Analysis of the singularity cells controlling the pattern formation of multicellular system

• Project Area: Singularity biology
• Project/Area Number: 18H05415
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: The University of Tokushima
• Principal Investigator: 堀川 一樹 徳島大学, 先端研究推進センター, 教授 (70420247)
• Co-Investigator(Kenkyū-buntansha): 竹本 龍也 徳島大学, 先端酵素学研究所, 教授 (30443899)
• Project Period (FY): 2018-06-29 – 2023-03-31
• Project Status: Granted (Fiscal Year 2022)
• Budget Amount: ¥128,960,000 (Direct Cost: ¥99,200,000、Indirect Cost: ¥29,760,000); Fiscal Year 2022: ¥25,870,000 (Direct Cost: ¥19,900,000、Indirect Cost: ¥5,970,000); Fiscal Year 2021: ¥26,910,000 (Direct Cost: ¥20,700,000、Indirect Cost: ¥6,210,000); Fiscal Year 2020: ¥25,480,000 (Direct Cost: ¥19,600,000、Indirect Cost: ¥5,880,000); Fiscal Year 2019: ¥24,050,000 (Direct Cost: ¥18,500,000、Indirect Cost: ¥5,550,000); Fiscal Year 2018: ¥26,650,000 (Direct Cost: ¥20,500,000、Indirect Cost: ¥6,150,000)
• Keywords: 自己組織化 / 生物物理 / cAMP / ゲノム編集 / 社会性アメーバ / 光メモリー / 細胞周期

Outline of Annual Research Achievements

これまでに1万細胞中10細胞ほどのリーダー細胞が信号伝達の起点として機能すること、これらは観察開始後4-6時間の間に出現することを見出している。これら少数個の細胞をイメージングにより同定するためのツールとして、超高親和性カルシウム指示薬のカラーバリアントの開発を試みた。具体的には、令和元年度に開発した緑色高感度カルシウムプローブ作成法を応用し、赤色並びに青色のカルシウムプローブであるR-GECOとB-GECOの改変を試みた。既存手法であるアミノ酸変異導入に加え、新たに分子全体の円順列変異体化を検討したところ、長鎖フレキシブルリンカーを用いた円順列変異体化により、従来は200 nM以上だった解離定数を30 nMまで向上させた超高親和性変異体を作成することに成功した。これら新規プローブを用いることで、従来は困難であった100 nM以下の細胞内Ca2+濃度変化を多色ライブイメージングできることに加え、cAMPやcGMPなど他のセカンドメッセンジャーとの同時多機能イメージングが可能になることを実証した。本技術を用いることで、1万細胞中10細胞ほどのリーダー細胞並びにフォロワー細胞を検出できるようになり、これらが信号伝達の起点として機能することを実証した。
また、細胞分裂回数カウンティングシステム開発のため、細胞周期特異的に発現変動する遺伝子と連動してゲノム編集機能が再構成されるシステムの構築を試みた。G1期特異的に加え、S/G2/M期それぞれで特異的に発現する分割Cas9系を構築することができた。

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

超高親和性カルシウムプローブや細胞分裂回数のメモリーモジュールはともにシンギュラリティ研究の推進に必須の技術である。さらにこれらの新規技術は、がん発生や神経変性の初期動態など、これまでは研究が困難だった未踏分野に対しても応用することが期待されることから、「順調に進捗している」と判断できる。

Strategy for Future Research Activity

これまで通り、総括班並びに各計画班と強固に連携し計画研究を遂行する。特に、総括班、A01-3班と共同でcAMATERASへの1細胞ピックアップ装置の実装を進め、リーダー細胞の分取と遺伝子解析を行う。取得されるscRNAseqデータをA02各班と共同で解析する。1細胞トラッキングの自動化を可能にするため、公募班との連携も進める。

Research Products

• [Journal Article] C-type Natriuretic Peptide?induced PKA Activation Promotes Endochondral Bone Formation in Hypertrophic Chondrocytes (2022)
  • Author(s): Hirota Keisho、Hirashima Tsuyoshi、Horikawa Kazuki、Yasoda Akihiro、Matsuda Michiyuki
  • Journal Title: Endocrinology Volume: 163 Pages: -
  • DOI: 10.1210/endocr/bqac005
  • Peer Reviewed / Open Access
• [Journal Article] Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka (2022)
  • Author(s): Beppu Kana、Tsutsumi Rie、Ansai Satoshi、Ochiai Nana、Terakawa Mai、Mori Marie、Kuroda Masashi、Horikawa Kazuki、Tomoi Takumi、Sakamoto Joe、Kamei Yasuhiro、Naruse Kiyoshi、Sakaue Hiroshi
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 601 Pages: 65～72
  • DOI: 10.1016/j.bbrc.2022.02.082
  • Peer Reviewed
• [Journal Article] PD-1 agonism by anti-CD80 inhibits T cell activation and alleviates autoimmunity (2022)
  • Author(s): Sugiura Daisuke、Okazaki Il-mi、Maeda Takeo K.、Maruhashi Takumi、Shimizu Kenji、Arakaki Rieko、Takemoto Tatsuya、Ishimaru Naozumi、Okazaki Taku
  • Journal Title: Nature Immunology Volume: 23 Pages: 399～410
  • DOI: 10.1038/s41590-021-01125-7
• [Journal Article] JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes (2021)
  • Author(s): Sakane Ayuko、Yano Taka-aki、Uchihashi Takayuki、Horikawa Kazuki、Hara Yusuke、Imoto Issei、Kurisu Shusaku、Yamada Hiroshi、Takei Kohji、Sasaki Takuya
  • Journal Title: Communications Biology Volume: 4 Pages: -
  • DOI: 10.1038/s42003-021-02080-7
  • Peer Reviewed / Open Access
• [Journal Article] FRET-Based Detection and Quantification of HIV-1 Virion Maturation (2021)
  • Author(s): Sarca Anamaria D.、Sardo Luca、Fukuda Hirofumi、Matsui Hiroyuki、Shirakawa Kotaro、Horikawa Kazuki、Takaori-Kondo Akifumi、Izumi Taisuke
  • Journal Title: Frontiers in Microbiology Volume: 12 Pages: -
  • DOI: 10.3389/fmicb.2021.647452
  • Peer Reviewed / Open Access
• [Journal Article] Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity (2021)
  • Author(s): Suzuki Akinobu Z.、Sakano Taichi、Sasaki Hirona、Watahiki Rei、Sone Masaki、Horikawa Kazuki、Furuta Toshiaki
  • Journal Title: Chemical Communications Volume: 57 Pages: 5630～5633
  • DOI: 10.1039/d1cc01405f
  • Peer Reviewed / Open Access
• [Journal Article] Exploring rare cellular activity in more than one million cells by a transscale scope (2021)
  • Author(s): Ichimura T.、Kakizuka T.、Horikawa K.、Seiriki K.、Kasai A.、Hashimoto H.、Fujita K.、Watanabe T. M.、Nagai T.
  • Journal Title: Scientific Reports Volume: 11 Pages: -
  • DOI: 10.1038/s41598-021-95930-7
  • Peer Reviewed / Open Access
• [Journal Article] Programmed death (PD)‐1/PD‐ligand 1 blockade mediates antiangiogenic effects by tumor‐derived CXCL10/11 as a potential predictive biomarker (2021)
  • Author(s): Mitsuhashi Atsushi、Kondoh Kensuke、Horikawa Kazuki、Koyama Kazuya、Nguyen Na Thi、Afroj Tania、Yoneda Hiroto、Otsuka Kenji、Ogino Hirokazu、Nokihara Hiroshi、Shinohara Tsutomu、Nishioka Yasuhiko
  • Journal Title: Cancer Science Volume: 112 Pages: 4853～4866
  • DOI: 10.1111/cas.15161
  • Peer Reviewed / Open Access
• [Journal Article] PD-1 preferentially inhibits the activation of low-affinity T cells (2021)
  • Author(s): Shimizu Kenji、Sugiura Daisuke、Okazaki Il-mi、Maruhashi Takumi、Takemoto Tatsuya、Okazaki Taku
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 118 Pages: e2107141118
  • DOI: 10.1073/pnas.2107141118
  • Open Access
• [Journal Article] The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function (2021)
  • Author(s): Hayashi Shinichi、Suzuki Hitomi、Takemoto Tatsuya
  • Journal Title: Developmental Biology Volume: 478 Pages: 155～162
  • DOI: 10.1016/j.ydbio.2021.07.006
• [Journal Article] An information-theoretic approach to infer the underlying interaction domain among elements from finite length trajectories in a noisy environment (2021)
  • Author(s): Basak Udoy S.、Sattari Sulimon、Hossain Md. Motaleb、Horikawa Kazuki、Komatsuzaki Tamiki
  • Journal Title: The Journal of Chemical Physics Volume: 154 Pages: -
  • DOI: 10.1063/5.0034467
  • Peer Reviewed
• [Journal Article] Transfer entropy dependent on distance among agents in quantifying leader-follower relationships (2021)
  • Author(s): Basak Udoy S.、Sattari Sulimon、Hossain Motaleb、Horikawa Kazuki、Komatsuzaki Tamiki
  • Journal Title: Biophysics and Physicobiology Volume: 18 Pages: 131～144
  • DOI: 10.2142/biophysico.bppb-v18.015
  • Peer Reviewed / Open Access
• [Journal Article] Inferring domain of interactions among particles from ensemble of trajectories (2020)
  • Author(s): Basak Udoy S.、Sattari Sulimon、Horikawa Kazuki、Komatsuzaki Tamiki
  • Journal Title: Physical Review E Volume: 102 Pages: -
  • DOI: 10.1103/physreve.102.012404
  • Peer Reviewed
• [Journal Article] Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2 (2020)
  • Author(s): Iida Hideaki、Furukawa Yoko、Teramoto Machiko、Suzuki Hitomi、Takemoto Tatsuya、Uchikawa Masanori、Kondoh Hisato
  • Journal Title: Genes to Cells Volume: 25 Pages: 242～256
  • DOI: 10.1111/gtc.12753
  • Peer Reviewed / Open Access
• [Journal Article] Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B‐ and Hirschsprung disease‐associated missense mutations of the RET gene (2020)
  • Author(s): Nakatani Taichi、Iwasaki Mitsuhiro、Yamamichi Atsuhiro、Yoshioka Yuta、Uesaka Toshihiro、Bitoh Yuko、Maeda Kosaku、Fukumoto Takumi、Takemoto Tatsuya、Enomoto Hideki
  • Journal Title: Development, Growth & Differentiation Volume: 62 Pages: 214～222
  • DOI: 10.1111/dgd.12664
  • Peer Reviewed / Open Access
• [Journal Article] Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2 (2020)
  • Author(s): Iida H, Furukawa Y, Teramoto M, Suzuki H, Takemoto T, Uchikawa M, Kondoh H.
  • Journal Title: Genes to Cells Volume: 25(4) Pages: 242-256
  • DOI: 10.1111 / gtc.12753
  • Peer Reviewed / Open Access
• [Journal Article] The absence of SOX2 in the anterior foregut alters the esophagus into trachea and bronchi in both epithelial and mesenchymal components (2020)
  • Author(s): Teramoto M, Sugawara R, Minegishi K, Uchikawa M, Takemoto T, Kuroiwa A, Ishii Y, Kondoh H.
  • Journal Title: Biology Open Volume: 9 (2) Pages: 558-566
  • DOI: 10.1242 / bio.048728
  • Peer Reviewed / Open Access
• [Journal Article] Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses (2019)
  • Author(s): Sugiura D, Maruhashi T, Okazaki I, Shimizu K, Maeda T, Takemoto T, Okazaki T.
  • Journal Title: Science Volume: 364 (6440) Pages: 558-566
  • DOI: 10.1126 / science.aav7062
  • Peer Reviewed / Open Access
• [Journal Article] Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage (2019)
  • Author(s): Yamamoto S, Uchida Y, Ohtani T, Nozaki E, Yin C, Gotoh Y, Yakushiji‐Kaminatsui N, Higashiyama T, Suzuki T, Takemoto T, Shiraishi Y, Kuroiwa A.
  • Journal Title: Development, growth & differentiation Volume: 61 (3) Pages: 228-251
  • DOI: 10.1111 / dgd.12601
  • Peer Reviewed / Open Access
• [Journal Article] Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation (2019)
  • Author(s): Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T
  • Journal Title: Anim Sci J Volume: 90(1) Pages: 55-61
  • DOI: 10.1111 / asj.13129
  • Peer Reviewed / Open Access
• [Journal Article] Cardiomyocytes cultured on mechanically compliant substrates, but not on conventional culture devices, exhibit prominent mitochondrial dysfunction due to reactive oxygen species and insulin resistance under high glucose (2018)
  • Author(s): Morishima M, Horikawa K, Funaki M
  • Journal Title: PLOS/ONE Volume: 13(8) Pages: e0201891
  • DOI: 10.1371 / journal.pone.0201891
  • Peer Reviewed / Open Access
• [Journal Article] Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation (2018)
  • Author(s): Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T
  • Journal Title: PLoS One Volume: 13(10) Pages: e0206360
  • DOI: 10.1371 / journal.pone.0206360
  • Peer Reviewed / Open Access
• [Journal Article] Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration (2018)
  • Author(s): Harada A, Maehara K, Ono Y, Taguchi H, Yoshioka K, Kitajima Y, Xie Y, Sato Y, Iwasaki T, “Nogami J, Okada S, Komatsu T, Semba Y, Takemoto T, Kimura H, Kurumizaka H, Ohkawa Y
  • Journal Title: Nat Commun Volume: 9(1) Pages: 1400-1400
  • DOI: 10.1038 / s41467-018-03845-1
  • Peer Reviewed / Open Access
• [Journal Article] Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos (2018)
  • Author(s): Nishio K, Tanihara F, Nguyen TV, Kunihara T, Nii M, Hirata M, Takemoto T, Otoi T
  • Journal Title: Reprod Domest Anim Volume: 53(2) Pages: 313-318
  • DOI: 10.1111 / rda.13106
  • Peer Reviewed / Open Access
• [Presentation] 興奮性多細胞システムのトランススケールイメージングで明らかになったらせん状信号波の形成機構 (2021)
  • Author(s): 堀川一樹
  • Organizer: 第60回日本生体医工学会・第36回日本生体磁気学会 合同大会
  • Invited
• [Presentation] Onset of the signaling wave at 10,000 cell population (2021)
  • Author(s): Kazuki Horikawa
  • Organizer: PacifiChem2021
  • Int'l Joint Research / Invited
• [Presentation] 大規模イメージングで明らかになった、cAMP信号波の螺旋コア形成機構 (2019)
  • Author(s): 堀川一樹
  • Organizer: 第42回日本分子生物学会
• [Presentation] “Singularity Biology : small elements change the function of the systems” (2019)
  • Author(s): Kazuki Horikawa
  • Organizer: ICSB2019
  • Int'l Joint Research
• [Presentation] Red fluorescent cAMP indicator with increased affinity and expanded dynamic range (2018)
  • Author(s): Kazuki Horikawa
  • Organizer: The 18th Annual Meeting of the Protein Science Society of Japan
• [Book] "Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically Modified Mice" in Electroporation Protocols (Methods in Molecular Biology) (2020)
  • Author(s): Tatsuya Takemoto
  • Total Pages: 6
  • Publisher: Humana Press
  • ISBN: 978-1-4939-9739-8
• [Book] 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 (2018)
  • Author(s): Kazuki Horikawa
  • Total Pages: 7
  • Publisher: Springer
  • ISBN: 978-981-13-2083-5
• [Book] 蛍光タンパク質ⅳ．cAMPプローブ, 生きてるものは全部観る！ イメージングの選び方・使い方100＋実験医学増刊 36 No.20 (2018)
  • Author(s): 堀川一樹
  • Total Pages: 2
  • Publisher: 羊土社
  • ISBN: 978-4-7581-0375-6