Sep. 2023, 第10回ＩＲＭＡＩＬサイエンスグラント「横河電機賞」　　　　　　　　　　　　　　　

May 2021, 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　　　　　　　　　　　　　　　

Jun. 2020, 日本細胞生物学会, CFS Award 2019　　　　　　　　　　　　　　　
藤岡容一朗、佐藤絢

Dec. 2019, 北海道科学技術奨励賞　　　　　　　　　　　　　　　
藤岡容一朗

Sep. 2019, 第8回北海道癌談話会奨励賞　　　　　　　　　　　　　　　
藤岡容一朗

Mar. 2019, 「第37回北海道医学会研究奨励賞」　　　　　　　　　　　　　　　
藤岡容一朗

Mar. 2019, 平成３０年度北海道大学大学院医学研究院「優秀論文賞」　　　　　　　　　　　　　　　
藤岡容一朗

Feb. 2019, 平成30年度コニカミノルタ画像科学奨励賞　　　　　　　　　　　　　　　
藤岡容一朗

Mar. 2015, 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　　　　　　　　　　　　　　　
藤岡容一朗

Jul. 2014, 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　　　　　　　　　　　　　　　
藤岡容一朗

Jun. 2014, 第66回日本細胞生物学会, 若手最優秀発表賞受賞　　　　　　　　　　　　　　　
藤岡容一朗

Mar. 2014, 北海道大学大学院医学研究科平成２５年度「優秀論文賞」　　　　　　　　　　　　　　　
藤岡容一朗

Intranasal administration of stem cell-derived exosome alleviates cognitive impairment against subarachnoid hemorrhage.
Shuho Gotoh, Masahito Kawabori, Sho Yamaguchi, Yo Nakahara, Erika Yoshie, Kohtarou Konno, Yuki Mizuno, Yoichiro Fujioka, Yusuke Ohba, Yuji Kuge, Masahiko Watanabe, Miki Fujimura
Experimental neurology, 386, 115143, 115143, Apr. 2025, [International Magazine]
English, Scientific journal, INTRODUCTION: Brain damage caused by subarachnoid hemorrhage (SAH) currently lacks effective treatment, leading to stagnation in the improvement of functional outcomes for decades. Recent studies have demonstrated the therapeutic potential of exosomes released from mesenchymal stem cells (MSC), which effectively attenuate neuronal apoptosis and inflammation in neurological diseases. Due to the challenge of systemic dilution associated with intravenous administration, intranasal delivery has emerged as a novel approach for targeting the brain. In this study, we investigate the effects of intranasally administered MSC-derived exosomes in a SAH animal model and elucidate their mode of action. METHODS: Exosomes were isolated from the cell supernatants of amnion-derived MSC. SAH was induced in 8-week-old Sprague-Dawley rats using an autologous blood prechiasmatic cistern injection model. A total of 1.2 × 1010 particles of exosomes in 200 μL of PBS or PBS alone were intranasally administered immediately and 24 h post-injury. Neurological function was assessed up to 7 days after injury, and histological analysis was performed to evaluate their anti-apoptotic and anti-inflammatory effects. The biodistribution of exosomes was assessed using PET/CT imaging of 64Cu labeled exosome. In vitro analyses were performed using primary glial cells and cell lines to evaluate the anti-inflammatory effects of the exosomes. RESULTS: Animals treated with exosomes exhibited significant improvement in cognitive function compared with PBS treated animal. Apoptotic cells and inflammation were reduced for the exosome group in the hippocampal CA1 area and in cortex, resulting in better neuronal cell survival. Blood brain barrier permeability was also preserved in the exosome group. Nuclear imaging revealed that exosomes were primarily transferred to the olfactory nerve and cerebrum; furthermore, exosomes were also observed in the trigeminal nerve and brainstem, where exosomes were co-localized with microglia and with endothelial cells. In vitro assessment showed that exosome administration ameliorated inflammation and prevented the death of glial cells. CONCLUSIONS: MSC-derived exosomes were successfully transferred into the brain through intranasal administration and alleviated brain damage following SAH.

BT-DNBS: a novel cyanine-based turn-on fluorescent probe with large Stokes shift for sensitive and selective detection of biothiols in live-cell imaging.
Shuai Zhang, Yoichiro Fujioka, Yusuke Ohba, Koji Yamada
RSC advances, 15, 1, 135, 141, 02 Jan. 2025, [International Magazine]
English, Scientific journal, Detecting biothiols like glutathione (GSH), homocysteine (Hcy), and cysteine (Cys) is key to understanding their roles in health and disease. We developed BT-DNBS, a cyanine-based turn-on fluorescent probe with a dinitrobenzenesulfonyl (DNBS) quencher group. Upon biothiol interaction, the quencher is cleaved, restoring fluorescence. The resulting probe BT-NH shows a maximum emission wavelength at 630 nm and a large Stokes shift (≈200 nm), enhancing detection accuracy. Low cytotoxicity and high time resolution make BT-DNBS suitable for live-cell imaging. Imaging of A431 cells confirmed intracellular biothiol detection, with NEM pre-treatment reducing fluorescence, verifying specificity. BT-DNBS holds promise for biomedical research, particularly in disease diagnostics.

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, 18 Feb. 2024, [International Magazine]
English, Scientific journal, 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, 17 Jan. 2024, [International Magazine]
English, Scientific journal, 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, Biophysical Society of Japan, 2024, [Peer-reviewed]
Scientific journal

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, Dec. 2023, [International Magazine]
English, Scientific journal, 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, Springer Science and Business Media LLC, 24 Jul. 2023
Scientific journal, 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, 28 Mar. 2023, [International Magazine]
English, Scientific journal, 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, Oct. 2022, [International Magazine]
English, Scientific journal, 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, Jul. 2022, [Domestic magazines]
English, Scientific journal, 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, 25 Jun. 2022, [Lead author, Corresponding author], [Domestic magazines]
English, Scientific journal, 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, Public Library of Science (PLoS), 03 Jun. 2022, [International Magazine]
English, Scientific journal, 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, Japan Society of Drug Delivery System, 25 Mar. 2022
Scientific journal

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, [International Magazine]
English, Scientific journal, 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, 15 Sep. 2020, [International Magazine]
English, Scientific journal, 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, 24 Feb. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 26 Dec. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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, 26 Dec. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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, 25 Apr. 2019, [Peer-reviewed], [Lead author], [Domestic magazines]
English, Scientific journal, 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, 31 Jan. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 13 Jun. 2018, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal

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, 01 May 2018, [Peer-reviewed]
English

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, Jan. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal

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, 48, Suppl., 139, 139, (一社)日本医学教育学会, Aug. 2017
Japanese

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, +, May 2017, [Peer-reviewed]
English, Scientific journal

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, Mar. 2017, [Peer-reviewed]
English, Scientific journal

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, Feb. 2017, [Peer-reviewed]
English, Scientific journal

Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells
Mika Horiguchi, Mari Fujioka, Takeshi Kondo, Yoichiro Fujioka, Xinxin Li, Kosui Horiuchi, Aya O. Satoh, Prabha Nepal, Shinya Nishide, Asuka Nanbo, Takanori Teshima, Yusuke Ohba
CELL STRUCTURE AND FUNCTION, 42, 1, 15, 26, 2017, [Peer-reviewed]
English, Scientific journal

Fluorescence bioimaging of intracellular signaling and its clinical application
Ohba Yusuke, Fujioka Yoichiro
Journal of Oral Biosciences, 58, 4, 113, 119, Nov. 2016, [Peer-reviewed]

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, Mar. 2016, [Peer-reviewed]
English, Scientific journal

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, 01 Feb. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, Feb. 2016, [Peer-reviewed]
English, Scientific journal

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, Jun. 2015, [Peer-reviewed]
English, Scientific journal

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, Apr. 2015, [Peer-reviewed], [Lead author]
English

[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, 87, 1, 91, 100, 日本生化学会, Feb. 2015, [Peer-reviewed]
Japanese

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, Dec. 2014, [Peer-reviewed]
English

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, Dec. 2014, [Peer-reviewed]
English, Scientific journal

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, Apr. 2014, [Peer-reviewed]
English

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, Apr. 2014, [Peer-reviewed]
English, Scientific journal

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, Jan. 2014, [Peer-reviewed]
English, Scientific journal

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, Dec. 2013, [Peer-reviewed]
English, Scientific journal

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, Nov. 2013, [Peer-reviewed], [Lead author]
English, Scientific journal

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, May 2013, [Peer-reviewed]
English, Scientific journal

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, 2013, [Peer-reviewed]
English, Scientific journal

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, Sep. 2012, [Peer-reviewed]
English, Scientific journal

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, Sep. 2011, [Peer-reviewed]
English, Scientific journal

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, Jan. 2011, [Peer-reviewed], [Lead author]
English, Scientific journal

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, Nov. 2009, [Peer-reviewed]
English, Scientific journal

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, Apr. 2009, [Peer-reviewed]
English, Scientific journal

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, Sep. 2007, [Peer-reviewed], [Lead author]
English, Scientific journal

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, 2007, [Peer-reviewed]
English

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, Jun. 2005, [Peer-reviewed]
English, Scientific journal

血管内皮細胞におけるSARS-CoV-2侵入機構の解析(Analysis of SARS-CoV-2 uptake mechanism in vascular endothelial cells)　　　　　　　　　　　　　　　
桜井 優弥, 間石 奈湖, 藤岡 容一郎, 大場 雄介, 武田 遼, 佐々木 道仁, 大場 靖子, 樋田 泰浩, 澤 洋文, 樋田 京子, 日本病理学会会誌, 113, 1, 369, 369, Feb. 2024
(一社)日本病理学会, English

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, [Lead author]

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, [Lead author]

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, [Lead author]

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, 01 Dec. 2017, [Lead author]
Japanese

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, Aug. 2017
(一社)日本医学教育学会, Japanese

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, May 2017
(一社)日本生理学会, Japanese

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, May 2017
(一社)日本細胞生物学会, Japanese

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, May 2017
(一社)日本細胞生物学会, Japanese

Fluorescence bioimaging of intracellular signaling and its clinical application
Yusuke Ohba, Yoichiro Fujioka, Journal of Oral Biosciences, 58, 4, 113, 119, 01 Nov. 2016
Japanese Association for Oral Biology, English, Book review

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, Jun. 2015
English, Summary international conference

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, 25 Feb. 2015
日本生化学会, Japanese

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, May 2014
(一社)日本細胞生物学会, Japanese

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, May 2014
(一社)日本細胞生物学会, Japanese

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, Mar. 2014
(一社)日本生理学会, Japanese

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, May 2013
(一社)日本細胞生物学会, Japanese

Galectin-9は細胞およびマウスにおけるインフルエンザAウイルスの感染を抑制する
服部ともえ, 服部ともえ, 丸山隼輝, 藤岡容一朗, 中山洋佑, 大場雄介, 仁木敏郎, 有川智博, 宮崎忠昭, 平島光臣, 喜田宏, 喜田宏, 日本ウイルス学会学術集会プログラム・抄録集, 60th, 2012

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
Japanese

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, May 2011
(一社)日本細胞生物学会, English

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, May 2011
(一社)日本細胞生物学会, English

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, May 2010
(一社)日本細胞生物学会, English

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, 28 Jul. 2007
Japanese

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, Feb. 2007
English, Summary international conference

Influenza A virus entry into host cells via calcium signaling-mediated endocytosis　　　　　　　　　　　　　　　
Yoichiro Fujioka
1st Material Symbiosis International symposium/3rd GI-CoRE/GSD International symposium/28th Pharmascience Forum “Joint Symposium for Young Researchers”, 30 Mar. 2023, English, Invited oral presentation
[Invited]

インフルエンザウイルス感染促進メカニズムの解明　　　　　　　　　　　　　　　
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, 14 Mar. 2023, Japanese

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　　　　　　　　　　　　　　　
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, 03 Mar. 2023, Japanese, Invited oral presentation
[Invited]

Molecular mechanisms of influenza virus entry into host cells　　　　　　　　　　　　　　　
Yoichiro Fujioka
Sialoglyco2022, 06 Sep. 2022, English, Invited oral presentation
[Invited]

新規エンドソーム構造に関する形態学的解析　　　　　　　　　　　　　　　
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, 03 Sep. 2022, Japanese, Oral presentation

インフルエンザウイルス感染伝播機構の解明　　　　　　　　　　　　　　　
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, 28 Jun. 2022, Japanese, Poster presentation

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　　　　　　　　　　　　　　　
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, 28 Jun. 2022, Japanese, Oral presentation

医学英語　　　　　　　　　　　　　　　
北海道大学

生理学　　　　　　　　　　　　　　　
北海道大学

医学研究法II　　　　　　　　　　　　　　　
北海道大学

蛍光イメージング入門　　　　　　　　　　　　　　　
Hokkaido University

フレッシュマンセミナー　　　　　　　　　　　　　　　
北海道大学

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
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2027
藤岡 容一朗
日本学術振興会, 基盤研究(B), 北海道大学, 24K02255

カルシウム波伝播によるウイルス感染促進機構の解明
科学研究費助成事業
28 Jun. 2024 - 31 Mar. 2026
藤岡 容一朗
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 24K22042

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
科学研究費助成事業 基盤研究(B)
Apr. 2021 - Mar. 2024
藤岡 容一朗
日本学術振興会, 基盤研究(B), 北海道大学, 21H02735

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
科学研究費助成事業 新学術領域研究(研究領域提案型)
Apr. 2021 - Mar. 2023
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。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 21H00413

Development of isotropic resolution microscopy using fluorescence polarization and visualization of cell membrane dynamics
Grants-in-Aid for Scientific Research
07 Oct. 2019 - 31 Mar. 2022
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.
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Hokkaido University, 19KK0198

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
科学研究費助成事業 新学術領域研究(研究領域提案型)
01 Apr. 2019 - 31 Mar. 2021
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。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 19H04823

Identification of key molecule for influenza virus entry
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
01 Apr. 2016 - 31 Mar. 2020
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (A), Hokkaido University, 16H06227

Investigation of a physiological role of endosome-mitochondrial interaction by fluorescence bioimaging
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2015 - 31 Mar. 2017
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 15K15023

Intracellular signaling machinery controlling endocytosis and uptake of extracellular materials
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2014 - 31 Mar. 2017
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 26293041

The mechanism of influenza virus internalization into host cells via calcium signaling-mediated endocytosis
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
01 Apr. 2014 - 31 Mar. 2016
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 26860254

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
科学研究費助成事業 特別研究員奨励費
2011 - 2013
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。
日本学術振興会, 特別研究員奨励費, 北海道大学, 11J04616