Electron microscopic analysis of lipid clusters in the inner leaflet of plasma membrane

• Project/Area Number: 15K08167
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General anatomy (including histology/embryology)
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Murate Motohide 国立研究開発法人理化学研究所, 開拓研究本部, 研究員 (30311369)
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 細胞膜 / 脂質二重層 / ホスファチジルセリン / ホスファチジルエタノールアミン / 免疫電子顕微鏡法 / ショウジョウバエ / スクランブラーゼ / フリッパーゼ / アポトーシス / 培養細胞 / スフィンゴミエリン / 非対称分布

Outline of Final Research Achievements

We examined transbilayer distribution of phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the plasma membrane of normal and lipid transfer protein-depleted Drosophila cells by freeze-fracture replica immunoelectron microscopy. Our results indicate that both lipids were considerably localized in the outer leaflet, in addition to the inner leaflet of normal cell plasma membranes, whereas they distributed almost restrictedly in the inner leaflet of mammalian cell plasma membranes as reported by our previous studies. This remarkable distribution of PS in the plasma membranes was disturbed in flippase-depleted cells, but it was not affected in scramblase-depleted cells. Futhermore, the depletion of neither scrambrase nor flippase influenced the transbilayer distribution of PE. These results suggest that the mechanism of formation and maintenance of lipid distribution between two leaflets is different in each species.

Academic Significance and Societal Importance of the Research Achievements

本研究により明らかにされたショウジョウバエ細胞における細胞膜脂質二重層の内外層における脂質の対称分布は、これまで哺乳類細胞を用いて解析され、また認識されてきた非対称分布とは大きく異なり、細胞膜の脂質分布の多様性を示すとともに、細胞膜研究に対する新たな認識を提供することができた。また、脂質の分布に影響を及ぼすと考えられている酵素を欠損させても、すべての脂質の分布に影響を及ぼすわけではないことを明らかにし、脂質分布の形成と維持の機構に関する知見を提供することができた。

Research Products

• [Journal Article] Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes. (2019)
  • Author(s): Inaba T., Murate M., Tomishige N., Lee Y.-F., Hullin-Matsuda F., Pollet B., Humbert N., Mely Y., Sako Y., Greimel P. and Kobayashi T.
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 5812-5812
  • DOI: 10.1038/s41598-019-42247-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Protein probes to visualize sphingomyelin and ceramide. (2018)
  • Author(s): Hullin-Matsuda F., Murate M. and Kobayashi T.
  • Journal Title: Chemistry and Physics of Lipids Volume: 216 Pages: 132-141
  • DOI: 10.1016/j.chemphyslip.2018.09.002
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Acute accumulation of free cholesterol induces the degradation of perilipin 2 and Rab18-dependent fusion of ER and lipid droplets in cultured human hepatocytes. (2016)
  • Author(s): Makino A, Hullin-Matsuda F, Murate M, Abe M, Tomishige N, Fukuda M, Yamashita S, Fujimoto T, Vidal H, Lagarde M, Delton I, Kobayashi T.
  • Journal Title: Mol Biol Cell Volume: 27 Issue: 21 Pages: 3293-3304
  • DOI: 10.1091/mbc.e15-10-0730
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Probing phosphoethanolamine-containing lipids in membranes with duramycin/cinnamycin and aegerolysin proteins. (2016)
  • Author(s): Hullin-Matsuda F., Makino A., Murate M., Kobayashi T.
  • Journal Title: Biochimie Volume: 130 Pages: 81-90
  • DOI: 10.1016/j.biochi.2016.09.020
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A novel spingomyeling/cholesterol specific probe reveals the dynamics of the membrane domain during virus and in Nieman-Pick type C (2016)
  • Author(s): Makino, A., Sako, Y. et al.
  • Journal Title: FASEB J. Volume: 31 Issue: 4 Pages: 1301-1322
  • DOI: 10.1096/fj.201500075r
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation. (2016)
  • Author(s): Inaba T, Kishimoto T, Murate M, Tajima T, Sakai S, Abe M, Makino A, Tomishige N, Ishitsuka R, Ikeda Y, Takeoka S, Kobayashi T.
  • Journal Title: Proc Natl Acad Sci USA Volume: 113 Issue: 28 Pages: 7834-7839
  • DOI: 10.1073/pnas.1603513113
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Revisiting transbilayer distribution of lipids in the plasma membrane. (2016)
  • Author(s): Murate M,, Kobayashi T.
  • Journal Title: Chem. Phys. Lipids Volume: 194 Pages: 58-71
  • DOI: 10.1016/j.chemphyslip.2015.08.009
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Visualization of the heterogeneous distribution of sphingomyelin associated with cytokinesis, cell polarity and sphingolipidosis (2015)
  • Author(s): Makino A, Abe M, Murate M, Inaba T, Yilmaz N, Hullin-Matsuda F, Kishimoto T, Schieber NL, Taguchi T, Arai H, Anderluh G, Parton RG, Kobayashi T.
  • Journal Title: FASEB J Volume: 29 Issue: 2 Pages: 477-493
  • DOI: 10.1096/fj.13-247585
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Transbilayer distribution of lipids at nano scale. (2015)
  • Author(s): Murate M, Abe M, Kasahara K, Iwabuchi K, Umeda M, Kobayashi T
  • Journal Title: J Cell Sci Volume: 128 Pages: 1627-1638
  • DOI: 10.1242/jcs.163105
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates (2015)
  • Author(s): Bhat HB, Ishitsuka R, Inaba T, Murate M, Abe M, Makino A, Kohyama-Koganeya A, Nagao K, Kurahashi A, Kishimoto T, Tahara M, Yamano A, Nagamune K, Hirabayashi Y, Juni N, Umeda M, Fujimori F, Nishibori K, Yamaji-Hasegawa A, Greimel P, Kobayashi T.
  • Journal Title: FASEB J Volume: 29(9) Issue: 9 Pages: 3920-34
  • DOI: 10.1096/fj.15-272112
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CARTS biogenesis requires VAP-lipid transfer protein complexes functioning at the endoplasmic reticulum-Golgi interface. (2015)
  • Author(s): Wakana Y., Kotake R., Oyama N., Murate M., Kobayashi T., Arasaki K., Inoue H., Tagaya M.
  • Journal Title: Mol. Biol Cell Volume: 26 Issue: 25 Pages: 4686-4699
  • DOI: 10.1091/mbc.e15-08-0599
  • Peer Reviewed / Open Access
• [Presentation] Phospholipase C beta1 induces membrane tubulation and is involved in caveolae formation. (2018)
  • Author(s): Kobayashi T., Inaba T., Kishimoto T., Murate M., Tajima T., Abe M., Makino A., Tomishige N. and Takeoka S.
  • Organizer: 第91回日本生化学会大会
  • Invited
• [Presentation] Transbilayer distribution of lipids in the plasma membrane revealed by immunoelectron microscopy. (2017)
  • Author(s): Murate M.
  • Organizer: University of Strasbourg-RIKEN Workshop on Membrane Lipidology
  • Int'l Joint Research
• [Presentation] Transbilayer distribution of lipids in the plasma membrane revealed by immuno-electron microscopy. (2016)
  • Author(s): Murate M.
  • Organizer: 第57回International Conference on the Bioscience of Lipids
  • Place of Presentation: フランス シャモニー
  • Year and Date: 2016-09-08
  • Int'l Joint Research
• [Presentation] Phospholipase C beta 1 induces membrane tubulation and is involved in caveolae formation. (2016)
  • Author(s): Inaba T., Kishimoto T., Murate M., Tajima T., Sakai S., Abe M., Makino A., Tomishige N., Ishitsuka R., Ikeda Y., Takeoka S., Kobayashi T.
  • Organizer: 第57回International Conference on the Bioscience of Lipids
  • Place of Presentation: フランス シャモニー
  • Year and Date: 2016-09-05
  • Int'l Joint Research
• [Presentation] 細胞膜の脂質二重層を構成する各種の脂質が示す非対称分布とその破綻 (2015)
  • Author(s): 村手源英, 阿部充宏, 笠原浩二, 岩渕和久, 梅田真郷, 小林俊秀
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Transbilayer lipid distribution in the plasma membrane at nano scale. (2015)
  • Author(s): Murate M., Abe M., Kasahara K., Iwabuchi K., Umeda M., Kobayashi T.
  • Organizer: 第11回GERLI Lipidomics Meeting
  • Place of Presentation: ビショフスハイム、フランス
  • Year and Date: 2015-10-25
  • Int'l Joint Research
• [Presentation] 細胞膜の脂質二重層を構成する様々な脂質が示す非対称分布の電子顕微鏡レベルでの解析 (2015)
  • Author(s): 村手源英, 阿部充宏, 笠原浩二, 岩渕和久, 梅田真郷, 小林俊秀
  • Organizer: 第67回日本細胞生物学会年会
  • Place of Presentation: タワーホール船堀（東京都江戸川区）
  • Year and Date: 2015-06-30
• [Presentation] Transbilayer and lateral distribution of lipids in the plasma membrane at nano scale. (2015)
  • Author(s): Murate M., Abe M., Kasahara K., Iwabuchi K., Umeda M., Kobayashi T.
  • Organizer: 第10回スフィンゴテラピィ研究会
  • Place of Presentation: ホテルアローレ（石川県加賀市）
  • Year and Date: 2015-06-16
• [Presentation] 細胞膜の内外層における構成脂質のナノメートルレベルでの局在解析 (2015)
  • Author(s): 村手源英, 阿部充宏, 岩渕和久, 梅田真郷, 小林俊秀
  • Organizer: 第57回日本脂質生化学会年会
  • Place of Presentation: 一橋大学一橋講堂（東京都千代田区）
  • Year and Date: 2015-05-28
• [Remarks] 理研 脂質の統合的理解
  • URL: http://lipidology.riken.jp/index.html
• [Remarks] 理化学研究所 脂質の統合的理解 研究プロジェクト
  • URL: http://lipidology.riken.jp/index.html
• [Remarks] 理化学研究所 脂質の統合的理解
  • URL: http://lipidology.riken.jp/index.html