MCT2. Lactate export was substantially suppressed to induce death along with lowering intracellular pH in MM cells by blockade of all three MCT molecules with -cyano-4-hydroxy cinnamate (CHC) or the MCT1 and MCT2 inhibitor AR-C155858 in combination with MCT4 knockdown, although only partially by knockdown of each MCT. CHC lowered intracellular pH and severely curtailed lactate secretion even when combined with metformin, which further lowered intracellular pH and enhanced cytotoxicity. Interestingly, an ambient acidic pH markedly enhanced CHC-mediated cytotoxicity, suggesting preferential targeting of MM cells in acidic MM bone lesions. Furthermore, treatment with CHC suppressed hexokinase II expression and ATP production to reduce side populations and colony formation. Finally, CHC caused downregulation of homing receptor CXCR4 and abrogated SDF-1-induced migration. Targeting tumor metabolism by MCT blockade therefore may become an effective therapeutic option for drug-resistant MM cells with elevated glycolysis.
Michihiro Nakamura, Koichiro Hayashi, Nakano Mutsuki, Kanadani Takafumi, Miyamoto Kazue, Toshinari Kori and Kazuki Horikawa : Identification of Polyethylene Glycol-Resistant Macrophages on Stealth Imaging In Vitro Using Fluorescent Organosilica Nanoparticles, ACS Nano, Vol.9, No.2, 1058-1071, 2015.
(Summary)
An in vitro imaging system to evaluate the stealth function of nanoparticles against mouse macrophages was established using fluorescent organosilica nanoparticles. Surface-functionalized organosilica nanoparticles with polyethylene glycol (PEG) were prepared by a one-step process, resulting in a brush-type PEG layer. A simultaneous dual-particle administration approach enabled us to evaluate the stealth function of nanoparticles with respect to single cells using time-lapse fluorescent microscopic imaging and flow cytometry analyses. Single-cell imaging and analysis revealed various patterns and kinetics of bare and PEGylated nanoparticle uptake. The PEGylated nanoparticles revealed a stealth function against most macrophages (PEG-sensitive macrophages); however, a stealth function against certain macrophages (PEG-insensitive macrophages) was not observed. We identified and characterized the PEG-resistant macrophages that could take up PEGylated nanoparticles at the same level as bare nanoparticles.
Kazuki Horikawa : Recent progress in the development of genetically encoded Ca2+ indicators., The Journal of Medical Investigation : JMI, Vol.62, No.1-2, 24-28, 2015.
(Summary)
Genetically encoded calcium indicators (GECIs) are powerful tools to monitor the dynamics of calcium ion (Ca(2+)) in living cells and organisms. With the help of GFP technology and DNA engineering, a dozen sets of GECIs have been developed so far. Their application has been widely extended into the analysis at the subcellular local, single and population of cell. In the past decades, GECIs have been dramatically improved in their performance and are becoming more and more useful for live imaging. In this review, the progress in the development of GECIs is discussed by introducing the history and emerging GECIs, which would help the selection of the appropriate GECI for a given application.
Kazunori Kanemaru, Hiroshi Sekiya, Ming Xu, Kaname Satoh, Nami Kitajima, Keitaro Yoshida, Yohei Okubo, Takuya Sasaki, Satoru Moritoh, Hidetoshi Hasuwa, Masaru Mimura, Kazuki Horikawa, Ko Matsui, Takeharu Nagai, Masamitsu Iino and Kenji F. Tanaka : In vivo visualization of subtle, transient, and local activity of astrocytes using an ultrasensitive Ca(2+) indicator., Cell Reports, Vol.8, No.1, 311-318, 2014.
(Summary)
Astrocytes generate local calcium (Ca(2+)) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca(2+) indicator yellow Cameleon-Nano 50 (YC-Nano50) in astrocytes. In these mice, we detected a unique pattern of Ca(2+) signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca(2+) signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology.
(Keyword)
Animals / Astrocytes / Calcium Signaling / Calcium-Binding Proteins / Fluorescence Resonance Energy Transfer / Mice / Somatosensory Cortex
Kazuki Horikawa and Takeharu Nagai : [Spatiotemporal regulation of Ca(2 +) dynamics in live cells revealed by Ca(2 +) imaging]., Clinical Calcium, Vol.23, No.4, 527-533, 2013.
(Summary)
Advance in the live imaging technology revealed that cells display a variety of Ca(2 +) dynamics, such as the wave, oscillation, blip, puff and spark. Accumulating evidences suggest that not only Ca(2 +) itself, but its spatio-temporal dynamics are the important information carrier in the physiological responses. Here, we will introduce the pattern of Ca(2 +) dynamics along with the latest version of high-performance Ca(2 +) probes.
Tomoki Matsuda, Kazuki Horikawa, Kenta Saito and Takeharu Nagai : Highlighted Ca2+ imaging with a genetically encoded 'caged' indicator., Scientific Reports, Vol.3, 1398, 2013.
(Summary)
Genetically encoded fluorescent indicators for bioimaging are powerful tools for visualizing biological phenomena in specified cell types or cellular compartments. However, available gene promoters or localization sequences are not applicable for visualizing all expression events. Furthermore, a visualization technique focusing on single cells or cellular compartments is required for characterizing specific cellular properties including individuality of cells in the cell population. To address these limitations, we developed a genetically encoded caged Ca(2+) indicator for which expression timing and location could be controlled. This indicator, PA-TNXL, comprises a Ca(2+)-binding protein and troponin between a photoactivatable FRET donor (PA-GFP) and a FRET quencher (dim variant of YFP). Ultraviolet irradiation activates the FRET Ca(2+) indicator. Using this indicator, we successfully imaged Ca(2+) dynamics in a given set of HeLa cells and cultured hippocampal neurons. This technology can be applied for developing other photoactivatable indicators, thereby opening a new area of biological research.
(Keyword)
calcium / Cells, Cultured / Fluorescence Resonance Energy Transfer / Green Fluorescent Proteins / HeLa Cells / Hippocampus / Humans / Recombinant Fusion Proteins / Troponin
Kohei Nishimura, Masamichi Ishiai, Kazuki Horikawa, Tatsuo Fukagawa, Minoru Takata, Haruhiko Takisawa and T Masato Kanemaki : Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks., Molecular Cell, Vol.47, No.4, 511-522, 2012.
(Summary)
DNA interstrand crosslinks (ICLs) are highly toxic lesions that stall the replication fork to initiate the repair process during the S phase of vertebrates. Proteins involved in Fanconi anemia (FA), nucleotide excision repair (NER), and translesion synthesis (TS) collaboratively lead to homologous recombination (HR) repair. However, it is not understood how ICL-induced HR repair is carried out and completed. Here, we showed that the replicative helicase-related Mcm family of proteins, Mcm8 and Mcm9, forms a complex required for HR repair induced by ICLs. Chicken DT40 cells lacking MCM8 or MCM9 are viable but highly sensitive to ICL-inducing agents, and exhibit more chromosome aberrations in the presence of mitomycin C compared with wild-type cells. During ICL repair, Mcm8 and Mcm9 form nuclear foci that partly colocalize with Rad51. Mcm8-9 works downstream of the FA and BRCA2/Rad51 pathways, and is required for HR that promotes sister chromatid exchanges, probably as a hexameric ATPase/helicase.
(Keyword)
Adenosine Triphosphatases / Animals / BRCA2 Protein / Cell Cycle Proteins / Cell Survival / Cells, Cultured / Chickens / Chromosome Aberrations / Cross-Linking Reagents / oxidative DNA damage / DNA Helicases / DNA Replication / DNA-Binding Proteins / Fanconi Anemia / Homologous Recombination / Mitomycin / Molecular Sequence Data / Recombinational DNA Repair / Sister Chromatid Exchange
The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.
Yoshiyuki Yamada, Takayuki Michikawa, Mitsuhiro Hashimoto, Kazuki Horikawa, Takeharu Nagai, Atsushi Miyawaki, Michael Häusser and Katsuhiko Mikoshiba : Quantitative comparison of genetically encoded Ca indicators in cortical pyramidal cells and cerebellar Purkinje cells., Frontiers in Cellular Neuroscience, Vol.5, 2011.
(Summary)
Genetically encoded Ca(2+) indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 ± 2°C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20 APs evoked at 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1-10 CSs at 10 Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide useful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano15 is suitable for detecting CSs.
We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.
(Keyword)
Animals / calcium / Dictyostelium discoideum / Fluorescent Dyes / Indicators and Reagents / knockout mice / Molecular Sequence Data / Neurons / Signal transduction of proinflammatory cytokine receptors / Zebrafish
Kenta Saito, Noriyuki Hatsugai, Kazuki Horikawa, Kentaro Kobayashi, Toru Matsu-Ura, Katsuhiko Mikoshiba and Takeharu Nagai : Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level., PLoS ONE, Vol.5, No.4, e9935, 2010.
(Summary)
The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+) imaging not only in single live cells but also in small living subjects.
Chie Satou, Yukiko Kimura, Tsunehiko Kohashi, Kazuki Horikawa, Hiroyuki Takeda, Yoichi Oda and Shin-ichi Higashijima : Functional role of a specialized class of spinal commissural inhibitory neurons during fast escapes in zebrafish., The Journal of Neuroscience, Vol.29, No.21, 6780-6793, 2009.
(Summary)
In teleost fish, the Mauthner (M) cell, a large reticulospinal neuron in the brainstem, triggers escape behavior. Spinal commissural inhibitory interneurons that are electrotonically excited by the M-axon have been identified, but the behavioral roles of these neurons have not yet been addressed. Here, we studied these neurons, named CoLo (commissural local), in larval zebrafish using an enhancer-trap line in which the entire population of CoLos was visualized by green fluorescent protein. CoLos were present at one cell per hemi-segment. Electrophysiological recordings showed that an M-spike evoked a spike in CoLos via electrotonic transmission and that CoLos made monosynaptic inhibitory connections onto contralateral primary motoneurons, consistent with the results in adult goldfish. We further showed that CoLos were active only during escapes. We examined the behavioral roles of CoLos by investigating escape behaviors in CoLo-ablated larvae. The results showed that the escape behaviors evoked by sound/vibration stimuli were often impaired with a reduced initial bend of the body, indicating that CoLos play important roles in initiating escapes. We obtained several lines of evidence that strongly suggested that the impaired escapes occurred during bilateral activation of the M-cells: in normal larvae, CoLo-mediated inhibitory circuits enable animals to perform escapes even in these occasions by silencing the output of the slightly delayed firing of the second M-cell. This study illustrates (1) a clear example of the behavioral role of a specialized class of interneurons and (2) the capacity of the spinal circuits to filter descending commands and thereby produce the appropriate behavior.
Wataru Tomosugi, Tomoki Matsuda, Tomomi Tani, Tomomi Nemoto, Ippei Kotera, Kenta Saito, Kazuki Horikawa and Takeharu Nagai : An ultramarine fluorescent protein with increased photostability and pH insensitivity., Nature Methods, Vol.6, No.5, 351-353, 2009.
(Summary)
We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca(2+) concentration and caspase-3 activation in the same apoptotic cells.
(Keyword)
Amino Acid Substitution / Animals / apoptosis / Calcium Signaling / Caspase 3 / Color / Dictyostelium discoideum / Escherichia coli / Fluorescence Resonance Energy Transfer / Green Fluorescent Proteins / HeLa Cells / Humans / Hydrogen-Ion Concentration / Luminescent Proteins / Molecular Sequence Data / Mutagenesis / Phagocytosis / Photobleaching / Protein Stability / Recombinant Fusion Proteins / Spectrometry, Fluorescence / Spectrophotometry, Ultraviolet / Transfection
Masashi Tanimoto, Yukiko Ota, Kazuki Horikawa and Yoichi Oda : Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish., The Journal of Neuroscience, Vol.29, No.9, 2762-2767, 2009.
(Summary)
Auditory perception in vertebrates depends on transduction of sound into neural signals in the inner ear hair cells (HCs) and on transmission of these signals to the brain through auditory (VIIIth) nerve afferents. To investigate the developmental acquisition of auditory inputs by the CNS, we have electrophysiologically and morphologically examined the process of acquisition of auditory responsiveness by zebrafish macular HCs and the Mauthner cells (M-cells) in vivo. The M-cells are a paired large reticulospinal neurons in the hindbrain; they receive direct inputs from the VIIIth nerve afferents and initiate an acoustic startle response. Whole-cell recordings from the M-cells showed that sound-evoked postsynaptic currents were first observed around 40 h postfertilization (hpf); during subsequent development, onset latency decreased and amplitude increased. The appearance and development of microphonic potentials in the inner ear coincided with those of the acoustic responses of the M-cell, whereas the functional auditory circuits from the macular HCs to the M-cell were already formed at 27 hpf. These results suggest that the functional maturation of inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by CNS neurons.
Saori Nagayoshi, Eriko Hayashi, Gembu Abe, Naoki Osato, Kazuhide Asakawa, Akihiro Urasaki, Kazuki Horikawa, Kazuho Ikeo, Hiroyuki Takeda and Koichi Kawakami : Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like., Development, Vol.135, No.1, 159-169, 2008.
(Summary)
Gene trap and enhancer trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an enhancer trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an enhancer trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene (synbl), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the Galpha(S) pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated enhancer trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate.
Kana Ishimatsu, Kazuki Horikawa and Hiroyuki Takeda : Coupling cellular oscillators: a mechanism that maintains synchrony against developmental noise in the segmentation clock., Developmental Dynamics, Vol.236, No.6, 1416-1421, 2007.
(Summary)
A unique feature of vertebrate segmentation is its strict periodicity, which is governed by the segmentation clock consisting of numerous cellular oscillators. These cellular oscillators, driven by a negative-feedback loop of Hairy transcription factor, are linked through Notch-dependent intercellular coupling and display the synchronous expression of clock genes. Combining our transplantation experiments in zebrafish with mathematical simulations, we review how the cellular oscillators maintain synchrony and form a robust system that is resistant to the effects of developmental noise such as stochastic gene expression and active cell proliferation. The accumulated evidence indicates that the segmentation clock behaves as a "coupled oscillators," a mechanism that also underlies the synchronous flashing seen in fireflies.
Hiroyuki Takeda and Kazuki Horikawa : [Basic principle of the segmentation clock: synchronization and robustness to noise]., Tanpakushitsu Kakusan Koso, Vol.52, No.3, 236-242, 2007.
Kazuki Horikawa, Kana Ishimatsu, Eiichi Yoshimoto, Shigeru Kondo and Hiroyuki Takeda : Noise-resistant and synchronized oscillation of the segmentation clock., Nature, Vol.441, No.7094, 719-723, 2006.
(Summary)
Periodic somite segmentation in vertebrate embryos is controlled by the 'segmentation clock', which consists of numerous cellular oscillators. Although the properties of a single oscillator, driven by a hairy negative-feedback loop, have been investigated, the system-level properties of the segmentation clock remain largely unknown. To explore these characteristics, we have examined the response of a normally oscillating clock in zebrafish to experimental stimuli using in vivo mosaic experiments and mathematical simulation. We demonstrate that the segmentation clock behaves as a coupled oscillator, by showing that Notch-dependent intercellular communication, the activity of which is regulated by the internal hairy oscillator, couples neighbouring cells to facilitate synchronized oscillation. Furthermore, the oscillation phase of individual oscillators fluctuates due to developmental noise such as stochastic gene expression and active cell proliferation. The intercellular coupling was found to have a crucial role in minimizing the effects of this noise to maintain coherent oscillation.
Kazuki Horikawa : PacifiChem2021, Honolulu, USA, Dec. 2021.
(Keyword)
transscale imaging
2.
Masaki Morishima, Kazuki Horikawa and Makoto Funaki : Mechanically Physiological Microenvironment Sensitizes Primary Cardiomyocytes to Glucotoxicity; New In Vitro Diabetic Heart Research Model, Diabetes, San Diego, Jun. 2017.
Takashi Uebanso, Takaaki Shimohata, Iba Hitomi, Nishimura Kazuya, Taniguchi Yuichi, Kazuki Horikawa, Mutsumi Nakahashi, Kazuaki Mawatari and Akira Takahashi : COMBINATION BETWEEN A FEW T3SS INJECTISOME AND A LOT EFFECTOR FOR KILLING HOST CELLS ON VIBRIO PARAHAEMOLYTICUS, 6th FEMS Microbiology Congress, Jun. 2015.
5.
Kazuki Horikawa : Japan Taiwan Joiunt symposium for Paradigm Innovation in Biology: novel strategy and thinking, Academia Sinica, Taipei, Taiwan, Jul. 2012.
Kazuki Horikawa : Red fluorescent cAMP indicator with increased affinity and expanded dynamic range, The 18th Annual Meeting of the Protein Science Society of Japan, Niigata, Japan,, Jun. 2018.
13.
Kazuki Horikawa : 1分子から個体レベルまでの多階層バイオイメージング, 公開シンポジウム 次世代バイオイメージングの研究の展望, Aug. 2017.
14.
Kazuki Horikawa : バイオイメージング拠点四国研究会, Nov. 2016.
15.
Kazuki Horikawa : 京都大学生命科学リトリート, Oct. 2016.
16.
Kazuki Horikawa : in vivo イメージング 研究会, Jul. 2016.
Et cetera, Workshop:
1.
Taishi Kakizuka, Yusuke Hara, Taro Ichimura, Takeharu Nagai and Kazuki Horikawa : 社会性アメーバにおける時空間自己組織化過程の多段階シンギュラリティ AMATERAS ver.1 を用いた定量トランススケール解析, 新学術領域研究「シンギュラリティ生物学」第5回領域会議, May 2021.
2.
Yusuke Hara, Kazuki Horikawa and Taishi Kakizuka : リエントリーからラセンへ:自発形成されるラセン波の起源, 新学術領域研究「シンギュラリティ生物学」第5回領域会議, May 2021.
3.
Kazuki Horikawa, Yusuke Hara and Taishi Kakizuka : AMATERAS観察で明らかになったラセン波の自発形成メカニズム, 新学術領域研究「シンギュラリティ生物学」第5回領域会議, May 2021.
Development of molecular probes and photo manipulation tools - Technologies for visualization and control of small number of biomolecules (Project/Area Number: 23115003 )
Spying minority in biological phenomena -Toward bridging dynamics between individual and ensemble processes- (Project/Area Number: 23115001 )
Development of multi-scale and functional imaging technique to quantify the fluctuation in intercellular signaling (Project/Area Number: 22770182 )