2018年10月, 第98回日本生理学会北海道地方会若手優秀発表賞　　　　　　　　　　　　　　　

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04月28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.Keywords: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.

Direct visualization of glucagon-like peptide-1 secretion by fluorescent fusion proteins.
Atsushi Tsuzuki, Yoichiro Fujioka, Aiko Yoshida, Sayaka Kashiwagi, Maho Amano, Tohru Hira, Akinobu Nakamura, Hideaki Miyoshi, Tatsuya Atsumi, Yusuke Ohba
Journal of diabetes investigation, 13, 7, 1134, 1139, 2022年04月04日, ［国内誌］
英語, 研究論文（学術雑誌）, Live-cell imaging with fluorescent proteins (FPs) is a powerful tool for investigating the exocytosis processes of hormones. However, the secretion process of glucagon-like peptide-1 (GLP-1) has not been visualized by FPs, which might be because tagging FPs inhibits GLP-1 synthesis through the post-translational processing from proglucagon. Here, we have developed FP-tagged GLP-1 by inserting FPs into the middle of GLP-1 and adding the proglucagon signal peptide. Confocal imaging confirmed that GLP-1 fused to FPs with high folding efficiency showed granular structure, in which secretory vesicle markers colocalized. The fluorescence intensity of FP in the culture supernatant from cells treated with KCl or forskolin was significantly increased compared with those from untreated cells. Furthermore, FP-tagged GLP-1 enables direct visualization of stimulation-dependent exocytosis of GLP-1 at a single granule resolution with total internal reflection fluorescence microscopy. FP-tagged GLP-1 might facilitate the screening of GLP-1 secretagogues and the discovery of new antidiabetic drugs.

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, ［査読有り］, ［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, ［査読有り］, ［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, ［査読有り］, ［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, ［査読有り］, ［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.

RANKLによるインテグリンα2の発現亢進はインテグリンβ2の細胞内輸送を介して細胞接着を亢進する
大場雄介, 山田珠希, 山田珠希, 山田珠希, 津田真寿美, 藤岡容一朗, 藤岡真理, 堀内浩水, 堀口美香, 佐藤絢, ネパール ブラバ, 王せい, 柏木彩花, 西出真也, 南保明日香, 芳賀永, 田中伸哉, 進藤正信, 日本生理学雑誌(Web), 79, 2, 49 (WEB ONLY), 49, 2017年05月
(一社)日本生理学会, 日本語

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定　　　　　　　　　　　　　　　
藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介, 日本細胞生物学会大会講演要旨集, 69回, 61, 61, 2017年05月
(一社)日本細胞生物学会, 日本語

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定
藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介, 日本細胞生物学会大会(Web), 69th, ROMBUNNO.T8‐04(P2‐028) (WEB ONLY), 61, 2017年05月
(一社)日本細胞生物学会, 日本語

新規Na/K測定法・操作法の開発によるエンドソーム機能制御メカニズムの解明
科学研究費助成事業
2022年04月01日 - 2025年03月31日
柏木 彩花
日本学術振興会, 若手研究, 北海道大学, 22K15362

Na,KポンプによるRas-PI3Kシグナル伝達の時空間的制御メカニズムの解明
科学研究費助成事業
2020年09月11日 - 2022年03月31日
柏木 彩花
すべての臓器に普遍的に発現するNa,K-ATPaseは細胞外物質の取り込み（エンドサイトーシス）を制御することが知られているが、その詳細なメカニズムは明らかでない。本研究はRas-phosphoinositide 3-kinase (PI3K)シグナル伝達が「いつ・どこで」起こるのかという時空間的制御の観点から、蛍光バイオイメージングの手法を用い、Na,K-ATPaseによるエンドサイトーシス制御メカニズムを解析した。その結果、エンドソーム上のNa,K-ATPaseがポンプ機能を介してRas-PI3K複合体のエンドソーム局在を抑制し、エンドサイトーシスを制御することが示唆された。
日本学術振興会, 研究活動スタート支援, 北海道大学, 20K22730