Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Physiology

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Physiology

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Profile and Settings

Profile and Settings

• Name (Japanese)

  Fujioka

• Name (Kana)

  Yoichiro

• Name

  201401048382456646

Alternate Names

http://kaken.nii.ac.jp/d/r/70597492.ja.html

Achievement

Research Interests

• 細胞生物   イメージング   エンドサイトーシス   シグナル伝達   インフルエンザウイルス   SARS-CoV-2   

Research Areas

• Life sciences / Molecular biology
• Life sciences / Cell biology
• Life sciences / Experimental pathology

Research Experience

• 2023/04 - Today 北海道大学大学院医学研究院細胞生理学教室 准教授
• 2018/02 - 2023/03 北海道大学大学院医学研究院細胞生理学教室 講師
• 2015/08 - 2018/02 北海道大学大学院医学研究院細胞生理学教室 助教
• 2014 Hokkaido University

Awards

• 2023/09 第10回ＩＲＭＡＩＬサイエンスグラント「横河電機賞」
  
• 2021/05 シンギュラリティ生物学第５回領域会議フラッシュトーク賞
  
• 2020/06 日本細胞生物学会 CFS Award 2019
   

  受賞者: 藤岡容一朗、佐藤絢
• 2019/12 北海道科学技術奨励賞
   

  受賞者: 藤岡容一朗
• 2019/09 第8回北海道癌談話会奨励賞
   

  受賞者: 藤岡容一朗
• 2019/03 「第37回北海道医学会研究奨励賞」
   

  受賞者: 藤岡容一朗
• 2019/03 平成３０年度北海道大学大学院医学研究院「優秀論文賞」
   

  受賞者: 藤岡容一朗
• 2019/02 平成30年度コニカミノルタ画像科学奨励賞
   

  受賞者: 藤岡容一朗
• 2015/03 第34回北海道大学大学院医学研究科・医学部 高桑榮松奨学基金奨励賞
   

  受賞者: 藤岡容一朗
• 2014/07 公益財団法人内藤記念科学振興財団 内藤コンファレンス ポスター賞
   

  受賞者: 藤岡容一朗
• 2014/06 第66回日本細胞生物学会 若手最優秀発表賞受賞
   

  受賞者: 藤岡容一朗
• 2014/03 北海道大学大学院医学研究科平成２５年度「優秀論文賞」
   

  受賞者: 藤岡容一朗

Published Papers

• Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.

  Yutaka Morishima, Masahito Kawabori, Kazuyoshi Yamazaki, Soichiro Takamiya, Sho Yamaguchi, Yo Nakahara, Hajime Senjo, Daigo Hashimoto, Sakiko Masuda, Yoichiro Fujioka, Yusuke Ohba, Yuki Mizuno, Yuji Kuge, Miki Fujimura

  International journal of molecular sciences 25 (4) 2024/02/18 

  Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.
• Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.

  Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi

  ACS applied materials & interfaces 16 (2) 2110 - 2119 2024/01/17 

  RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.
• Strength in numbers: Unleashing the potential of trans-scale scope AMATERAS for massive cell quantification

  Taro Ichimura, Taishi Kakizuka, Yuki Sato, Yoichiro Fujioka, Yusuke Ohba, Kazuki Horikawa, Takeharu Nagai

  Biophysics and Physicobiology 2024 [Refereed]
  
• Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles.

  Shunyi Li, Hiroki Toriumi, Daisuke Takahashi, Tomoko Kamasaki, Yoichiro Fujioka, Satoru Nagatoishi, Jinting Li, Yiwei Liu, Takanatsu Hosokawa, Kouhei Tsumoto, Yusuke Ohba, Yoshiki Katayama, Daisuke Murakami, Koji Hase, Takeshi Mori

  Biomaterials 303 122381 - 122381 2023/12 

  Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.
• Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

  Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Saori Suzuki, Marie Kato, Zannatul Ferdous, Hiromi Mouri, Kenji Shishido, Naoko Misawa, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Mai Suganami, Mika Chiba, Ryo Yoshimura, So Nakagawa, Jiaqi Wu, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Ayaka Sakamoto, Naoko Yasuhara, Takashi Irie, Ryoko Kawabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, Monira Begum, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Akatsuki Saito, Yuri L. Tanaka, Erika P. Butlertanaka, Maya Shofa, Kaori Tabata, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara

  Communications Biology 6 (1) 2023/07/24 

  Abstract The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.
• 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 2023/03/28 

  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.
• Stimulation of the mitochondrial calcium uniporter mitigates chronic heart failure-associated ventricular arrhythmia in mice.

  Hikaru Hagiwara, Masaya Watanabe, Yoichiro Fujioka, Takahide Kadosaka, Takuya Koizumi, Taro Koya, Motoki Nakao, Rui Kamada, Taro Temma, Kazufumi Okada, Jose Antonio Moreno, Ohyun Kwon, Hisakata Sabe, Yusuke Ohba, Toshihisa Anzai

  Heart rhythm 19 (10) 1725 - 1735 2022/10 

  BACKGROUND: An aberrant increase in the diastolic calcium concentration ([Ca2+]i) level is a hallmark of heart failure (HF) and the cause of delayed afterdepolarization and ventricular arrhythmia (VA). Although mitochondria play a role in regulating [Ca2+]i, whether they can compensate for the [Ca2+]i abnormality in ventricular myocytes is unknown. OBJECTIVE: We investigated whether enhanced Ca2+ uptake of mitochondria may compensate for an abnormal increase in the [Ca2+]i of ventricular myocytes in HF to effectively mitigate VA. METHODS: We used a HF mouse model, in which myocardial infarction was induced by permanent left anterior descending coronary artery ligation. The mitochondrial Ca2+ uniporter was stimulated by kaempferol. Ca2+ dynamics and membrane potential were measured using an epifluorescence microscope, a confocal microscope, and the perforated patch-clamp technique. VA was induced in the Langendorff-perfused hearts, and the hemodynamic parameters were measured using a microtip transducer catheter. RESULTS: Protein expression of the mitochondrial Ca2+ uniporter, as assessed by its subunit expression, did not change between HF and sham mice. Treatment of cardiomyocytes with kaempferol, isolated from HF mice at 28 days after coronary ligation, reduced the appearance of aberrant diastolic [Ca2+]i waves and sparks and spontaneous action potentials. Kaempferol effectively reduced the VA occurring in Langendorff-perfused hearts. Intravenous administration of kaempferol did not markedly affect the left ventricular hemodynamic parameters. CONCLUSION: The effects of kaempferol in HF of mice implied that mitochondria may have the potential to compensate for abnormal [Ca2+]i. Mechanisms involved in mitochondrial Ca2+ uptake may provide novel targets to treat HF-associated VA.
• 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 2022/07 

  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.
• 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 2022/06/25 

  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.
• Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses.

  Tomokazu Tamura, Shiho Torii, Kentaro Kajiwara, Itsuki Anzai, Yoichiro Fujioka, Kisho Noda, Shuhei Taguwa, Yuhei Morioka, Rigel Suzuki, Yuzy Fauzyah, Chikako Ono, Yusuke Ohba, Masato Okada, Takasuke Fukuhara, Yoshiharu Matsuura

  PLoS pathogens 18 (6) e1010593 - e1010593 2022/06/03 

  Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E^rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.
• Imaging technology that enables simultaneous visualization of weak interaction interfaces and cell responses

  Aiko Yoshida, Yoichiro Fujioka, Maho Amano, Yusuke Ohba

  Drug Delivery System 37 (2) 102 - 111 0913-5006 2022/03/25
• Schlafen family member 11 indicates favorable prognosis of patients with head and neck cancer following platinum-based chemoradiotherapy.

  Seijiro Hamada, Satoshi Kano, Junko Murai, Takayoshi Suzuki, Nayuta Tsushima, Takatsugu Mizumachi, Masanobu Suzuki, Tsuyoshi Takashima, Daiki Taniyama, Naoya Sakamoto, Yoichiro Fujioka, Yusuke Ohba, Akihiro Homma

  Frontiers in oncology 12 978875 - 978875 2022 

  Recently, Schlafen family member 11 (SLFN11) has been reported to increase the sensitivity of cancer cells to DNA-damaging agents, including platinum derivatives; thus, SLFN11 may be a predictive biomarker for platinum-based chemoradiotherapy (CRT). In this study, we examined whether SLFN11 expression was associated with the therapeutic outcome of platinum-based CRT in head and neck squamous cell carcinoma (HNSCC). We performed immunohistochemical analyses for SLFN11 expression in 161 HNSCC tissues from patients who had been administered cisplatin-based CRT and examined the correlation between SLFN11 expression and progression-free survival (PFS). Additionally, SLFN11 expression was examined in 10 paired samples obtained before and after CRT in patients with local failure. Furthermore, in vitro experiments were performed using several HNSCC cell lines and isogenic SLFN11-knockout cells to assess the association between SLFN11 expression and drug sensitivity. PFS was found to be significantly better in the SLFN11-positive group than in the SLFN11-negative group among the 161 patients (5-year PFS: 78.8% vs. 52.8%, respectively, p < 0.001). Similar results were observed for the PFS at each primary site. The percentage of SLFN11 positivity was lower in tumor samples from patients with local failure after CRT than that in the corresponding primary tumors before CRT in 8 of 10 cases. Results of the in vitro assay demonstrated that SLFN11-knockout cells exhibited reduced sensitivity to DNA-damaging agents but not to the non-DNA-damaging agent docetaxel. Our findings suggest that SLFN11 may serve as a potential biomarker for predicting the response of HNSCC patients to platinum-based CRT.
• A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

  Tadasuke Tsukiyama, Juqi Zou, Jihoon Kim, Shohei Ogamino, Yuki Shino, Takamasa Masuda, Alessandra Merenda, Masaki Matsumoto, Yoichiro Fujioka, Tomonori Hirose, Sayuri Terai, Hidehisa Takahashi, Tohru Ishitani, Keiichi I Nakayama, Yusuke Ohba, Bon-Kyoung Koo, Shigetsugu Hatakeyama

  Nature communications 11 (1) 4586 - 4586 2020/09/15 

  Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.
• Calcium Wave Promotes Cell Extrusion.

  Yasuto Takeuchi, Rika Narumi, Ryutaro Akiyama, Elisa Vitiello, Takanobu Shirai, Nobuyuki Tanimura, Keisuke Kuromiya, Susumu Ishikawa, Mihoko Kajita, Masazumi Tada, Yukinari Haraoka, Yuki Akieda, Tohru Ishitani, Yoichiro Fujioka, Yusuke Ohba, Sohei Yamada, Yoichiroh Hosokawa, Yusuke Toyama, Takaaki Matsui, Yasuyuki Fujita

  Current biology : CB 30 (4) 670 - 681 2020/02/24 [Refereed][Not invited]
   

  When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.
• 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 0386-7196 2019/12/26 [Refereed][Not invited]
   

  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 0386-7196 2019/12/26 [Refereed][Not invited]
   

  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 0386-7196 2019/04/25 [Refereed][Not invited]
   

  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.
• Antibody-free digital influenza virus counting based on neuraminidase activity.

  Kazuhito V Tabata, Yoshihiro Minagawa, Yuko Kawaguchi, Mana Ono, Yoshiki Moriizumi, Seiya Yamayoshi, Yoichiro Fujioka, Yusuke Ohba, Yoshihiro Kawaoka, Hiroyuki Noji

  Scientific reports 9 (1) 1067 - 1067 2019/01/31 [Refereed][Not invited]
   

  There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.
• 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 1931-3128 2018/06/13 [Refereed][Not invited]
   

  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.
• Dermoscopic evaluation for skin grafts after surgery; neo-vascularization correlates with survival of skin grafts: A prospective study

  Shinya Kitamura, Teruki Yanagi, Yuka Inamura-Takashima, Keisuke Imafuku, Hiroo Hata, Yoichiro Fujioka, Yusuke Ohba, Hiroshi Shimizu

  Journal of Dermatological Science 90 (2) 213 - 216 1873-569X 2018/05/01 [Refereed][Not invited]
  
• Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

  Asuka Nanbo, Junki Maruyama, Masaki Imai, Michiko Ujie, Yoichiro Fujioka, Shinya Nishide, Ayato Takada, Yusuke Ohba, Yoshihiro Kawaoka

  PLoS pathogens 14 (1) e1006848  1553-7366 2018/01 [Refereed][Not invited]
   

  Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.
• 北海道大学における医学英語の取り組みと、学生の英語学習に対するモチベーションに関するアンケート調査

  小野澤 真弘, Houman Goudarzi, 武冨 貴久子, 稲葉 直子, 藤岡 容一朗, 川久保 和道, 折茂 達也, 金野 陽輔, 坊垣 暁之, 伊藤 智城, 本間 理央, 北市 雄士, 村上 壮一, 村上 学, 川畑 秀伸, 小華和 柾志, 大滝 純司

  医学教育 (一社)日本医学教育学会 48 (Suppl.) 139 - 139 0386-9644 2017/08
• Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

  Shunsuke Kon, Kojiro Ishibashi, Hiroto Katoh, Sho Kitamoto, Takanobu Shirai, Shinya Tanaka, Mihoko Kajita, Susumu Ishikawa, Hajime Yamauchi, Yuta Yako, Tomoko Kamasaki, Tomohiro Matsumoto, Hirotaka Watanabe, Riku Egami, Ayana Sasaki, Atsuko Nishikawa, Ikumi Kameda, Takeshi Maruyama, Rika Narumi, Tomoko Morita, Yoshiteru Sasaki, Ryosuke Enoki, Sato Honma, Hiromi Imamura, Masanobu Oshima, Tomoyoshi Soga, Jun-ichi Miyazaki, Michael R. Duchen, Jin-Min Nam, Yasuhito Onodera, Shingo Yoshioka, Junichi Kikuta, Masaru Ishii, Masamichi Imajo, Eisuke Nishida, Yoichiro Fujioka, Yusuke Ohba, Toshiro Sato, Yasuyuki Fujita

  NATURE CELL BIOLOGY 19 (5) 530 - + 1465-7392 2017/05 [Refereed][Not invited]
   

  Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
• Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells

  Sayaka Saitoh, Takeshi Maruyama, Yuta Yako, Mihoko Kajita, Yoichiro Fujioka, Yusuke Ohba, Nobuhiro Kasai, Natsu Sugama, Shunsuke Kon, Susumu Ishikawa, Takashi Hayashi, Tomohiro Yamazaki, Masazumi Tada, Yasuyuki Fujita

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (12) E2327 - E2336 0027-8424 2017/03 [Refereed][Not invited]
   

  Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.
• Leukemogenic kinase FIP1L1-PDGFRA and a small ubiquitin-like modifier E3 ligase, PIAS1, form a positive cross-talk through their enzymatic activities

  Makoto Ibata, Junko Iwasaki, Yoichiro Fujioka, Koji Nakagawa, Stephanie Darmanin, Masahiro Onozawa, Daigo Hashimoto, Yusuke Ohba, Shigetsugu Hatakeyama, Takanori Teshima, Takeshi Kondo

  CANCER SCIENCE 108 (2) 200 - 207 1347-9032 2017/02 [Refereed][Not invited]
   

  Fusion tyrosine kinases play a crucial role in the development of hematological malignancies. FIP1L1-PDGFRA is a leukemogenic fusion kinase that causes chronic eosinophilic leukemia. As a constitutively active kinase, FIP1L1-PDGFRA stimulates downstream signaling molecules, leading to cellular proliferation and the generation of an anti-apoptotic state. Contribution of the N-terminal FIP1L1 portion is necessary for FIP1L1-PDGFRA to exert its full transforming activity, but the underlying mechanisms have not been fully characterized. We identified PIAS1 as a FIP1L1-PDGFRA association molecule by yeast two-hybrid screening. Our analyses indicate that the FIP1L1 portion of FIP1L1-PDGFRA is required for efficient association with PIAS1. As a consequence of the association, FIP1L1-PDGFRA phosphorylates PIAS1. Moreover, the kinase activity of FIP1L1-PDGFRA stabilizes PIAS1. Therefore, PIAS1 is one of the downstream targets of FIP1L1-PDGFRA. Moreover, we found that PIAS1, as a SUMO E3 ligase, sumoylates and stabilizes FIP1L1-PDGFRA. In addition, suppression of PIAS1 activity by a knockdown experiment resulted in destabilization of FIP1L1-PDGFRA. Therefore, FIP1L1-PDGFRA and PIAS1 form a positive cross-talk through their enzymatic activities. Suppression of sumoylation by ginkgolic acid, a small molecule compound inhibiting a SUMO E1-activating enzyme, also destabilizes FIP1L1-PDGFRA, and while the tyrosine kinase inhibitor imatinib suppresses FIP1L1-PDGFRA-dependent cell growth, ginkgolic acid or siRNA of PIAS1 has a synergistic effect with imatinib. In conclusion, our results suggest that sumoylation by PIAS1 is a potential target in the treatment of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.
• 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 0386-7196 2017 [Refereed][Not invited]
   

  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 Forster 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.
• Fluorescence bioimaging of intracellular signaling and its clinical application

  Ohba Yusuke, Fujioka Yoichiro

  Journal of Oral Biosciences 58 (4) 113 - 119 1880-3865 2016/11 [Refereed][Not invited]
  
• Receptor activator of NF-kappa B ligand induces cell adhesion and integrin alpha 2 expression via NF-kappa B 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  2045-2322 2016/03 [Refereed][Not invited]
   

  Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-kappa B 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 alpha 2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin alpha 2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-kappa B signaling mediated integrin alpha 2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin beta 1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin alpha 2 and endocytosis. Moreover, the RANK-integrin alpha 2 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.
• A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).

  Sayaka Yamamoto, Yuta Yako, Yoichiro Fujioka, Mihoko Kajita, Takeshi Kameyama, Shunsuke Kon, Susumu Ishikawa, Yusuke Ohba, Yusuke Ohno, Akio Kihara, Yasuyuki Fujita

  Molecular biology of the cell 27 (3) 491 - 9 1939-4586 2016/02/01 [Refereed][Not invited]
   

  At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.
• 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  2045-2322 2016/02 [Refereed][Not invited]
   

  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 Forster 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.
• Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

  Tadasuke Tsukiyama, Akimasa Fukui, Sayuri Terai, Yoichiro Fujioka, Keisuke Shinada, Hidehisa Takahashi, Terry P. Yamaguchi, Yusuke Ohba, Shigetsugu Hatakeyama

  MOLECULAR AND CELLULAR BIOLOGY 35 (11) 2007 - 2023 0270-7306 2015/06 [Refereed][Not invited]
   

  Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/beta-catenin pathway. Here, we show that RNF43 suppresses both Wnt/beta-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/beta-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/beta-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/beta-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/beta-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
• Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane

  Yoichiro Fujioka, Asuka Nanbo, Shin-ya Nishide, Yusuke Ohba

  ANALYTICAL SCIENCES 31 (4) 267 - 274 0910-6340 2015/04 [Refereed][Not invited]
   

  In response to extracellular stimuli, cells display a variety of behaviors, including proliferation, differentiation, morphological changes and migration. The analysis of the spatiotemporal regulation of signal transduction in living cells is needed for a better understanding of such behaviors, and such investigations have been greatly accelerated by the development of fluorescent protein-based biosensors. Currently, by using these biosensors a range of molecular actions, including lipid metabolism, protein activation, and ion dynamics, can be visualized in living cells. We recently reported that intracellular calcium, with its relevant downstream signaling pathways consisting of the small GTPase Ras and the lipid kinase phoshoinositide-3-kinase (PI3K), can be exploited in an efficient incorporation of influenza A viruses into host cells via endocytosis using a set of biosensors based on fluorescent proteins and the principle of Forster resonance energy transfer. Here, we focus this review on fluorescent protein-based biosensors that have been utilized in our recent research reports.
• [The Ras-PI3K signaling is involved in the regulation of endocytosis and virus internalization].

  Fujioka Y, Ohba Y

  Seikagaku. The Journal of Japanese Biochemical Society 日本生化学会 87 (1) 91 - 100 0037-1017 2015/02 [Refereed][Not invited]
  
• Calcium signaling mediated influenza virus entry into host cells.

  Y. Fujioka, M. Tsuda, A. Nanbo, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki, Y. Ohba

  MOLECULAR BIOLOGY OF THE CELL 25 1059-1524 2014/12 [Refereed][Not invited]
  
• Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors

  Koji Terada, Takahiro Horinouchi, Yoichiro Fujioka, Tsunehito Higashi, Prabha Nepal, Mika Horiguchi, Sarita Karki, Chizuru Hatate, Akimasa Hoshi, Takuya Harada, Yosuke Mai, Yusuke Ohba, Soichi Miwa

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 (51) 35283 - 35295 0021-9258 2014/12 [Refereed][Not invited]
   

  Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated 5KR mutant) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated 4KR mutant), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.
• Lysosomal interaction of Akt with Phafin2: a critical step in the induction of autophagy

  Masayuki Noguchi, Mami Matsuda-Lennikov, Noroyuki Hirata, Manabu Hashimoto, Kohki Kimura, Tatsuma Edamura, Futoshi Suizu

  FASEB JOURNAL 28 (1) e79795  0892-6638 2014/04 [Refereed][Not invited]
  
• Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

  Mohamed K. Hassan, Hidemichi Watari, Alaa-eldin Salah-eldin, Ahmed S. Sultan, Zainab Mohamed, Yoichiro Fujioka, Yusuke Ohba, Noriaki Sakuragi

  PLOS ONE 9 (4) e94213  1932-6203 2014/04 [Refereed][Not invited]
   

  This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5 degrees C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.
• Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

  Mami Matsuda-Lennikov, Futoshi Suizu, Noriyuki Hirata, Manabu Hashimoto, Kohki Kimura, Tadashi Nagamine, Yoichiro Fujioka, Yusuke Ohba, Toshihiko Iwanaga, Masayuki Noguchi

  PLOS ONE 9 (1) 1932-6203 2014/01 [Refereed][Not invited]
   

  Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3) P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3) P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.
• Beneficial innate signaling interference for antibacterial responses by a Toll-like receptor-mediated enhancement of the MKP-IRF3 axis

  Hideo Negishi, Kosuke Matsuki, Nobuyasu Endo, Hana Sarashina, Shoji Miki, Atsushi Matsuda, Keiko Fukazawa, Naoko Taguchi-Atarashi, Hiroaki Ikushima, Hideyuki Yanai, Junko Nishio, Kenya Honda, Yoichiro Fujioka, Yusuke Ohba, Tetsuo Noda, Shun'ichiro Taniguchi, Eisuke Nishida, Yongliang Zhang, Hongbo Chi, Richard A. Flavell, Tadatsugu Taniguchi

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (49) 19884 - 19889 0027-8424 2013/12 [Refereed][Not invited]
   

  A major function of innate immune receptors is to recognize pathogen-associated molecular patterns and then evoke immune responses appropriate to the nature of the invading pathogen(s). Because innate immune cells express various types of these receptors, distinct combinations of signaling pathways are activated in response to a given pathogen. Although the conventional wisdom is that these signaling pathways cooperate with one another to ensure an effective host response, a more nuanced view recognizes antagonism between the individual pathways, where the attenuation of a signaling pathway(s) by others may shape the immune response. In this study, we show that, on Listeria monocytogenes infection, Toll-like receptor-triggered MyD88 signaling pathways suppress type I IFN gene induction, which is detrimental to macrophage bactericidal activity. These pathways target and suppress the IFN regulatory factor 3 (IRF3) transcription factor that is activated by the stimulator of IFN genes-TANK-binding kinase-1 kinase pathway. We also provide evidence for the involvement of the MAPK phosphatase family members, which renders IRF3 hypophosphorylated on Toll-like receptor signaling by enhancing the formation of an MAPK phosphatase-IRF3-TANK-binding kinase-1 ternary complex. This study, therefore, reveals a hitherto unrecognized and important contribution of a beneficial innate signaling interference against bacterial infections.
• A Ca2+-dependent signalling circuit regulates influenza A virus internalization and infection

  Yoichiro Fujioka, Masumi Tsuda, Asuka Nanbo, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba

  NATURE COMMUNICATIONS 4 2763  2041-1723 2013/11 [Refereed][Not invited]
   

  Various viruses enter host cells via endocytosis, but the molecular mechanisms underlying the specific internalization pathways remain unclear. Here we show that influenza A viruses (IAVs) enter cells via redundant pathways of clathrin-mediated and clathrin-independent endocytosis, with intracellular Ca2+ having a central role in regulation of both pathways by activating a signalling axis comprising RhoA, Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and phospholipase C (PLC). IAV infection induces oscillations in the cytosolic Ca2+ concentration of host cells, the prevention of which markedly attenuates virus internalization and infection. The small GTPase RhoA is found both to function downstream of the virus-induced Ca2+ response and itself to induce Ca2+ oscillations in a manner dependent on Rho-kinase and subsequent PIP5K-PLC signalling. This signalling circuit regulates both clathrin-mediated and clathrin-independent endocytosis during virus infection and seems to constitute a key mechanism for regulation of IAV internalization and infection.
• Inhibition of influenza A virus infection by Galectin-9

  Tomoe Hattori, Tomohiro Arikawa, Yoichiro Fujioka, Junki Maruyama, Yousuke Nakayama, Yusuke Ohba, Toshiro Niki, Tadaaki Miyazaki, Mitsuomi Hirashima, Hiroshi Kida

  JAPANESE JOURNAL OF VETERINARY RESEARCH 61 (1-2) 5 - 18 0047-1917 2013/05 [Refereed][Not invited]
   

  Galectin-9 (Gal-9) inhibited the infection of H1N1, H3N2 and H5N1 influenza A viruses in vitro and in vivo. Fifty percent effective doses (ED50) of Gal-9 were 0.1-0.5 mu M depending on virus strains in the plaque reduction assay. Gal-9 but not Gal-1 bound to the virus particles of A/Puerto Rico/8/34 (H1N1) (PR/8), resulting in inhibition of virus attachment to the host cells. Lactose but not sucrose inhibited the binding of Gal-9 to the viruses. Endogenous Gal-9 expression was detected and increased with the course of infection with influenza A viruses in mice. Fifty percent of Gal-9-transgenic mice survived after the challenge with PR/8, while all of the wild-type mice died. Gal-9 treatment of mice affected diminishing influenza virus replication in the lungs, body weight loss and the expression level of inflammatory cytokines. Combined administration of Gal-9 and oseltamivir was more effective than the use of single compound in mouse model. The present results indicate that Gal-9 is a candidate compound for influenza A virus infection therapy.
• Fluorescent protein-based biosensors and their clinical applications

  Yusuke Ohba, Yoichiro Fujioka, Shigeyuki Nakada, Masumi Tsuda

  Progress in Molecular Biology and Translational Science 113 313 - 348 1877-1173 2013 [Refereed][Not invited]
   

  Green fluorescent protein and its relatives have shed their light on a wide range of biological problems. To date, with a color palette consisting of fluorescent proteins with different spectra, researchers can "paint" living cells as they desire. Moreover, sophisticated biosensors engineered to contain single or multiple fluorescent proteins, including FRET-based biosensors, spatiotemporally unveil molecular mechanisms underlying physiological processes. Although such molecules have contributed considerably to basic research, their abilities to be used in applied life sciences have yet to be fully explored. Here, we review the molecular bases of fluorescent proteins and fluorescent protein-based biosensors and focus on approaches aimed at applying such proteins to the clinic. © 2013 Elsevier Inc.
• Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

  Shigeki Chiba, Muhammad Baghdadi, Hisaya Akiba, Hironori Yoshiyama, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Yoichiro Fujioka, Yusuke Ohba, Jacob V. Gorman, John D. Colgan, Mitsuomi Hirashima, Toshimitsu Uede, Akinori Takaoka, Hideo Yagita, Masahisa Jinushi

  NATURE IMMUNOLOGY 13 (9) 832 - 842 1529-2908 2012/09 [Refereed][Not invited]
   

  The mechanisms by which tumor microenvironments modulate nucleic acid mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.
• DJ-1 associates with synaptic membranes

  Yukiko Usami, Taku Hatano, Satoshi Imai, Shin-ichiro Kubo, Shigeto Sato, Shinji Saiki, Yoichiro Fujioka, Yusuke Ohba, Fumiaki Sato, Manabu Funayama, Hiroto Eguchi, Kaori Shiba, Hiroyoshi Ariga, Jie Shen, Nobutaka Hattori

  NEUROBIOLOGY OF DISEASE 43 (3) 651 - 662 0969-9961 2011/09 [Refereed][Not invited]
   

  Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons. Although many reports have suggested that genetic factors are implicated in the pathogenesis of PD, molecular mechanisms underlying selective dopaminergic neuronal degeneration remain unknown. DJ-1 is a causative gene for autosomal recessive form of PARK7-linked early-onset PD. A number of studies have demonstrated that exogenous DJ-1 localizes within mitochondria and the cytosol, and functions as a molecular chaperon, as a transcriptional regulator, and as a cell protective factor against oxidative stress. However, the precise subcellular localization and function of endogenous DJ-1 are not well known. The mechanisms by which mutations in DJ-1 contributes to neuronal degeneration also remain poorly understood. Here we show by immunocytochemistry that DJ-1 distributes to the cytosol and membranous structures in a punctate appearance in cultured cells and in primary neurons obtained from mouse brain. Interestingly. DJ-1 colocalizes with the Golgi apparatus proteins GM130 and the synaptic vesicle proteins such as synaptophysin and Rab3A. Forster resonance energy transfer analysis revealed that a small portion of DJ-1 interacts with synaptophysin in living cells. Although the wild-type DJ-1 protein directly associates with membranes without an intermediary protein, the pathogenic L166P mutation of DJ-1 exhibits less binding to synaptic vesicles. These results indicate that DJ-1 associates with membranous organelles including synaptic membranes to exhibit its normal function. (C) 2011 Elsevier Inc. All rights reserved.
• The Ras-PI3K Signaling Pathway Is Involved in Clathrin-Independent Endocytosis and the Internalization of Influenza Viruses

  Yoichiro Fujioka, Masumi Tsuda, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba

  PLOS ONE 6 (1) e16324  1932-6203 2011/01 [Refereed][Not invited]
   

  Background: Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype. Methodology/Principal Findings: Here, we identify the Ras-phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrinin-dependent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras-PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes. Conclusions/Significance: Taken together, these results demonstrate that the Ras-PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.
• Visualization of Ras-PI3K interaction in the endosome using BiFC

  Kaori Tsutsumi, Yoichiro Fujioka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba

  CELLULAR SIGNALLING 21 (11) 1672 - 1679 0898-6568 2009/11 [Refereed][Not invited]
   

  Recent studies indicate the importance of spatiotemporal regulation in the diversity and specificity of intracellular signaling. Here, we show that Ras-PI3K signaling plays an important role in the local regulation of phosphatidylinositol metabolism in the endosome through live-cell imaging by using a bimolecular fluorescence complementation technique, in which molecular interaction is indicated by fluorescence emission. Using several possible combinations of Ras and the Ras-binding domain, we identified an optimal set of probe molecules that yielded the most significant increase in fluorescence intensity between the active and inactive forms of Ras. This combination revealed that, among the Ras effectors tested, phosphatidy-linositol 3-kinase (PI3K) was specifically implicated in signaling in the endosome. We also found that full length PI3K was recruited to the endosome in EGF- and Ras-dependent manners, which appears to be essential for the activation of PI3K in this compartment. Taken together, these findings demonstrate that the spatiotemporal regulation of Ras-PI3K signaling may dictate the activation of PI3K and subsequent downstream signaling in the endosome. (C) 2009 Elsevier Inc. All rights reserved.
• SGS3 and RDR6 interact and colocalize in cytoplasmic SGS3/RDR6-bodies

  Naoyoshi Kumakura, Atsushi Takeda, Yoichiro Fujioka, Hiroyasu Motose, Ryo Takano, Yuichiro Watanabe

  FEBS LETTERS 583 (8) 1261 - 1266 0014-5793 2009/04 [Refereed][Not invited]
   

  Suppressor of gene silencing 3 (SGS3) is involved in RNA-dependent RNA polymerase 6 (RDR6)-dependent small-interfering RNA (siRNA) pathways in Arabidopsis. However, the roles of SGS3 in those pathways are unclear. Here, we show that SGS3 interacts and colocalizes with RDR6 in cytoplasmic granules. Interestingly, the granules containing SGS3 and RDR6 (named SGS3/RDR6-bodies) were distinct from the processing bodies where mRNAs are decayed and/or stored. Microscopic analyses and complementation experiments using SGS3-deletion mutants suggested that proper localization of SGS3 is important for its function. These results provide novel insights into RDR6-dependent siRNA formation in plants.
• Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in arabidopsis

  Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe

  PLANT AND CELL PHYSIOLOGY 48 (9) 1243 - 1253 0032-0781 2007/09 [Refereed][Not invited]
   

  There has been much recent research on the contribution of microRNA (miRNA) in plant organogenesis and hormone action. In plants, it has been reported that Dicer-like 1 (DCL1), HYPONASTIC LEAVES1 (HYL1). and SERRATE (SE) are involved in the production of miRNAs. The means by which miRNAs are processed and transported is. not well understood in detail, however. In this study, we investigated the intracellular localization and intermolecular interaction of these molecules using imaging techniques, including bimolecular fluorescence complementation and fluorescence resonance energy transfer techniques, making use of various enhanced fluorescent proteins. We found that DCL1, HYL1 and SE formed bodies which localized in the nuclei. We were also able to locate the miRNA primary transcript using an MS2-tagged method on these bodies. It appears very likely that the observed DCL1-HYL1-SE nuclear body is involved in miRNA production. Co-expression of SmD3 or SmB proteins revealed the localization of DCL1-HYL1-SE complexes in the SmD3/SmB nuclear bodies.
• Biogenesis of small RNAs in plants and their biological significance

  Yuichiro Watanabe, Yukio Kurihara, Yuko Tagami, Yoichiro Fujioka, Naoko Inaba, Maki Utsumi

  PLANT AND CELL PHYSIOLOGY 48 S7 - S7 0032-0781 2007 [Refereed][Not invited]
  
• Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

  M Kobayashi, Y Fujioka, T Mori, M Terashima, H Suzuki, Y Shimada, T Saito, ZY Wang, T Nozawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 (6) 1130 - 1136 0916-8451 2005/06 [Refereed][Not invited]
   

  Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LM-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or-by the core light-harvesting complexes. The. results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.

MISC

• フラビウイルスの粒子産生における分泌型NS1タンパク質の役割

  田村友和, 田村友和, 鳥居志保, 鳥居志保, 梶原健太郎, 安齋樹, 藤岡容一朗, 野田暉翔, 田鍬修平, 森岡佑平, 森岡佑平, 鈴木理滋, YUZY Fauzyah, 小野慎子, 小野慎子, 大場雄介, 岡田雅人, 福原崇介, 福原崇介, 松浦善治, 松浦善治  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• Visualizing virus entry into host cells

  藤岡容一朗, 天野麻穂, 大場雄介  医学のあゆみ  280-  (9)  2022
• インフルエンザウイルスの宿主細胞侵入メカニズム

  藤岡容一朗, 天野麻穂, 大場雄介  創薬研究者がこれだけは知っておきたい最新のウイルス学  146  -153  2021
• Pathogen dynamics in host cells

  藤岡容一朗, 天野麻穂, 大場雄介  実験医学  39-  (2)  2021
• 少数性生物学ってなんだ?少数の要素を計測する―ウイルス何個で“感染”するか?

  藤岡容一朗, 田端和仁, 田端和仁, 大場雄介, 野地博行, 野地博行  実験医学  35-  (19)  3197‐3203  2017/12/01  [Not refereed][Not invited]
  
• PBL初学者に対する臨床実習におけるPBL導入の試み

  川久保 和道, 村上 壮一, 北市 雄士, 小野澤 真弘, 坊垣 暁之, 金野 陽輔, 折茂 達也, 藤岡 容一郎, 稲場 直子, 本間 理央, 武冨 貴久子, 桂田 武彦, 森川 賢一, 工藤 俊彦, 中積 宏之, 小華和 柾志, 川畑 秀伸, 坂本 直哉, 大滝 純司  医学教育  48-  (Suppl.)  206  -206  2017/08  [Not refereed][Not invited]
  
• 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  2017/05  [Not refereed][Not invited]
  
• 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  2017/05  [Not refereed][Not invited]
  
• 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  2017/05  [Not refereed][Not invited]
  
• Fluorescence bioimaging of intracellular signaling and its clinical application

  Yusuke Ohba, Yoichiro Fujioka  Journal of Oral Biosciences  58-  (4)  113  -119  2016/11/01  [Not refereed][Not invited]
   

  Background Fluorescent proteins have continued to shed light on cell biology since the cDNA of wild type green fluorescent protein was first isolated. Nowadays, these remarkable proteins are useful tools, not only in basic research, but also in clinical medicine. Highlight By taking advantage of fluorescent protein-based technologies, we identified a signaling network critical for influenza virus internalization and infection. In addition, we developed a highly sensitive biosensor for monitoring kinase activity that utilizes energy transfer between fluorescent proteins. This has led to a high-performance clinical test that enables the prediction of future therapeutic responses and the risk of acquired drug resistance for each individual patient before beginning molecular target therapy. Conclusion Technologies that utilize fluorescent proteins, such as the biosensor presented here, should find increasing applications in clinical medicine.
• IDENTIFICATION OF FIP1L1-PDGFRA ASSOCIATING MOLECULE THAT LOCATES IN THE NUCLEUS AND AUGMENTS THE ACTIVITY OF FIP1L1-PDGFRA

  T. Kondo, M. Ibata, J. Iwasaki, Y. Fujioka, K. Nakagawa, S. Darmanin, M. Onozawa, D. Hashimoto, Y. Ohba, S. Hatakeyama, T. Teshima  HAEMATOLOGICA  100-  327  -327  2015/06  [Not refereed][Not invited]
  
• Ras‐PI3Kシグナルによるエンドサイトーシスとウイルス粒子取り込みの制御機構

  藤岡容一朗, 大場雄介  生化学  87-  (1)  91  -100  2015/02/25  [Not refereed][Not invited]
  
• インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構

  藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  66回-  103  -103  2014/05  [Not refereed][Not invited]
  
• インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構

  藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  66回-  146  -146  2014/05  [Not refereed][Not invited]
  
• Ras-PI3Kシグナルが制御する外来因子取込み機構の解析

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本生理学雑誌  76-  (2)  81  -81  2014/03  [Not refereed][Not invited]
  
• Ras-PI3Kシグナルが制御する外来因子取込み機構の解析

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 大場 雄介  日本細胞生物学会大会講演要旨集  65回-  153  -153  2013/05  [Not refereed][Not invited]
  
• RANKLはインテグリンα2の発現とエンドサイトーシスを介したインテグリンの細胞内輸送を亢進する

  我妻孝則, 津田真寿美, 山田珠希, 藤岡容一朗, 芳賀永, 戸塚靖則, 進藤正信, 大場雄介  日本分子生物学会年会プログラム・要旨集(Web)  35th-  3P-0363 (WEB ONLY)  2012  [Not refereed][Not invited]
  
• RANKLは口腔癌細胞のインテグリンα2の発現と細胞接着を亢進する(RANKL upregulates integrin α2 expression and cell adhesion in oral cancer cells)

  大場 雄介, 山田 珠希, 藤岡 容一朗, 甲斐原 拓真, 戸塚 泰則, 進藤 正信, 津田 真寿美  日本細胞生物学会大会講演要旨集  63回-  156  -156  2011/05  [Not refereed][Not invited]
  
• 染色体・核・遺伝子発現・シグナル伝達 インフルエンザウイルスは、カルシウムシグナル伝達によって、Ras-PI3K-仲介エンドサイトーシスを活性化する(Signal transduction/Chromosome/Cell nucleus/Gene expression Influenza viruses activate Ras-PI3K-mediated endocytosis via calcium signaling)

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  63回-  112  -112  2011/05  [Not refereed][Not invited]
  
• メンブレントラフィック Ras-PI3Kシグナル経路がエンドサイトーシスとインフルエンザ感染を調節する(Membrane traffic The Ras-PI3K signaling pathway mediates endocytosis and the incorporation of influenza viruses)

  藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介  日本細胞生物学会大会講演要旨集  62回-  117  -117  2010/05  [Not refereed][Not invited]
  
• 植物のta-siRNA及びvsiRNA合成に関わるSGS3/RDR6-bodyの解析

  熊倉直祐, 竹田篤史, 藤岡容一郎, 渡辺雄一郎  日本RNA学会年会要旨集  11th-  2009
• 植物におけるsmall RNA生成/制御の場の解析

  藤岡容一朗, 内海真希, 岩崎信太郎, 大場雄介, 渡辺雄一郎  日本RNA学会年会要旨集  9th-  138  2007/07/28  [Not refereed][Not invited]
  
• Structure for thermostability of photosynthetic reaction center from thermophilic purple sulfur bacterium, Thermochromatium tepidum.

  M. Kobayashi, Y. Shimada, Y. Fujioka, H. Suzuki, Z. Wang, T. Nozawa  PHOTOSYNTHESIS RESEARCH  91-  (2-3)  142  -142  2007/02  [Not refereed][Not invited]
  

Presentations

• Influenza A virus entry into host cells via calcium signaling-mediated endocytosis  [Invited]

  Yoichiro Fujioka

  1st Material Symbiosis International symposium/3rd GI-CoRE/GSD International symposium/28th Pharmascience Forum “Joint Symposium for Young Researchers”  2023/03
• インフルエンザウイルス感染促進メカニズムの解明  [Not invited]

  小澤史弥, 藤岡容一朗, 吉田藍子, 釜崎とも子, 酒井信明, 柏木彩花, 天野麻穂, 大場雄介

  令和４年度北大細胞生物研究集会  2023/03
• 蛍光イメージングで紐解く、細胞の物質取り込み機構の研究  [Invited]

  藤岡容一朗

  2022年度日本分光学会北海道支部シンポジウム  2023/03
• Molecular mechanisms of influenza virus entry into host cells  [Invited]

  Yoichiro Fujioka

  Sialoglyco2022  2022/09
• 新規エンドソーム構造に関する形態学的解析  [Not invited]

  釜崎とも子, 佐藤絢, 藤岡容一朗, 酒井信明, 吉田藍子, 柏木彩花, 天野麻穂, 大場雄介

  第102回北海道医学大会生理系分科会／日本生理学会北海道地方会  2022/09
• インフルエンザウイルス感染伝播機構の解明  [Not invited]

  小澤史弥, 藤岡容一朗, 吉田藍子, 柏木彩花, 天野麻穂, 大場雄介

  第74回日本細胞生物学会大会  2022/06
• オプトジェネティクスを用いたEGFRシグナル制御機構の解析  [Not invited]

  鬼塚洋之進, 藤岡容一朗, 吉田藍子, 柏木彩花, 天野麻穂, 大場雄介

  第74回日本細胞生物学会大会  2022/06

Teaching Experience

蛍光イメージング入門蛍光イメージング入門 Hokkaido University

Association Memberships

• THE PHYSIOLOGICAL SOCIETY OF JAPAN   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   JAPAN SOCIETY FOR CELL BIOLOGY   日本バイオイメージング学会   

Research Projects

• ウイルスが感染時に共通して利用する宿主細胞側マシナリーの同定

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2027/03 

  Author : 藤岡 容一朗
• ウイルス粒子の個性に注目した細胞取り込み機構と応答スペクトルの解析

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 藤岡 容一朗
• カルシウムシグナルが駆動するウイルス感染のシンギュラリティ現象の解析

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2021/04 -2023/03 

  Author : 藤岡 容一朗

   

  ヒト生体内での感染症発症機序を理解することは、新しい切り口での感染症対策を講じるためにも重要である。しかし、これまでのウイルス研究は、十分にウイルス量の増えた段階を想定した条件下で実験が行われていたため、生体内での感染拡大の機構はほとんどわかっていない。我々はこれまでに粒子数を厳密に制御した定量的ウイルス感染実験系を構築し、ある一定の粒子数が細胞に曝露すると感染細胞数が爆発的に増えることを見出している。これをウイルス感染のシンギュラリティ現象と定義し、本研究においてその分子メカニズムの解明に挑んだ。R3年度はカルシウムバイオセンサーであるO-GECOを恒常的に発現する細胞株を樹立し、ウイルス感染後のカルシウムダイナミクスを細胞集団レベルで詳細に解析した。その結果、感染細胞でカルシウム濃度上昇が生じた後、近接する細胞においてカルシウム濃度上昇が伝播する現象、"propagating-wave"が発生することを見出した。このpropagating-waveはA型インフルエンザウイルスの複数の亜型において認められたことから、A型インフルエンザウイルスに共通する現象であると示唆される。また、興味深いことに、2次元培養環境下よりも3次元培養環境下で上皮層を形成した細胞集団において、このwaveの発生が多く観察された。カルシウム伝播の速度を定量したところ、300 um2/sec程度であることから、細胞外に分泌された小分子を介してカルシウム濃度上昇が促されると考えられる。各種小分子の阻害薬存在下でカルシウムイメージングしたところ、propagating-waveの発生を抑制する複数の阻害薬を同定した。さらに、この阻害薬によりウイルス感染が抑制されたことから、propagating-waveの発生が感染に重要であることが示された。
• Development of isotropic resolution microscopy using fluorescence polarization and visualization of cell membrane dynamics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/10 -2022/03 

  Author : Ohba Yusuke

   

  A goniometric biosensor was developed by adding two types of membrane localizing sequences at two sites on the green fluorescent protein. The cell membrane and the fluorescent protein chromophore are fixed at a position orthogonal to each other. A similar biosensor was also developed for the red fluorescent protein. In addition, a total-reflection polarized fluorescence microscope was set up to excite samples with regulated anisotropic light and to detect emission lights through spectroscopy based on the polarization. Using these techniques, we established a method to capture changes in the angle of the membrane as changes in fluorescence intensity and succeeded in visualizing the very early process of receptor-mediated endocytosis. In addition, using the total reflection microscope we developed in this study, we succeeded for the first time in the world in direct capturing the secretion of the incretin GLP-1 from L cells.
• ウイルス感染超初期過程におけるウイルス－宿主共生機構の解析

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  Date (from‐to) : 2019/04 -2021/03 

  Author : 藤岡 容一朗

   

  これまでに申請者は、ある一定数以上の粒子数のインフルエンザウイルスが細胞に曝露するとインフルエンザウイルス感染が爆発的に広がる現象を見出している。また、本研究においてに20粒子以上のウイルスに曝露された細胞では、細胞内カルシウム濃度が上昇すること、および20粒子以下の 場合は細胞内カルシウム濃度は変化しないことを明らかにし、粒子数の多寡で細胞応答が劇的に変化することを見出した。すなわち、少数のウイルス粒子感染時にはカルシウムシグナル非依存的な感染メカニズムが存在すると考えられる。また、インフルエンザウイルスの感染成立をモニターするためのレポーター遺伝子を作製し、そのレポーターを安定的に発現する細胞株を樹立した。この細胞を用いることで、生きた細胞においてウイルス感染成立を評価することが可能になった。今後は、少数のウイルス粒子に曝露された際に感染成立する細胞と成立しない細胞の違いを明らかにするために、細胞内カルシウム濃度上昇を生じないにも関わらず感染成立した細胞を分取して1細胞解析を行い 、その感染成立メカニズムを解明する。また、ウイルス粒子を細胞に曝露する際、ウイルス様粒子(Virus like particle)存在下では感染効率が上昇することも見出した 。このことから、ウイルスRNAを含まない不完全な粒子が感染成立に寄与することが示唆された。すなわち、多数のウイルス粒子の中に感染性を有する粒子が一つ存在する、もしくは複数の粒子が協調してはじめて感染が成立するという2 つの仮説が立てられるため、今後どちらの仮説が正しいか検証を行う。 以上により、我々が日々の生活を営む「現実の世界」で生じるウイルス感染現象の理解を目標とする。
• Identification of key molecule for influenza virus entry

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)

  Date (from‐to) : 2016/04 -2020/03 

  Author : Fujioka Yoichiro

   

  We have discovered that the Ca2+ channel, a transmembrane protein that allows Ca2+ to move across the plasma membrane, is the key receptor molecule for influenza virus infections. Furthermore, treating human cells with a calcium channel blocker (CCB), which is commonly used as anti-hypertension drug, significantly suppressed virus infections.
• Investigation of a physiological role of endosome-mitochondrial interaction by fluorescence bioimaging

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2015/04 -2017/03 

  Author : Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.

   

  We have reported that the complex of small G protein Ras and its target molecule PI3K is localized in the endosomes and controls endocytosis and viral particle internalization. However, the molecular mechanism by which the Ras-PI3K complex is localized is the endosome remains unknown. In this study, the amino acid sequence responsible for the above phenomenon was identified. By screening the binding protein for the sequence, a mitochondrial protein was identified. Knockdown of this mitochondrial protein inhibited the translocation of Ras-PI3K complex to the endosome, endocytosis, and mitochondrial-endosome interaction, suggesting that binding of PI3K and the factor regulates endocytosis through interaction between organelles.
• Intracellular signaling machinery controlling endocytosis and uptake of extracellular materials

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2014/04 -2017/03 

  Author : Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.

   

  Endosome is a multifunctional platform through the emission and the regulation of a variety of signal transduction pathways. However, the relationship between signaling dynamics and cell function via the endosomes in living cells has yet to be analyzed in detail. In this study, we demonstrated that angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R)-signaling through a series of events including heterodimerization of AT1R and AT2R, internalization via endocytosis of the complex, change in the molecular orientation of receptors in the complex. We also clarified that activation of both receptors and and phosphorylation of serine residues at the C-terminus of AT2R by protein kinase C (PKC) are indispensable for this function.
• The mechanism of influenza virus internalization into host cells via calcium signaling-mediated endocytosis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2014/04 -2016/03 

  Author : Fujioka Yoichiro

   

  We have reported that Ras-PI3K signaling mediates endocytosis, and influenza viruses exploit this pathway to expedite their efficient incorporation into cells. Moreover, calcium signaling, triggered by the viruses, has been identified as an upstream regulator of Ras-PI3K signaling. In this study, we explored in detail the mechanism by which calcium signaling is activated upon the viral infection. First, we performed high-speed imaging experiments with fluorescently labeled virus particles to gain further insight into the spatiotemporal dynamics of Ca2+ responses upon virus entry. Immediately after infection, localized and modest Ca2+ elevations, prior to the aforementioned robust Ca2+ release from endoplasmic reticulum, were detected in the regions where the labeled particles were adsorbed. Moreover, we identified the membrane protein, which is involved in the Ca2+ elevation by the viruses.
• Ras-PI3Kシグナルによるエンドサイトーシスとウイルス感染制御機構の解明

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  Date (from‐to) : 2011 -2013 

  Author : 藤岡 容一朗

   

  我々はこれまでにRasの標的因子であるPI3Kのエンドゾーム移行がエンドサイトーシスを制御することを明らかとしており、インフルエンザをはじめとする複数種のウイルスは宿主細胞のRas-PI3Kシグナル活性化を介してエンドサイトーシスを促進し、この亢進したエンドサイトーシスに便乗して巧妙に侵入することを報告している。昨年度までに、"PI3Kに特徴的な配列に結合する因子が、Ras-PI3K複合体の局在制御を介してエンドサイトーシスによる物質取り込みやウイルスの宿主細胞侵入に関与する"という仮説のもと、この配列を欠損したPI3KのRas結合領域とRasの複合体の細胞内挙動を追跡したところ、増殖因子刺激依存的なエンドゾームへの移行は見られずゴルジ装置付近に蓄積した。また、上記アミノ酸配列にエピトープタグもしくは蛍光タンパク質を融合して培養細胞に発現させたところ、エンドサイトーシスとウイルス感染が抑制された。以上から、上記仮説を支持するデータが得られており、本年度は、この配列に結合する因子を北海道大学大学院生命科学院小布施力史教授との共同研究によりMS/MS解析を用いて網羅的に探索した。その結果、ユビキチン化関連タンパク質、細胞骨格関連タンパク質をはじめとする、40個の候補因子が同定された。それら因子をクローニングし、免疫沈降法を用いてPI3Kとの相互作用を検討した。PI3Kとの結合が確認された因子に対して、蛍光タンパク質を融合し培養細胞に発現させ、蛍光顕微鏡による観察を行ったところ、エンドゾームへの局在が確認された因子を特定した。以上の結果から、特定された因子は上記の仮説に関与することが示唆され、現在はRNA干渉を用いた発現抑制を行い、Ras-PI3Kのエンドゾーム移行、およびエンドサイトーシスへの関与を検討している。最終的には、エンドサイトーシスの緻密な制御機構の解明につなげたい。

Industrial Property Rights

• 特願2021-125781:シュードタイプウイルス集団及びその製造方法  2021年/07/30