研究者データベース

岸本 拓磨(キシモト タクマ)
遺伝子病制御研究所 疾患制御研究部門
助教

基本情報

所属

  • 遺伝子病制御研究所 疾患制御研究部門

職名

  • 助教

学位

  • 博士(医学)(2006年03月 北海道大学)

J-Global ID

研究分野

  • ライフサイエンス / 細胞生物学

職歴

  • 2017年01月 - 現在 北海道大学 遺伝子病制御研究所 助教
  • 2017年01月 - 現在 北海道大学 大学院生命科学院 助教
  • 2014年04月 - 2016年12月 杏林大学 医学部 助教
  • 2011年04月 - 2014年03月 理化学研究所 基礎科学特別研究員
  • 2010年04月 - 2011年03月 理化学研究所 特別研究員
  • 2006年08月 - 2010年03月 米国カルフォルニア大学バークレー校 博士研究員

学歴

  • 2002年04月 - 2006年03月   北海道大学   医学研究科   癌医学専攻
  • 2000年04月 - 2002年03月   北海道大学   工学研究科   分子化学専攻
  • 1996年04月 - 2000年03月   北海道大学   工学部   応用化学科

所属学協会

  • 酵母遺伝学フォーラム   日本脂質生化学会   日本細胞生物学会   日本生化学会   

研究活動情報

論文

  • Kaori Kanemaru, Makoto Shimozawa, Manabu Kitamata, Rikuto Furuishi, Hinako Kayano, Yui Sukawa, Yuuki Chiba, Takatsugu Fukuyama, Junya Hasegawa, Hiroki Nakanishi, Takuma Kishimoto, Kazuya Tsujita, Kazuma Tanaka, Toshiki Itoh, Junko Sasaki, Takehiko Sasaki, Kiyoko Fukami, Yoshikazu Nakamura
    Nature Communications 13 1 2347 - 2347 2022年05月 
    Abstract Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.
  • Tetsuo Mioka, Tian Guo, Shiyao Wang, Takuma Tsuji, Takuma Kishimoto, Toyoshi Fujimoto, Kazuma Tanaka
    Journal of cell science 135 5 2022年03月01日 
    Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.
  • Takuma Kishimoto, Tetsuo Mioka, Eriko Itoh, David E Williams, Raymond J Andersen, Kazuma Tanaka
    Molecular biology of the cell mbcE20110699  2021年05月26日 [査読有り]
     
    Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.
  • Takuma Kishimoto, Nario Tomishige, Motohide Murate, Reiko Ishitsuka, Hubert Schaller, Yves Mély, Kazumitsu Ueda, Toshihide Kobayashi
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020年03月12日 [査読有り][通常論文]
     
    During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.
  • Mamoru Miyasaka, Tetsuo Mioka, Takuma Kishimoto, Eriko Itoh, Kazuma Tanaka
    PloS one 15 7 e0236520  2020年 [査読有り][通常論文]
     
    In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.
  • Tetsuo Mioka, Konomi Fujimura-Kamada, Nahiro Mizugaki, Takuma Kishimoto, Takamitsu Sano, Hitoshi Nunome, David E. Williams, Raymond J. Andersen, Kazuma Tanaka
    Molecular Biology of the Cell 29 10 1203 - 1218 2018年05月15日 [査読有り][通常論文]
     
    Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.
  • Asami Makino, Mitsuhiro Abe, Reiko Ishitsuka, Motohide Murate, Takuma Kishimoto, Shota Sakai, Francoise Hullin-Matsuda, Yukiko Shimada, Takehiko Inaba, Hideyuki Miyatake, Hideko Tanaka, Atsushi Kurahashi, Chan-Gi Pack, Rinshi S. Kasai, Shuku Kubo, Nicole L. Schieber, Naoshi Dohmae, Naoya Tochio, Kyoji Hagiwara, Yutaka Sasaki, Yoko Aida, Fumihiro Fujimori, Takanori Kigawa, Kozo Nishibori, Robert G. Parton, Akihiro Kusumi, Yasushi Sako, Gregor Anderluh, Makoto Yamashita, Tetsuyuki Kobayashi, Peter Greimel, Toshihide Kobayashi
    FASEB JOURNAL 31 4 1301 - 1322 2017年04月 [査読有り][通常論文]
     
    We identified a novel, nontoxicmushroomprotein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM-and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C. =
  • Kishimoto T, Ishitsuka R, Kobayashi T
    Biochimica et biophysica acta 1861 8 Pt B 812 - 829 2016年08月 [査読有り][通常論文]
  • Inaba T, Kishimoto T, Murate M, Tajima T, Sakai S, Abe M, Makino A, Tomishige N, Ishitsuka R, Ikeda Y, Takeoka S, Kobayashi T
    Proceedings of the National Academy of Sciences of the United States of America 113 28 7834 - 7839 2016年07月 [査読有り][通常論文]
     
    Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase C beta 1 (PLC beta 1) as a new candidate. PLC beta 1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLC beta 1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLC beta 1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLC beta 1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLC beta 1: plasma membrane remodeling, and in particular, caveolae formation.
  • Aoyagi K, Ohara-Imaizumi M, Itakura M, Torii S, Akimoto Y, Nishiwaki C, Nakamichi Y, Kishimoto T, Kawakami H, Harada A, Takahashi M, Nagamatsu S
    Diabetes 65 6 1648 - 1659 2016年06月 [査読有り][通常論文]
     
    VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic beta-cells is largely unknown. To elucidate the physiological role of VAMP7 in beta-cells, we generated pancreatic beta-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient beta-cells showed defective autophagosome formation and reduced mitochondria! function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient beta-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre beta-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in beta-cells.
  • Hema Balakrishna Bhat, Reiko Ishitsuka, Takehiko Inaba, Motohide Murate, Mitsuhiro Abe, Asami Makino, Ayako Kohyama-Koganeya, Kohjiro Nagao, Atsushi Kurahashi, Takuma Kishimoto, Michiru Tahara, Akinori Yamano, Kisaburo Nagamune, Yoshio Hirabayashi, Naoto Juni, Masato Umeda, Fumihiro Fujimori, Kozo Nishibori, Akiko Yamaji-Hasegawa, Peter Greimel, Toshihide Kobayashi
    FASEB JOURNAL 29 9 3920 - 3934 2015年09月 [査読有り][通常論文]
     
    Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family-pleurotolysin A2 (PlyA2; K-D = 12 nM), ostreolysin (Oly; K-D = 1.3 nM), and erylysin A (EryA; K-D = 1.3 nM)-strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K-D = 41 mu M), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.
  • Yoshio Yamauchi, Noriyuki Iwamoto, Maximillian A. Rogers, Sumiko Abe-Dohmae, Toyoshi Fujimoto, Catherine C. Y. Chang, Masato Ishigami, Takuma Kishimoto, Toshihide Kobayashi, Kazumitsu Ueda, Koichi Furukawa, Ta-Yuan Chang, Shinji Yokoyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 39 23464 - 23477 2015年09月 [査読有り][通常論文]
     
    Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.
  • Yuko Arita, Shinichi Nishimura, Reiko Ishitsuka, Takuma Kishimoto, Junichi Ikenouchi, Kumiko Ishii, Masato Umeda, Shigeki Matsunaga, Toshihide Kobayashi, Minoru Yoshida
    CHEMISTRY & BIOLOGY 22 5 604 - 610 2015年05月 [査読有り][通常論文]
     
    Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3 beta-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.
  • Lee S, Uchida Y, Wang J, Matsudaira T, Nakagawa T, Kishimoto T, Mukai K, Inaba T, Kobayashi T, Molday RS, Taguchi T, Arai H
    The EMBO journal 34 5 669 - 688 2015年03月 [査読有り][通常論文]
     
    P-4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P-4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2(-/-) mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2(+/+) mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.
  • Asami Makino, Mitsuhiro Abe, Motohide Murate, Takehiko Inaba, Neval Yilmaz, Francoise Hullin-Matsuda, Takuma Kishimoto, Nicole L. Schieber, Tomohiko Taguchi, Hiroyuki Arai, Gregor Anderluh, Robert G. Parton, Toshihide Kobayashi
    FASEB JOURNAL 29 2 477 - 493 2015年02月 [査読有り][通常論文]
     
    Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation ofSM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.
  • Hema Balakrishna Bhat, Takuma Kishimoto, Mitsuhiro Abe, Asami Makino, Takehiko Inaba, Motohide Murate, Naoshi Dohmae, Atsushi Kurahashi, Kozo Nishibori, Fumihiro Fujimori, Peter Greimel, Reiko Ishitsuka, Toshihide Kobayashi
    JOURNAL OF LIPID RESEARCH 54 10 2933 - 2943 2013年10月 [査読有り][通常論文]
     
    A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply) A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-beta-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding.(jlr) Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.
  • Takuma Kishimoto, Yidi Sun, Christopher Buser, Jian Liu, Alphee Michelot, David G. Drubin
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 44 E979 - E988 2011年11月 [査読有り][通常論文]
     
    During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.
  • Ji-Young Youn, Helena Friesen, Takuma Kishimoto, William M. Henne, Christoph F. Kurat, Wei Ye, Derek F. Ceccarelli, Frank Sicheri, Sepp D. Kohlwein, Harvey T. McMahon, Brenda J. Andrews
    MOLECULAR BIOLOGY OF THE CELL 21 17 3054 - 3069 2010年09月 [査読有り][通常論文]
     
    BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161-Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.
  • Kenzi Nakano, Takaharu Yamamoto, Takuma Kishimoto, Takehiro Noji, Kazuma Tanaka
    MOLECULAR BIOLOGY OF THE CELL 19 4 1783 - 1797 2008年04月 [査読有り][通常論文]
     
    Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50 Delta mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3 Delta/dnf1 Delta dnf2 Delta mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1 Delta fpk2 Delta mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.
  • Kishimoto T, Yamamoto T, Tanaka K
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 6 Suppl 767 - 775 2006年05月 [査読無し][通常論文]
  • T Kishimoto, T Yamamoto, K Tanaka
    MOLECULAR BIOLOGY OF THE CELL 16 12 5592 - 5609 2005年12月 [査読有り][通常論文]
     
    Specific changes in membrane lipid composition are implicated in actin cytoskeletal organization, vesicle formation, and control of cell polarity. Cdc50p, a membrane protein in the endosomal/trans-Golgi network compartments, is a noncatalytic subunit of Drs2p, which is implicated in translocation of phospholipids across lipid bilayers. We found that the cdc50 Delta mutation is synthetically lethal with mutations affecting the late steps of ergosterol synthesis (erg2 to erg6). Defects in cell polarity and actin organization were observed in the cdc50 Delta erg3 Delta mutant. In particular, actin patches, which are normally found at cortical sites, were assembled intracellularly along with their assembly factors, including Las17p, Abp1p, and Sla2p. The exocytic SNARE Snc1p, which is recycled by an endocytic route, was also intracellularly accumulated, and inhibition of endocytic internalization suppressed the cytoplasmic accumulation of both Las17p and Snc1p. Simultaneous loss of both phospholipid asymmetry and sterol structural integrity could lead to accumulation of endocytic intermediates capable of initiating assembly of actin patches in the cytoplasm.
  • Nishimura K, Tsuciya H, Takada K, Kishimoto T, Imai H, Mikami Y, Munekata M
    Human cell 16 4 217 - 229 2003年12月 [査読有り][通常論文]

講演・口頭発表等

  • ステロール分子動態可視化法による細胞膜ステロール維持機構の遺伝学的解析  [通常講演]
    第55回 酵母遺伝学フォーラム 2022年09月 口頭発表(一般)
  • 細胞膜ステロール活性化を介した細胞内ステロールホメオシタシス機構の解析  [通常講演]
    岸本拓磨, 賈 子木, 田中一馬
    2022年06月 ポスター発表
  • 細胞膜ステロール活性化の制御機構による細胞膜秩序の維持に関する解析  [通常講演]
    岸本拓磨, 賈 子木, 田中一馬
    2022年06月 口頭発表(一般)
  • 細胞膜リン脂質非対称性は膜ステロールの保持に重要な役割を持つ  [通常講演]
    岸本拓磨, 田中一馬
    第94回 日本生化学会 2021年11月 ポスター発表
  • 細胞膜リン脂質非対称性の生理的意義の解明  [通常講演]
    岸本拓磨, 田中一馬
    第54回酵母遺伝学フォーラム研究報告会 2021年08月 口頭発表(一般)
  • 細胞膜ステロール活性化を制御する出芽酵母Sfk1および関連タンパク質の機能解析  [通常講演]
    岸本拓磨, 賈 子木, 田中一馬
    第63回 日本脂質生化学会 2021年06月 口頭発表(一般)
  • 岸本拓磨
    第93回日本生化学会大会 2020年09月 シンポジウム・ワークショップパネル(公募)
  • リン脂質非対称性による細胞膜インテグリティ制御機構の解析
    岸本拓磨, 三岡哲生, 田中一馬
    第61回日本脂質化学会大会 2019年07月 口頭発表(一般)
  • 岸本拓磨, 三岡哲生, 田中一馬
    第19回日本蛋白質科学会年会 第71回日本細胞生物学会大会 合同年次大会 2019年06月 ポスター発表
  • リン脂質の非対称性の崩壊は細胞膜の完全性に異常を引き起こす
    岸本拓磨, 三岡哲夫, 田中一馬
    60th The International Conference on the Bioscience of Lipids 2019年06月 ポスター発表
  • 岸本 拓磨, 三岡 哲生, 田中 一馬
    第91回日本生化学会大会 2018年09月 口頭発表(一般)
  • 岸本拓磨, 三岡哲生, 田中一馬
    第51回酵母遺伝学フォーラム 2018年09月 口頭発表(一般)
  • 岸本 拓磨, 三岡 哲生, 田中 一馬
    第55回生化学会北海道支部例会 2018年07月 口頭発表(一般)
  • コレステロールの細胞膜二層間動態と膜変形機構におけるフィードバック制御機構  [通常講演]
    岸本 拓磨, 小林俊秀
    第54回日本生化学会北海道支部例会 2017年07月 口頭発表(一般)
  • コレステロールの細胞膜二層間動態と低分子量GTPaseCdc42 によるフィードバック制御機構
    岸本拓磨, 永松信哉, 植田和光, 小林俊秀
    第67回日本細胞生物会 2015年06月 シンポジウム・ワークショップパネル(公募)
  • コレステロール細胞膜二層間動態が及ぼす細胞膜変形機構
    岸本拓磨 永松信哉 植田和光 小林俊秀
    第57回日本脂質生化学会 2015年05月 口頭発表(一般)

その他活動・業績

  • リン脂質非対称性により制御される細胞膜ステロールの保持機構
    岸本拓磨, 田中一馬 生化学 94 (1) 82 -86 2022年02月 [査読有り][招待有り]
  • リン脂質非対称性によるステロールコントロールを介した 細胞膜インテグリティ制御機構の解明
    岸本拓磨, 田中一馬 日本脂質生化学研究 62 81 -82 2020年05月 [査読無し][通常論文]
  • 生体膜ダイナミクスと脂質 細胞質分裂、極性形成、スフィンゴ脂質蓄積症におけるスフィンゴミエリンの不均一な膜分布の可視化
    牧野 麻美, 阿部 充宏, 村手 源英, 稲葉 岳彦, Neval Yilmaz, Hullin-Matsuda Francoise, 岸本 拓磨, 田口 友彦, 新井 洋由, 小林 俊秀 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2W11 -p 2015年12月 [査読無し][通常論文]
  • 稲葉岳彦, 岸本拓磨, 岸本拓磨, 田島拓也, 牧野麻美, 阿部充宏, 村手源英, 石塚玲子, 池田康夫, 武岡真司, 小林俊秀 日本細胞生物学会大会要旨集 67th 161 2015年06月05日 [査読無し][通常論文]
  • 稲葉岳彦, 岸本拓磨, 田島拓也, 牧野麻美, 阿部充宏, 村手源英, 石塚玲子, 池田康夫, 武岡真司, 小林俊秀 脂質生化学研究 56 38 -39 2014年05月28日 [査読無し][通常論文]
  • 脳の脂質と疾患
    岸本拓磨, 稲葉岳彦, 酒井翔太, 小林俊秀 最新精神医学 17 (3) 201 -210 2012年 [査読無し][招待有り]
  • 岸本 拓磨, 山本 隆晴, 田中 一馬 蛋白質核酸酵素 51 (6) 767 -775 2006年05月

受賞

  • 2021年08月 酵母遺伝学フォーラム 第54回研究報告会 会長賞
     細胞膜リン脂質非対称性の生理的意義の解明
  • 2011年07月 内藤記念科学振興財団 第30回内藤カンファレンス優秀ポスター賞
     Dynamic behaviors of lipid domain and actin cytoskeleton in plasma membrane during HeLa cell spreading. 
    受賞者: 岸本拓磨 小林俊秀

共同研究・競争的資金等の研究課題

  • 細胞膜ステロールの維持に関わる因子の機能解明と新たな因子の探索
    日本学術振興会:科学研究費助成 基盤研究(C)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 岸本 拓磨
  • ゴルジ体膜における脂質非対称性の生理的意義の解明
    日本学術振興会:科学研究費助成 基盤研究(C)
    研究期間 : 2019年04月 -2024年03月 
    代表者 : 田中 一馬
  • 出芽酵母可視化スクリーニングによる活 性化ステロール分布領域制御因子の特定 と制御機構の解明
    公益財団法人 発酵研究所:一般研究助成
    研究期間 : 2021年04月 -2023年03月 
    代表者 : 岸本 拓磨
  • 日本学術振興会:科学研究費助成 基盤研究(C)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 岸本 拓磨
  • インスリン分泌における細胞膜脂質非対称の生理学的意義の解明 -コレステロールの二重膜間輸送が及ぼす影響-
    日本私立学校振興・共済事業団:学術研究振興資金(若手研究者奨励金)
    研究期間 : 2016年04月 -2017年03月 
    代表者 : 岸本 拓磨
  • ライブセルイメージングによるインスリン分泌における細胞膜コレステロールドメイン機能の解明
    小野医学研究財団:研究奨励助成
    研究期間 : 2016年04月 -2017年03月 
    代表者 : 岸本 拓磨
  • 生体膜脂質ドメインの構成とダイナミクスの分子解析
    日本学術振興会:科学研究費助成 基盤研究(B)
    研究期間 : 2013年04月 -2017年03月 
    代表者 : 小林 俊秀
  • HeLa細胞の細胞伸展における細胞膜上の脂質ドメインとアクチン細胞骨格の動的挙動
    内藤記念科学振興財団:内藤記念特定研究助成金
    研究期間 : 2011年04月 -2012年03月 
    代表者 : 岸本 拓磨
  • 脂質分布と膜物性の同時可視化で迫る糸状仮足形成における脂質ラフト動態と機能の解明
    日本学術振興会:科学研究費助成 若手研究(B)
    代表者 : 岸本 拓磨

教育活動情報

主要な担当授業

  • 生命システム科学基礎論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 生命システム, 生命機能, 研究方法論, 研究技術論
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 生命システム, 生命機能, 研究方法論, 研究技術論


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