研究活動情報

• Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.
  Aya O Satoh; Yoichiro Fujioka; Sayaka Kashiwagi; Aiko Yoshida; Mari Fujioka; Hitoshi Sasajima; Asuka Nanbo; Maho Amano; Yusuke Ohba
  Cell reports, 42, 3, 112229, 112229, 2023年03月28日, ［国際誌］
  英語, 研究論文（学術雑誌）, Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.
• A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.
  Yoichiro Fujioka; Sayaka Kashiwagi; Aiko Yoshida; Aya O Satoh; Mari Fujioka; Maho Amano; Yohei Yamauchi; Yusuke Ohba
  Cell structure and function, 47, 1, 43, 53, 2022年06月25日, ［国内誌］
  英語, 研究論文（学術雑誌）, The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.
• Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins.
  Sayaka Kashiwagi; Yoichiro Fujioka; Aya O Satoh; Aiko Yoshida; Mari Fujioka; Prabha Nepal; Atsushi Tsuzuki; Ozora Aoki; Sarad Paudel; Hitoshi Sasajima; Yusuke Ohba
  Cell structure and function, 44, 2, 183, 194, 2019年12月26日, ［国内誌］
  英語, 研究論文（学術雑誌）, The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.Key words: fluorescent protein, organelle, fusion protein, mitochondria.
• Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.
  Sayaka Kashiwagi; Yoichiro Fujioka; Takeshi Kondo; Aya O Satoh; Aiko Yoshida; Mari Fujioka; Hitoshi Sasajima; Maho Amano; Takanori Teshima; Yusuke Ohba
  Cell structure and function, 44, 2, 195, 204, 2019年12月26日, ［国内誌］
  英語, 研究論文（学術雑誌）, The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.
• A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.
  Yoichiro Fujioka; Aya O Satoh; Kosui Horiuchi; Mari Fujioka; Kaori Tsutsumi; Junko Sasaki; Prabha Nepal; Sayaka Kashiwagi; Sarad Paudel; Shinya Nishide; Asuka Nanbo; Takehiko Sasaki; Yusuke Ohba
  Cell structure and function, 44, 1, 61, 74, 2019年04月25日, ［国内誌］
  英語, 研究論文（学術雑誌）, Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.
• A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.
  Yoichiro Fujioka; Shinya Nishide; Toyoyuki Ose; Tadaki Suzuki; Izumi Kato; Hideo Fukuhara; Mari Fujioka; Kosui Horiuchi; Aya O Satoh; Prabha Nepal; Sayaka Kashiwagi; Jing Wang; Mika Horiguchi; Yuko Sato; Sarad Paudel; Asuka Nanbo; Tadaaki Miyazaki; Hideki Hasegawa; Katsumi Maenaka; Yusuke Ohba
  Cell host & microbe, 23, 6, 809, 818, 2018年06月13日, ［国際誌］
  英語, 研究論文（学術雑誌）, Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.
• Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells.
  Mika Horiguchi; Mari Fujioka; Takeshi Kondo; Yoichiro Fujioka; Xinxin Li; Kosui Horiuchi; Aya O Satoh; Prabha Nepal; Shinya Nishide; Asuka Nanbo; Takanori Teshima; Yusuke Ohba
  Cell structure and function, 42, 1, 15, 26, 2017年02月02日, ［国内誌］
  英語, 研究論文（学術雑誌）, Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Förster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.
• Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers.
  Tamaki Yamada; Masumi Tsuda; Takanori Wagatsuma; Yoichiro Fujioka; Mari Fujioka; Aya O Satoh; Kosui Horiuchi; Shinya Nishide; Asuka Nanbo; Yasunori Totsuka; Hisashi Haga; Shinya Tanaka; Masanobu Shindoh; Yusuke Ohba
  Scientific reports, 6, 23545, 23545, 2016年03月24日, ［国際誌］
  英語, 研究論文（学術雑誌）, Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin β1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.
• Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase C.
  Takayuki Inuzuka; Yoichiro Fujioka; Masumi Tsuda; Mari Fujioka; Aya O Satoh; Kosui Horiuchi; Shinya Nishide; Asuka Nanbo; Shinya Tanaka; Yusuke Ohba
  Scientific reports, 6, 21613, 21613, 2016年02月09日, ［国際誌］
  英語, 研究論文（学術雑誌）, Angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R) signaling. However, the precise molecular mechanism of AT2R-mediated AT1R inhibition remains poorly understood. Here, we characterized the local and functional interaction of AT2R with AT1R. AT2R colocalized and formed a complex with AT1R at the plasma membrane, even in the absence of AII. Upon AII stimulation, the spatial arrangement of the complex was modulated, as confirmed by Förster resonance energy transfer (FRET) analysis, followed by AT2R internalization along with AT1R. AT2R internalization was specifically observed only in the presence of AT1R; AT2R alone could not be internalized. The AT1R-specific inhibitor losartan completely inhibited both the conformational change and the internalization of AT2R with AT1R, whereas the AT2R-specific inhibitor PD123319 partially hindered these phenomena, demonstrating that the activation of both receptors was indispensable for these effects. In addition, treatment with the protein kinase C (PKC) inhibitors inhibited the ligand-dependent accumulation of AT2R but not that of AT1R in the endosomes. A mutation in the putative phosphorylation sites of AT2R also abrogated the co-internalization of ATR2 with AT1R and the inhibitory effect of ATR2 on AT1R. These data suggest that AT2R inhibits ligand-induced AT1R signaling through the PKC-dependent pathway.

• ミトコンドリアポアタンパク質を介したミトコンドリア-エンドソーム間相互作用によるエンドサイトーシス制御機構
  佐藤絢; 藤岡容一朗; 笹島仁; 南保明日香; 大場雄介, 日本細胞生物学会大会(Web), 71st, 2019年
• FRETイメージングを利用した炎症反応の概日リズム解析
  西出真也; 藤岡容一朗; 堀内浩水; 佐藤絢; ネパール プラバ; 柏木彩花; 王セイ; 吉田藍子; パウデル サラド; 南保明日香; 大場雄介, 日本生理学雑誌(Web), 80, 1, 2018年
• アンジオテンシンII-アンジオテンシンII受容体シグナル伝達の時空間制御機構
  藤岡容一朗; 犬塚貴之; 藤岡真理; 佐藤絢; 堀内浩水; 西出真也; 南保明日香; 大場雄介, 日本生理学雑誌(Web), 79, 2, 2017年
• PI3K由来ペプチドによるインフルエンザウイルス感染の抑制
  佐藤絢; 藤岡容一朗; 堀内浩水; 藤岡真理; パウデル サラド; 西出真也; 南保明日香; 大場雄介, 日本細胞生物学会大会(Web), 69th, 2017年
• Ras-PI3K複合体によるエンドサイトーシスの制御因子の探索と機能解析
  堀内浩水; 藤岡容一朗; 佐藤絢; NEPAL Prabha; WANG Jing; 堀口美香; PAUDEL Sarad; 西出真也; 南保明日香; 小布施力史; 大場雄介, 日本細胞生物学会大会(Web), 69th, 2017年
• Ras-PI3K複合体の時空間制御を介したエンドサイトーシス調節因子の探索
  堀内浩水; 藤岡容一朗; 佐藤絢; NEPAL Prabha; 堀口美香; WANG Jing; 西出真也; 南保明日香; 小布施力史; 大場雄介, 日本細胞生物学会大会(Web), 68th, 2016年
• Ras-PI3Kシグナルを介したエンドサイトーシス制御機構におけるミトコンドリアポアタンパク質の機能解析
  佐藤絢; 藤岡容一朗; 堀内浩水; 西出真也; NEPAL Prabha; 堀口美香; WANG Jing; 南保明日香; 大場雄介, 日本細胞生物学会大会(Web), 68th, 2016年
• 蛍光バイオセンサーを用いた時計タンパク質BMAL1-CLOCKの細胞内動態の解析
  西出真也; 藤岡容一朗; 堀内浩水; 堀口美香; 佐藤絢; NEPAL Prabha; WANG Jing; 南保明日香; 大場雄介, 日本細胞生物学会大会(Web), 68th, 2016年
• インフルエンザウイルス宿主細胞侵入を制御する宿主側因子の同定
  藤岡容一朗; 西出真也; 佐藤絢; 堀内浩水; NEPAL Prabha; 堀口美香; WANG Sei; 南保明日香; 大場雄介, 日本細胞生物学会大会(Web), 68th, 2016年

• ミトコンドリア-エンドソーム間相互作用によるエンドソーム成熟化メカニズムの解明
  科学研究費助成事業
  2020年04月01日 - 2023年03月31日
  佐藤 絢
  申請者らはこれまでに、ミトコンドリアとエンドソームの相互作用がエンドソームの成熟化を促進すること、およびこの現象にはEGF刺激依存的なphosphoinositide 3-kinase (PI3K) とミトコンドリアポアタンパク質であるvoltage-dependent anion channel 2 (VDAC2) の結合が必要であるという予備データを得ている。しかし、VDAC2がどのようにして二つのオルガネラの相互作用を制御し、エンドソームの成熟化を促進するのか、その分子メカニズムは未知である。そこで本研究は、VDAC2-PI3K間結合を介したミトコンドリア-エンドソーム間相互作用によるエンドソーム成熟化促進の分子メカニズムの解明を目的とした。昨年度はこの成果を論文にまとめて投稿し、また、bioRxivにも論文を掲載した (doi: https://doi.org/10.1101/2021.01.18.427063)。当該年度は、VDAC2の物質透過性変異体が実際に物質透過が抑制されているかの検証や、適切なコントロールを置いた実験、オルガネラ間相互作用上でのVDAC2-PI3K間結合の可視化などを求める査読者からのコメントに対する実験を行った。ミトコンドリアのカルシウムイメージングによるVDAC2の物質透過性の検証や、split GFPを用いたVDAC2-PI3K間結合の可視化を検討し、得られた成果を論文に追加して再び投稿した。
  日本学術振興会, 若手研究, 北海道大学, 20K16115
• 蛍光偏光を用いた等方性分解能顕微鏡の開発と細胞膜動態の可視化
  科学研究費助成事業
  2019年10月07日 - 2022年03月31日
  大場 雄介; 藤岡 容一朗; 佐藤 絢
  緑色蛍光タンパク質の２箇所に膜局在配列を一つずつ付加することで細胞膜と蛍光タンパク質の発色団が常に直行する位置に固定される角度センサーを作製した。また、赤色蛍光タンパク質でも同様のセンサーを作製した。加えて、任意の角度の偏光での励起と発した光を偏光に基づき分光する全反射偏光蛍光顕微鏡をセットアップした。これらを用いて膜の角度の変化を蛍光強度の変化として捉えることができる手法を確立し、受容体依存性エンドサイトーシス超初期過程を可視化することに成功した。が確立できた。また、開発した全反射顕微鏡を用いて、インクレチンGLP-1がL細胞から分泌される瞬間を直接捉えることに世界で始めて成功した。
  日本学術振興会, 国際共同研究加速基金(国際共同研究強化(B)), 北海道大学, 19KK0198
• バイオイメージングによるエンドソーム―ミトコンドリア相互作用の生理的意義の解明
  科学研究費助成事業
  2015年04月01日 - 2017年03月31日
  大場 雄介; 南保 明日香; 西出 真也; 藤岡 容一朗; 藤岡 真理; 堀内 浩水; 佐藤 絢
  我々は低分子量Gタンパク質Rasと標的分子PI3K複合体がエンドソームに局在し、エンドサイトーシスとウイルス粒子取込みを制御することを報告した。しかし、Ras-PI3K複合体がエンドソームに局在する分子機構は不明である。本研究では上記現象の原因となるアミノ酸配列を同定した。またその結合タンパク質のスクリーニングによりミトコンドリアタンパク質を同定した。このミトコンドリアタンパク質のノックダウンはRas-PI3Kのエンドソーム移行、エンドサイトーシス、ミトコンドリア―エンドソーム相互作用を阻害したので、PI3Kとの結合がオルガネラ間相互作用を介してエンドサイトーシスを調節することが示唆された。
  日本学術振興会, 挑戦的萌芽研究, 北海道大学, 15K15023
• エンドサイトーシスと外来因子取込を制御する細胞内シグナル伝達マシナリー
  科学研究費助成事業
  2014年04月01日 - 2017年03月31日
  大場 雄介; 南保 明日香; 西出 真也; 藤岡 容一朗; 藤岡 真理; 堀内 浩水; 佐藤 絢
  エンドゾームは自ら積極的にシグナルを発することで多様な機能を発揮するプラットフォームである。しかし、生きた細胞でのダイナミクスと細胞機能との関連の詳細は不明な点が多い。本研究では、アンギオテンシンII（AII）2型受容体（AT2R）が、1型受容体（AT1R）シグナル伝達を負に調節するためには、AT1RとAT2Rのヘテロ二量体化とそのエンドサイトーシスによる内在化、さらに複合体内の受容体間分子配向変化が重要であることを見出した。また、この機能には両方の受容体の活性化が不可欠であること、プロテインキナーゼC（PKC）によるAT2RのC末端のセリン残基のリン酸化が重要であることも明らかにした。
  日本学術振興会, 基盤研究(B), 北海道大学, 26293041