Updated on 2026/03/11

写真a

 
Akikazu Fujita
 
Organization
Research Field in Veterinary Medicine, Agriculture, Fisheries and Veterinary Medicine Area Joint faculty of Veterinary Medicine Joint Department of Veterinary Medicine Professor
Title
Professor
Degree
Ph.D (1996.3 Osaka Prefecture University)
External link

Research Interests

  • 脂質，ナノスケール，電子顕微鏡，エンドサイトーシス

  • 電子顕微鏡

  • 超微細構造

  • 脂質

  • 細胞膜

  • 細胞内情報伝達

  • ラフト

  • カベオラ

  • Autophagy

  • Ultra fine structure

  • Signal transduction

  • Raft

  • Plasma membrane

  • Lipid

  • Electron microscopy

  • Caveolae

Research Areas

細胞生物, Anatomy, Cell biology, Veterinary medical science

Education

  • 1992.4 - 1996.3    Osaka Prefecture University   Graduate School of Agriculture and Life Science

  • 1985.4 - 1992.3    Osaka Prefecture University

Research History

Professional Memberships

  • 2015.10    日本獣医学会

  • 2015.10    顕微鏡学会

  • 2015.10    日本細胞生物学会

  • 2015.10    日本組織細胞化学会

  • 2015.10    日本解剖学会

  • 2015.10    日本生化学会

  • 2011    Association of Veterinary Science researchmap

  •    解剖学会 researchmap

  •    組織細胞化学会 researchmap

  •    細胞生物学会 researchmap

  •    生化学会 researchmap

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Committee Memberships

Studying abroad experiences

  • 2003.10 - 2004.8   The University of Queensland  

  • 1997.8 - 1997.10   University of Oslo   Post doctor

  • 1994.7 - 1995.8   Georgetown University   Research fellow

Qualification acquired

  • Veterinarian

 

Papers

  • Mineoka N, Konishi R, Nakashima Y, Muramoto M, Fukuda K, Kuriyama S, Masatani T, Fujita A .  Regulation of macroautophagy and microautophagic lipophagy by phosphatidylserine synthase Cho1 and external ethanolamine. .  Biochimica et biophysica acta. Molecular and cell biology of lipids1871 ( 2 ) 159702   2025.10

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    DOI： 10.1016/j.bbalip.2025.159702

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  • Moe Muramoto, Nanaru Mineoka, Kayoko Fukuda, Sayuri Kuriyama, Tatsunori Masatani, Akikazu Fujita .  Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy. .  Biochimica et biophysica acta. Biomembranes1866 ( 4 ) 184308 - 184308   2024.4Reviewed International journal

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    Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

    DOI： 10.1016/j.bbamem.2024.184308

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  • Rikako Konishi, Kayoko Fukuda, Sayuri Kuriyama, Tatsunori Masatani, Xuenan Xuan, Akikazu Fujita .  Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis. .  Histochemistry and cell biology160 ( 4 ) 279 - 291   2023.7Reviewed International journal

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    Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

    DOI： 10.1007/s00418-023-02218-0

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  • Moe Muramoto, Yuki Yamakuchi, Rikako Konishi, Shiomi Koudatsu, Hiromu Tomikura, Kayoko Fukuda, Sayuri Kuriyama, Yuna Kurokawa, Tatsunori Masatani, Hisanori Tamaki, Akikazu Fujita .  Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation .  Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids1867 ( 9 ) 159184 - 159184   2022.6Reviewed International journal

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    Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.

    DOI： 10.1016/j.bbalip.2022.159184

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  • 藤田 秋一 .  膜脂質の疎水性領域を可視化する逆転レプリカ法の開発 .  上原記念生命科学財団研究報告集35   1 - 5   2021.12

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    Language：Japanese   Publisher：(公財)上原記念生命科学財団  

  • 1. Yamakuchi, Y., Kurokawa, Y., Konishi, R., Fukuda, K., Masatani, T., Fujita, A. .  Selective increment of phosphatidylserine on the autophagic body membrane in yeast’s vacuole. .  FEBS Lett.595 ( 17 ) 2197 - 2207   2021.8Reviewed International journal

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    In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

    DOI： 10.1002/1873-3468.14167

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  • 2. Koudatsu, S., Masatani, T., Konishi, R., Asada, M., Hakimi, H., Kurokawa, Y., Tomioku, K., Kaneko, O., Fujita, A. .  Glycosphingolipid GM3 is localized in both exoplasmic and cytoplasmic leaflets of Plasmodium falciparum malaria parasite plasma membrane. .  Sci. Rep.11 ( 1 ) 14890 - 14890   2021.7Reviewed International journal

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    Lipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes.

    DOI： 10.1038/s41598-021-94037-3

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  • 3. Kurokawa, Y., Konishi, R., Yoshida, A., Tomioku, K., Tanabe, K., Fujita, A. .  The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane. .  Histochem. Cell Biol.156 ( 2 ) 109 - 121   2021.5Reviewed International journal

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    Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze-fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze-fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

    DOI： 10.1007/s00418-021-01989-8

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  • Rikako Konishi, Yuna Kurokawa, Kanna Tomioku, Tatsunori Masatani, Xuenan Xuan, Akikazu Fujita .  Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii. .  European journal of cell biology100 ( 2 ) 151149 - 151149   2021.3Reviewed International journal

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    Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1's gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.

    DOI： 10.1016/j.ejcb.2020.151149

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  • Rikako Konishi, Yuna Kurokawa, Kanna Tomioku, Tatsunori Masatani, Xuenan Xuan, Akikazu Fujita .  Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii .  Eur. J. Cell Biol.100   151149   2021.1Reviewed

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  • 藤田 秋一 .  The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane .  Hitochemistry and Cell Biology-   2021Reviewed

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  • Microautophagy in the yeast vacuole depends on the activities of phosphatidylinositol 4-kinases, Stt4p and Pik1p .  Biochim. Biophys. Acta. Biomembrane.1862   183416   2020.7Reviewed

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    DOI： 10.1016/j.bbamem.2020.183416

    Other Link： https://www.ncbi.nlm.nih.gov/pubmed/32726584

  • 1. Kurokawa, Y., Masatani, T., Konishi R, Tomioku, K., Xuan, X., Fujita, A. .  Nanoscale analysis reveals no domain formation of glycosylphosphatidylinositol-anchored protein SAG1 in the plasma membrane of living Toxoplasma gondii. .  Histochem. Cell Biol.   2019.9Reviewed

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    Glycosylphosphatidylinositol (GPI)-anchored proteins typically localise to lipid rafts. GPI-anchored protein microdomains may be present in the plasma membrane; however, they have been studied using heterogeneously expressed GPI-anchored proteins, and the two-dimensional distributions of endogenous molecules in the plasma membrane are difficult to determine at the nanometre scale. Here, we used immunoelectron microscopy using a quick-freezing and freeze-fracture labelling (QF-FRL) method to examine the distribution of the endogenous GPI-anchored protein SAG1 in Toxoplasma gondii at the nanoscale. QF-FRL physically immobilised molecules in situ, minimising the possibility of artefactual perturbation. SAG1 labelling was observed in the exoplasmic, but not cytoplasmic, leaflets of T. gondii plasma membrane, whereas none was detected in any leaflet of the inner membrane complex. Point pattern analysis of SAG1 immunogold labelling revealed mostly random distribution in T. gondii plasma membrane. The present method obtains information on the molecular distribution of natively expressed GPI-anchored proteins and demonstrates that SAG1 in T. gondii does not form significant microdomains in the plasma membrane.

    DOI： 10.1007/s00418-019-01814-3.

    Other Link： https://www.ncbi.nlm.nih.gov/pubmed/31542792

  • 3. Kurokawa, Y., Konishi, R., Yoshida, A., Fujii, E., Tomioku, K., Futagami, T., Tamaki, H., Tanabe, K., Fujita, A. .  Essential and distinct roles of phosphatidylinositol 4-kinases, Pik1p and Stt4p, in yeast autophagy. .  Biochim. Biophys. Acta. Mol. Cell Biol. Lipids. 1864   1214 - 1225   2019.9Reviewed

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    Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome-vacuole fusion in yeast cells, respectively.

    DOI： 10.1016/j.bbalip.2019.05.004.

  • Kurokawa Y, Konishi R, Yoshida A, Tomioku K, Futagami T, Tamaki H, Tanabe K, Fujita A .  Essential and distinct roles of phosphatidylinositol 4-kinases, Pik1p and Stt4p, in yeast autophagy. .  Biochimica et biophysica acta. Molecular and cell biology of lipids1864 ( 9 ) 1214 - 1225   2019.9

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    DOI： 10.1016/j.bbalip.2019.05.004

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  • 2. Tsuji, T., Cheng, J., Tatematsu, T., Ebata, A., Kamikawa, H., Fujita, A., Gyobu, S., Segawa, K., Arai, H., Taguchi, T., Nagata, S., Fujimoto, T. .  Predominant localization of phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent redistribution. .  Proc. Natl. Acad. Sci. U S A.116   13368 - 13373   2019.7Reviewed

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    TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca2+ ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca2+-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca2+-dependent phospholipid scrambling in the ER.

    DOI： 10.1073/pnas.

    Other Link： https://www.ncbi.nlm.nih.gov/pubmed/31217287

  • 4. Kurokawa, Y., Yoshida, A., Fujii, E., Tomioku, K., Hayashi, H., Tanabe, K., Fujita, A. .  Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling. .  Traffic20   82 - 95   2019.1Reviewed

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    Phosphatidylinositol 4-phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P-binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP-binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze-fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome-lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.

    DOI： 10.1111/tra.12623

    Other Link： https://www.ncbi.nlm.nih.gov/pubmed/30426618

  • 5. Tsuji, T., Fujita, A., Fujimoto, T. .  Immunoelectron Microscopy of Gangliosides. .  Methods Mol. Biol.1804   231 - 239   2018.11Reviewed

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    Because chemical fixatives like aldehydes do not work on most lipid molecules in the membrane, small-scale lipid distribution cannot be identified by immunoelectron microscopy in cells fixed by conventional methods. Here we describe a method for physically stabilizing membranes through quick-freezing and freeze-fracture replica formation and for specifically labeling gangliosides for electron microscopy. This method enables the ultrahigh-resolution mapping of membrane lipids including gangliosides within the two-dimensional plane of membranes.

    DOI： 10.1007/978-1-4939-8552-4_11.

  • 1. Tomioku, K.N., Shigekuni, M., Hayashi, H., Yoshida, A., Futagami, T., Tamaki, H., Tanabe, K., Fujita, A. .  Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells. .  Eur. J. Cell Biol.97   269 - 278   2018.3Reviewed

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    In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.

    DOI： 10.1016/j.ejcb.2018.03.007

  • Yoshida A, Hayashi H, Tanabe K, Fujita A .  Segregation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate into distinct microdomains on the endosome membrane .  Biochim Biophys Acta1859 ( 10 ) 1880 - 1890   2017.7Reviewed

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    Phosphatidylinositol 4-phosphate (PtdIns(4)P) is the immediate precursor of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which is located on the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cellular functions. Although PtdIns(4)P and PtdIns(4,5)P2 have been reported to localize to multiple intracellular compartments and to each function as regulatory molecules, their generation, regulation and functions in most intracellular compartments are not well-defined. To analyze PtdIns(4)P and PtdIns(4,5)P2 distributions, at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P and PtdIns(4,5)P2 on the freeze-fracture replica of intracellular biological membranes. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P was localized to the cytoplasmic leaflet of Golgi apparatus and vesicular-shaped structures. The PtdIns(4,5)P2 labeling was observed in the cytoplasmic leaflet of the mitochondrial inner membrane and vesicular-shaped structures. Double labeling of PtdIns(4)P and PtdIns(4,5)P2 with endosome markers illustrated that PtdIns(4)P and PtdIns(4,5)P2 were mainly localized to the late endosome/lysosome and early endosome, respectively. PtdIns(4)P and PtdIns(4,5)P2 were colocalized in some endosomes, with the two phospholipids separated into distinct microdomains on the same endosomes. This is the first report showing, at a nanoscale, segregation of PtdIns(4)P- and PtdIns(4,5)P2-enriched microdomains in the endosome, of likely importance for endosome functionality.

    DOI： 10.1016/j.bbamem.2017.06.014

    Other Link： https://www.ncbi.nlm.nih.gov/pubmed/28648675

  • Yoshida, A., Shigekuni, M., Tanabe, K., Fujita, A. .  Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane. .  Biochim. Biophys. Acta. 1858 ( 6 ) 1298 - 1305   2016.3Reviewed

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    Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10s, began to increase at 30s and largely increased to 341.9% at 40s, and then returned to the initial level at 130s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas.

    DOI： 10.1016/j.bbamem.2016.03.011

  • Henry, Y., Morikawa, Y., Oe, N., Ikeda, N., Fujita, A., Takei, K., Minogue, S., Tatabe, K. .  PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes. .  Mol. Biol. Cell27 ( 6 ) 990 - 1001   2016.1Reviewed

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    Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain-containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P2 was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P2.

    DOI： 10.1091/mbc.E15-08-0564

  • Fujita, A., Inanobe, A., Hibino, H., Nielsen, S., Ottersen, OP., Kurachi, Y. .  Clustering of Kir4.1 at the specialized compartments of the lateral membrane in ependymal cells of rat brain .  Cell Tissue Res359 ( 2 ) 627 - 634   2015.12Reviewed

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  • Cheng, J., Fujita, A., Yamamoto, H., Tatematsu, T., Kakuta, S., Obara, K., Ohsumi, Y., Fujimoto, T. .  Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries. .  Nat. Commun.5   3207   2014.2Reviewed

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  • Fujita, A., Fujimoto, T., Ozato-Sakurai, N., Suzuki, H. .  A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy. .  J. Electron. Microsc. (Tokyo)61 ( 6 ) 441 - 446   2012.11Reviewed

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  • Ozato-Sakurai, N., Fujita, A., Fujimoto, T. .  The distribution of phosphatidy linositol 4,5-bisphosphate in acinar cells of rat pancreas revealed with the freeze-fracture replica labeling method. .  PLoS One. 6   e23567   2011.8Reviewed

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  • Kawai, Y., Hamazaki, Y., Fujita, H., Fujita, A., Sato, T., Furuse, M., Fujimoto, T., Jetten, A.M., Agata, Y., Minato, N. .  Claudin-4 induction by E-protein activity in later stages of CD4/8 double-positive thymocytes to increase positive selection efficiency. .  Proc. Natl. Acad. Sci. U S A. 108   4075 - 4080   2011.3Reviewed

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  • Fujita, A., Cheng, J., Fujimoto, T. .  Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution. .  Nat. Protoc. 5   661 - 669   2010.3Reviewed

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  • Fujita, A., Fujimoto T. .  High resolution molecular localization by freeze-fracture replica labeling. .  Methods Mol. Biol.657   205 - 216   2010Reviewed

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  • Fujita, A., Cheng, J., Tauchi-Sato, K., Takenawa, T., Fujimoto, T. .  A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique. .  Proc. Natl. Acad. Sci. U S A. 106   9256 - 9261   2009.6Reviewed

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  • Cheng, J., Fujita, A., Ohsaki, Y., Suzuki, M., Shinohara, Y., Fujimoto, T. .  Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets. .  Histochem. Cell Biol.132   281 - 291   2009.6Reviewed

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  • Fujita, A., Cheng, J., Fujimoto, T. .  Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton. .  Biochim. Biophys. Acta. 1791   388 - 396   2009.1Reviewed

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  • Ohsaki, Y., Cheng, J., Suzuki, M., Fujita, A., Fujimoto, T. .  Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100. .  J. Cell Sci. 121   2415 - 2422   2008.7Reviewed

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  • Kurahashi, M., Niwa, Y., Cheng, J., Ohsaki, Y., Fujita, A., Goto, H., Fujimoto, T., Torihashi, S. .  Platelet-derived growth factor signals play critical roles in differentiation of longitudinal smooth muscle cells in mouse embryonic gut. .  Neurogastroenterol. Motil. 20   521 - 531   2008.5Reviewed

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  • Nakajima, H., Goto, H., Azuma, Y.T., Fujita, A., Takeuchi, T. .  Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum. .  J. Neurochem. 103   2428 - 2438   2007.12Reviewed

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  • Fujita, A., Fujimoto, T. .  Quantitative retention of membrane lipids in the freeze-fracture replica. .  Histochem. Cell Biol. 128   385 - 389   2007.9Reviewed

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  • Nakajima, H., Amano, W., Fujita, A., Fukuhara, A., Azuma, Y.T., Hata, F., Inui, T., Takeuchi, T. .  The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death. .  J. Biol. Chem. 282   26562 - 26574   2007.9Reviewed

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  • Fujita, A., Cheng, J., Hirakawa, M., Furukawa, K., Kusunoki, S., Fujimoto, T. .  Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. .  Mol. Biol. Cell. 18   2112 - 2122   2007.6Reviewed

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  • Tsuiki, E., Fujita, A., Ohsaki, Y., Cheng, J., Irie, T., Yoshikawa, K., Senoo, H., Mishima, K., Kitaoka, T., Fujimoto, T. .  All-trans-retinol generated by rhodopsin photobleaching induces rapid recruitment of TIP47 to lipid droplets in the retinal pigment epithelium. Invest. .  Ophthalmol. Vis. Sci. 48   2858 - 2867   2007.6Reviewed

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  • Cheng, J., Ohsaki, Y., Tauchi-Sato, K., Fujita, A., Fujimoto, T. .  Cholesterol depletion induces autophagy. .  Biochem. Biophys. Res. Comm.351   246 - 252   2006.10Reviewed

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  • Ohsaki, Y., Cheng, J., Fujita, A., Tokumoto, T., Fujimoto, T. .  Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein .  B. Mol. Biol. Cell. 17   2674 - 2683   2006.6Reviewed

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  • Mukai, K., Takeuchi, T., Toyoshima, M., Satoh, Y., Fujita, A., Shintani, N., Hashimoto, H., Baba, A., Hata, F. .  PACAP- and PHI-mediated sustained relaxation in circular muscle of gastric fundus: findings obtained in PACAP knockout mice. .  Regul. Pept. 133   54 - 61   2006.1Reviewed

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  • Takeuchi, T., Fujinami, K., Goto, H., Fujita, A., Taketo, M.M., Manabe, T., Matsui, M., Hata, F. .  Roles of M2 and M4 muscarinic receptors in regulating acetylcholine release from myenteric neurons of mouse ileum. .  J. Neurophysiol. 93   2841 - 2848   2005.5Reviewed

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  • Okishio, Y., Takeuchi, T., Fujita, A., Suenaga, K., Fujinami, K., Munakata, S., Hata, F. .  Examination of the role of cholinergic myenteric neurons with the impairment of neural reflexes in the ileum of c-kit mutant mice. .  J. Smooth Muscle Res.41   49 - 60   2005.2Reviewed

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  • Okishio, Y., Takeuchi, T., Fujita, A., Suenaga, K., Fujinami, K., Munakata, S., Takewaki, T. , Hata, F. .  Ascending contraction and descending relaxation in the distal colon of mice lacking interstitial cells of Cajal. .  J. Smooth Muscle Res. 41   163 - 174   2005.1Reviewed

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  • Waseda, K., Takeuchi, T., Ohta, M., Okishio, Y., Fujita, A., Hata, F., Takewaki, T. .  Participation of ATP in nonadrenergic, noncholinergic relaxation of longitudinal muscle of wistar rat jejunum. .  J. Pharmacol. Sci. 97   91 - 100   2005.1Reviewed

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  • Kirkham, M., Fujita, A., Chadda, R., Nixon, S.J., Kurzchalia, T.V., Sharma, D.K., Pagano, R.E., Hancock, J.F., Mayor, S., Parton, R.G. .  Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. .  J. Cell Biol.168   465 - 476   2005.1Reviewed

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  • Fujita, A., Okishio, Y., Fujinami, K., Nakagawa, M., Takeuchi, T., Takewaki, T., Hata, F. .  Role of the interstitial cells distributed in the myenteric plexus in neural reflexes in the mouse ileum. .  J. Pharmacol. Sci.96   483 - 492   2004.12Reviewed

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  • Hibino, H., Fujita, A., Iwai, K., Yamada, M., Kurachi, Y. .  Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes. .  J. Biol. Chem. 279   44065 - 44073   2004.10Reviewed

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  • Takeuchi, T., Kushida, M., Hirayama, N., Kitayama, M., Fujita, A., Hata, F. .  Mechanisms involved in carbachol-induced Ca2+ sensitization of contractile elements in rat proximal and distal colon. .  Br. J. Pharmacol. 142   657 - 666   2004.6Reviewed

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  • Takeuchi. T., Fujinami, K., Fujita, A., Okishio, Y., Takewaki, T., Hata, F. .  Essential role of the interstitial cells of Cajal in nitric oxide-mediated relaxation of longitudinal muscle of the mouse ileum. .  J. Pharmacol. Sci. 95   71 - 80   2004.5Reviewed

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  • Takeuchi, T., Yamazaki, Y., Negoro, T., Fujinami, K., Mukai, K., Fujita, A., Takewaki, T., Hata, F. .  Changes in mechanism of PACAP-induced relaxation in longitudinal muscle of the distal colon of Wistar rats with age. .  Regul. Pept. 118   1 - 9   2004.4Reviewed

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  • Takeuchi, T., Kushida, M., Kitayama, M., Fujita, A., Hata, F. .  Origin of ATP for Ca2+-induced contraction in the guinea-pig femoral artery. .  Naunyn. Schmiedebergs Arch. Pharmacol. 369   350 - 357   2004.3Reviewed

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  • Hibino, H., Higashi-Shingai, K., Fujita, A., Iwai, K., Ishii, M., Kurachi, Y. .  Expression of an inwardly rectifying K+ channel, Kir5.1, in specific types of fibrocytes in the cochlear lateral wall suggests its functional importance in the establishment of endocochlear potential. .  Eur. J. Neurosci. 19   76 - 84   2004.1Reviewed

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  • 藤田 秋一，置塩 豊，竹内 正吉，畑 文明 .  マウス腸管運動調節におけるカハールの介在細胞の役割 .  日薬理誌123   170 - 178   2004Reviewed

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  • Kushida, M., Takeuchi, T., Fujita, A., Hata, F. .  Dependence of Ca2+-induced contraction on ATP in alpha-toxin-permeabilized preparations of rat femoral artery. .  J. Pharmacol. Sci. 93   171 - 179   2003.10Reviewed

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  • Ishii, M., Fujita, A., Iwai, K., Kusaka, S., Higashi, K., Inanobe, A., Hibino, H., Kurachi, Y. .  Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina. .  Am. J. Physiol. Cell Physiol.285   C260 - C267   2003.8Reviewed

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  • Takeuchi, T., Kitayama, M., Kushida, M., Fujita, A., Hata, F. .  Essential role of ATP synthesized by creatine kinase in contraction of alpha-toxin permeabilized preparations of tonic type smooth muscle. .  J. Pharmacol. Sci. 92   374 - 380   2003.8Reviewed

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  • Fujita, A., Takeuchi, T., Hanai, J., Hata, F. .  Expression of the small conductance Ca2+-activated K+ channel, SK3, in the olfactory ensheating glial cells of rat brain. .  Cell Tiss. Res.313   187 - 193   2003.8Reviewed

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  • Fujita, A., Takeuchi, T., Hanai, J., Hata, F. .  Localization of Ca2+-activated K+ channel, SK3, in fibroblast-like cells forming gap junctions with smooth muscle cells in the mouse small intestine. .  J. Pharmacol. Sci.92   35 - 42   2003.5Reviewed

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  • Takeuchi, T., Fujita, A., Kushida, M., Hata, F. .  The site where newly synthesized ATP is necessary for tension development in alpha-toxin permeabilized preparations of rat proximal colon. .  J. Pharmacol. Sci.91   277 - 284   2003.4Reviewed

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  • Chachin, M., Yamada, M., Fujita, A., Matsuoka, T., Matsushita, K., Kurachi, Y. .  Nateglinide, a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety, specifically inhibits pancreatic β-cell-type KATP channels. .  J. Pharmacol. Exp. Ther. 304, 1025-1032, Mar, 2003.304   1025 - 1032   2003.3Reviewed

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  • Yamaji, M., Ohta, M., Yamazaki, Y., Fujinami, K., Fujita, A., Takeuchi, T., Hata, F., Takewaki, T. .  A possible role of neurotensin in NANC relaxation of longitudinal muscle of the jejunum and ileum of Wistar rats. .  Br. J. Pharmacol. 137   629 - 636   2002.11Reviewed

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  • Mukai, K., Satoh, Y., Fujita, A., Takeuchi, T., Shintani, N., Hashimoto, H., Baba, A., Hata, F. .  PAC1 receptor-mediated relaxation of longitudinal muscle of the mouse proximal colon. .  Jpn. J. Pharmacol. 90   97 - 100   2002.9Reviewed

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  • Inanobe, A., Fujita, A., Ito, M., Tomoike, H., Inageda, K., Kurachi, Y. .  Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses. .  Am. J. Physiol. Cell Physiol. 282   C1396 - C1403   2002.6Reviewed

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  • Fujita, A., Horio, Y., Higashi, K., Mouri, T., Hata, F., Takeguchi, N., Kurachi, Y. .  Specific localization of an inwardly rectifying K+ channel, Kir4.1, at the apical membrane of rat gastric parietal cells; its possible involvement in K+ recycling for the H+/K+-pump. .  J. Physiol. (Lond) 540   85 - 92   2002.4Reviewed

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  • Tanemoto, M., Fujita, A., Higashi, K., Kurachi, Y. .  PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain. .  Neuron34   387 - 397   2002.4Reviewed

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  • Matsushita, K., Kinoshita, K., Matsuoka, T., Fujita, A., Fujikado, T., Tano, Y., Nakamura, H., Kurachi, Y. .  Intramolecular interaction of SUR2 subtypes for intracellular ADP-Induced differential control of KATP channels. .  Circ. Res.90   554 - 561   2002.3Reviewed

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  • Takeuchi, T., Sumiyoshi, M., Kitayama, M., Hirayama, N., Fujita, A., Hata, F. .  Origin of Ca2+ necessary for carbachol-induced contraction in longitudinal muscle of the proximal colon of rats. .  Jpn. J. Pharmacol.87   309 - 317   2001.12Reviewed

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  • Fujita, A., Takeuchi, T., Saitoh, N., Hanai, J., Hata, F. .  Expression of Ca2+-activated K+ channels, SK3, in the interstitial cells of Cajal in the gastrointestinal tract. .  Am. J. Physiol. Cell Physiol. 281   C1727 - C1733   2001.11Reviewed

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  • Fujita, A., Hattori,Y., Takeuchi, T., Kamata, Y., Hata, F. .  NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells. .  Neuroreport12   3599 - 3602   2001.11Reviewed

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  • Satoh, Y., Uchida, M., Fujita, A., Nishio, H., Takeuchi, T., Hata, F. .  Possible role of orexin A in NANC inhibitory response of longitudinal muscle of the mouse small intestine. .  Eur. J. Pharmacol. 428   337 - 342   2001.10Reviewed

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  • Higashi, K., Fujita, A., Inanobe, A., Tanemoto, M., Doi, K., Kubo, T., Kurachi, Y. .  An inwardly rectifying K+ channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain. .  Am. J. Physiol. Cell Physiol.281   C922 - C931   2001.9Reviewed

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  • Takeuchi, T., Fujita, A., Roumy, M., Zajac, J-M., Hata, F. .  Effect of 1Dme, a neuropeptide FF analog, on acetylcholine release from myenteric plexus of guinea pig ileum. .  Jpn. J. Pharmacol. ( 86 ) 417 - 422   2001.8Reviewed

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  • Takeuchi, T., Sugimoto, K., Morimoto, H., Fujita, A., Hata, F. .  Mechanism of a nitric oxide donor, NOR1-induced relaxation in longitudinal muscle of rat proximal colon. .  Jpn. J. Pharmacol. 86   390 - 398   2001.8Reviewed

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  • Kusaka, S., Inanobe, A., Fujita, A., Makino, Y., Tanemoto, M., Matsushita, K., Tano, K., Kurachi, Y. .  Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium. .  J. Physiol. (Lond)531   27 - 36   2001.2Reviewed

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  • Matsuoka, T., Matsushita, K., Katayama, Y., Fujita, A., Inageda, K., Tanemoto, M., Inanobe, A., Yamashita, S., Matsuzawa, Y., Kurachi, Y. .  C-terminal tails of sulfonylurea receptors control ADP-induced activation and diazoxide modulation of ATP-sensitive K+ channels. .  Circ. Res. 87   873 - 880   2000.11Reviewed

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  • Takeuchi, T., Negoro, T., Yamaji, M., Yamazaki, Y., Fujita, A., Nishio, H., Takewaki, T., Takatsuji, K., Hata, F. .  Increase in participation of vasoactive intestinal peptide in relaxation of the distal colon of wistar rats with age. .  Br. J. Pharmacol. 131   942 - 948   2000.11Reviewed

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  • Katayama, Y., Fujita, A., Ohe, T., Findlay, I., Kurachi, Y. .  Inhibitory effects of vesnarinone on cloned cardiac delayed rectifier K+ channels expressed in a mammalian cell line. .  J. Pharmacol. Exp. Ther. 294   339 - 346   2000.7Reviewed

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  • Hibino, H., Inanobe, A., Tanemoto, M., Fujita, A., Doi, K., Kubo, T., Hata, Y., Takai, Y., Kurachi, Y. .  Anchoring proteins confer G protein sensitivity to an inward-rectifier K+ channel through the GK domain. .  EMBO J. 19   78 - 83   2000.1Reviewed

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  • Inanobe, A., Horio, Y., Fujita, A., Tanemoto, M., Hibino, H., Inageda, K., Kurachi, Y. .  Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis. .  J. Physiol. (Lond) 521   19 - 30   1999.11Reviewed

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  • Fujita, A., Horio, Y., Nielsen, S., Nagelhus, E.A., Hata, F., Ottersen, O.P., Kurachi, Y. .  High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach. .  FEBS Let. 459   305 - 309   1999.10Reviewed

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  • Kusaka, S., Horio, Y., Fujita, A., Matsushita, K., Inanobe, A., Gotow, T., Uchiyama, Y., Tano, Y., Kurachi, Y. .  Expression and polarized distribution of an inwardly rectifying K+ channel, Kir4.1, in rat retinal pigment epithelium. .  J. Physiol. (Lond) 520   373 - 381   1999.10Reviewed

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  • Hibino, H., Horio, Y., Fujita, A., Inanobe, A., Doi, K., Gotow, T., Uchiyama, Y., Kubo, T., Kurachi, Y. .  Expression of an inwardly rectifying K+ channel, Kir4.1, in the satelite cells of rat cochlear ganglia. .  Am. J. Physiol. Cell Physiol.277   C638 - C644   1999.10Reviewed

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    Language：English   Publishing type：Research paper (scientific journal)  

  • Repunte, V.P., Nakamura, H., Fujita, A., Horio, Y., Findlay, I., Pott, L., Kurachi, Y. .  Extracellular links in Kir subunits control the unitary conductance of SUR/Kir6.0 ion channels. .  EMBO J.18   3317 - 3324   1999.6Reviewed

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  • Nagelhus, E.A., Horio, Y., Inanobe, A., Fujita, A., Haug, F-M., Nielsen, S., Kurachi, Y., Ottersen, O.P. .  Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. .  Glia26   47 - 54   1999.3Reviewed

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    Language：English   Publishing type：Research paper (scientific journal)  

  • Murahashi, T., Fujita, A., Kitazawa, T. .  Ca2+-induced Ca2+ desensitization of myosin light chain phosphrylation and contraction in phasic smooth muscle. .  Mol. Cell. Biochem. 190   91 - 98   1999.1Reviewed

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  • Kim, D., Fujita, A., Horio, Y., Kurachi, Y. .  Cloning and functional expression of a novel cardiac two-pore background K+ channel (cTBAK-1). .  Circ. Res. 82   513 - 518   1998.3Reviewed

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    Language：English   Publishing type：Research paper (scientific journal)  

  • Fujita, A., Takeuchi, T., Ishii,T., Nishio, H., Hata, F. .  Cooperation of ATP and norepinephrine in inducing contraction in guinea pig vas deferens is not associated with change in intracellular Ca2+ level. .  Jpn. J. Pharmacol. 70   273 - 276   1996.3Reviewed

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    Language：English   Publishing type：Research paper (scientific journal)  

  • Takeuchi, T., Fujita, A., Ishii, T., Nishio, H., Hata, F. .  Necessity of newly synthesized ATP by creatine kinase for contraction permeabilized longitudinal muscle preparations of rat proximal colon. .  J. Pharmacol. Exper. Ther.275   429 - 434   1995.10Reviewed

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    Language：English   Publishing type：Research paper (scientific journal)  

  • Fujita, A., Takeuchi, T., Nakajima, H., Nishio, H., Hata, F. .  Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens. .  J. Pharmacol. Exper. Ther.274   555 - 561   1995.7Reviewed

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  • Nagao, T., Fujita, A., Takeuchi, T., Hata, F. .  Changes in neuronal contribution to contractile responses of vas deferens of young and adult guinea pigs. .  J. Auton. Nerv. Syst.50   87 - 92   1994.12Reviewed

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  • Maehara, T., Fujita, A., Suthamnatpong, N., Takeuchi, T., Hata, F. .  Differences in relaxant effects of cyclic GMP on skinned muscle preparations from the proximal and distal colon of rats. .  Eur. J. Pharmacol.261   163 - 170   1994.8Reviewed

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  • Fujita, A., Takeuchi, T., Hata, F., Yagasaki, O. .  Role of PGE2 in neurotransmission from pre- to post-ganglionic hypogastric nerves of guinea pigs. .  Jpn. J. Pharmacol.58   61 - 66   1992.1Reviewed

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Books

  • Veterinary Biochemistry

    Akikazu Fujita（ Role： Contributor ,  Lipid structure and biomembrane）

    2016.4 

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    Total pages：13   Language：Japanese Book type：Textbook, survey, introduction

  • ナノメートルレベルでの膜脂質局在解析

    藤田 秋一，藤本 豊士（ Role： Joint author）

    「分子から個体へと進化する脂質生物学」, 実験医学，羊土社，東京  2010 

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    Language：Japanese Book type：Scholarly book

  • 脂質の組織化学

    藤田 秋一, 大崎 雄樹, 藤本 豊士（ Role： Joint author）

    「組織細胞化学２００９」, 日本組織細胞化学会  2009 

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    Language：Japanese Book type：Scholarly book

  • 凍結レプリカ標識法による膜脂質の分布解析

    藤田 秋一, 藤本 豊士（ Role： Joint author）

    電子顕微鏡で読み解く生命のなぞ, 藤本豊士, 山本章嗣（監修）, 秀潤社  2008 

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    Language：Japanese Book type：Scholarly book

  • 蛋白質と脂質を見るための電子顕微鏡法

    藤田 秋一, 藤本 豊士（ Role： Joint author）

    「組織細胞化学２００８」, 日本組織細胞化学会  2008 

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    Language：Japanese Book type：Scholarly book

  • 免疫電顕法

    藤本 豊士，藤田 秋一（ Role： Joint author）

    「組織細胞化学2006」，日本組織細胞化学会編，学際企画，京都  2006 

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    Language：Japanese Book type：Scholarly book

  • SDS処理凍結割断レプリカ標識法（藤本法）

    藤田 秋一，藤本 豊士（ Role： Joint author）

    「染色・バイオイメージング実験ハンドブック」，高田邦昭，斉藤尚亮，川上速人 編, 羊土社，東京  2006 

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    Language：Japanese Book type：Scholarly book

  • 超薄切片を用いた免疫電顕法

    藤田 秋一，藤本 豊士（ Role： Joint author）

    「組織細胞化学2005」，日本組織細胞化学会編，学際企画，京都  2005 

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    Language：Japanese Book type：Scholarly book

  • カリウムイオン

    倉智 嘉久，藤田 秋一（ Role： Joint author）

    「NEW薬理学」田中千賀子，加藤隆一 編，南光堂，東京  2001 

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    Language：Japanese Book type：Scholarly book

  • チャネル遺伝子発現システムとその解析法

    藤田 秋一，藤田 聡，倉智 嘉久（ Role： Joint author）

    「新パッチクランプ実験技術法」，岡田泰伸 編，吉岡書店，京都  2001 

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    Language：Japanese Book type：Scholarly book

  • 膜電流系の受容体機構

    藤田 秋一，倉智 嘉久（ Role： Joint author）

    「新不整脈」，井上博 編，南光堂，東京  2001 

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    Language：Japanese Book type：Scholarly book

  • Regulation of Ion Channels by Membrane Proteins

    Fujita, A., Hibino, H. and Kurachi, Y.（ Role： Joint author）

    THE CELL PHYSIOLOGY SOURCE BOOK, Third Edition, edited by Sperelakis, N. ,Academic Press, San Diego  2000 

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    Language：English Book type：Scholarly book

  • 膜の構造と透過性

    倉智 嘉久，藤田 秋一（ Role： Joint author）

    『分子・細胞の生物学II-細胞』石川春律，高井義美，月田承一郎 編，「現代医学の基礎」，岩波書店，東京  2000 

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    Language：Japanese Book type：Scholarly book

  • The Inward Rectifying K+ channels in heart

    Tanemoto, M., Fujita, A. and Kurachi, Y. （ Role： Joint author）

    PHYSIOLOGY AND PATHOLOGY OF THE HEART, Fourth Edition, edited by Terzic, A. And Kurachi, Y., Kluwer Academic Publishers, Boston  2000 

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    Language：English Book type：Scholarly book

  • Inward rectifying and ATP-sensitive K+ channels in the ventricular myocardium. Potassium channels in cardiovascular biology, edited by Archer, S.L. and Rush, N.J.

    Fujita, A. and Kurachi, Y.（ Role： Joint author）

    Plenum Press, New York  1999 

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    Language：English Book type：Scholarly book

  • 神経細胞イオンチャネル・受容体と細胞内分布

    倉智 嘉久、藤田 秋一（ Role： Joint author）

    「神経精神疾患理解のための病態生理」中村重信 編，神経精神疾患state of arts，医学のあゆみ，医歯薬出版社，東京  1999 

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    Language：Japanese Book type：Scholarly book

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MISC

  • 膜脂質分子をナノレベルで可視化する

    藤田 秋一，藤本 豊士

    顕微鏡,日本顕微鏡学会   44   206 - 209   2009

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure. Reviewed

    Ohsaki Y, Cheng J, Suzuki M, Shinohara Y, Fujita A, Fujimoto T.

    Biochim. Biophys. Acta.   1791   399 - 407   2009

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    Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • 分子の局在を見る：凍結レプリカ標識法による膜脂質の分布解析

    藤田 秋一，藤本 豊士

    細胞工学   26 ( 3 )   314 - 319   2007

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • カベオラ，ラフトと細胞内輸送

    藤本 豊士，藤田 秋一

    The Lipid   17   17 - 24   2006

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • エンドサイトーシスとその微細構造

    藤田 秋一

    細胞   38   36 - 39   2006

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • 電子顕微鏡を用いた細胞内取込み現象の可視化

    藤田 秋一

    顕微鏡   41   57 - 60   2006

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • 膜4回貫通型Two-pore domain K+ チャネル

    藤田 秋一

    「イオンチャネルの最前線」、倉智嘉久 編，医学のあゆみ   201   1043 - 1048   2002

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • チャネル遺伝子発現システムとその解析法

    藤田 秋一，藤田 聡，倉智 嘉久

    「新パッチクランプ実験技術法」，岡田泰伸 編，吉岡書店、京都   260 - 272   2001

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • チャネル・トランスポートのstructure-functionと薬物設計

    藤田 秋一，松岡 哲郎，松下 賢治，倉智 嘉久

    日薬理誌   118   177 - 186   2001

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • SAP family proteins. Reviewed

    Fujita, A. and Kurachi, Y.

    Biochem. Biophy. Res. Comm.   269   1 - 6   2000

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    Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • イオンチャネルの概念と最近の動向

    倉智 嘉久，藤田 秋一

    「チャネロパチー」，岡本幸市 編，CLINICAL NEUROSCIENCE   18   256 - 261   2000

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • K+チャネル作用薬

    倉智 嘉久，藤田 秋一

    「最先端創薬」，長尾拓ほか編，蛋白質核酸酵素   45   1023 - 1031   2000

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • Molecular Aspects of KATP channels in the Cardiovascular System and K+ channel openers. Basic and Therapeutic Relevance of K+ channel Regulation, edited by A.C. Sartorelli, W.C. Bowman, M.J. Curtis and S.G. Amara, Reviewed

    Fujita, A. and Kurachi, Y.

    Pharmacol. Therapeut   85   39 - 53   2000

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    Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • ATP感受性カリウムチャネル

    藤田 秋一，倉智嘉久

    脳の科学   20   101 - 103   1998

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

  • イオンチャネルカップル受容体

    藤田 秋一，倉智嘉久

    内分泌・糖尿病科   7   301 - 307   1998

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    Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Presentations

  • Localization of glycosphingolipid GM3 in Plasmodium falciparum malaria parasite  

    2022.11 

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    Event date： 2022.11

    Language：Japanese   Presentation type：Poster presentation  

    Country：Japan  

  • 向達汐美、正谷達謄、小西里可子、黒川夕奈、山口優希、富奥甘奈、福田佳代子、ハキミ　ハッサン、麻田正仁、金子　修、藤田秋一   ヒトmalaria原虫Plasmodium falciparumの細胞膜ではラフト主成分GM3は内葉と外葉の両方に局在する   Invited

    第94回　日本生化学大会  2021.11 

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    Event date： 2021.11

    Language：Japanese   Presentation type：Poster presentation  

    Venue：Web開催  

  • 黒川夕奈、小西里可子、福田佳代子、正谷達謄、藤田秋一   急速凍結・凍結割断レプリカ標識（QF-FRL）法を用いた酵母細胞膜におけるPI(4,5)P2の局在解析による、PI(4,5)P2生成の制御機構の解明   Invited

    第94回　日本生化学大会  2021.11 

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    Event date： 2021.11

    Language：Japanese   Presentation type：Oral presentation (general)  

    Venue：Web開催  

  • 山口優希、黒川夕奈、向達汐美、富奥甘奈、藤田秋一   酵母細胞でのautophagosome膜およびautophagic body膜における膜脂質の微細分布解析  

    第94回　日本生化学大会  2021.11 

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    Event date： 2021.11

    Language：Japanese   Presentation type：Poster presentation  

    Venue：Web開催  

  • Selective increment of phosphatidylserine in the autophagic body membrane in the yeast vacuole  

    2022.11 

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    Event date： 2022.11

    Language：Japanese   Presentation type：Oral presentation (general)  

    Country：Japan  

  • 藤田秋一，程晶磊，藤本豊士   免疫電顕法を用いたホスホイノシタイドの微細局在  

    日本平滑筋学会総会  日本平滑筋学会総会

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    Event date： 2013.8

    Language：Japanese  

    Venue：旭川  

    国内学会

  • 山口 優希, 黒川 夕奈, 向達 汐美, 福田 佳代子, 正谷 達謄, 藤田 秋一   酵母細胞でのautophagosome膜およびautophagic body膜における膜脂質の微細分布解析  

    日本生化学会大会プログラム・講演要旨集  2021.11  (公社)日本生化学会

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    Language：Japanese  

  • 黒川 夕奈, 小西 里可子, 富奥 甘奈, 藤田 秋一   急速凍結・凍結割断レプリカ標識(QF-FRL)法を用いた酵母細胞膜におけるPI(4,5)P2の局在解析による、PI(4、5)P2生成の制御機構の解明  

    日本生化学会大会プログラム・講演要旨集  2021.11  (公社)日本生化学会

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    Language：Japanese  

  • 向達 汐美, 正谷 達謄, 小西 里可子, 黒川 夕奈, 山口 優希, 富奥 甘奈, 福田 佳代子, ハキミ・ハッサン , 麻田 正仁, 金子 修, 藤田 秋一   ヒトmalaria原虫Plasmodium falciparumの細胞膜ではラフト主成分の糖脂質GM3は内葉と外葉の両方に局在する  

    日本生化学会大会プログラム・講演要旨集  2021.11  (公社)日本生化学会

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    Language：Japanese  

  • 向達 汐美, 正谷 達謄, 小西 里可子, 黒川 夕奈, 山口 優希, 富奥 甘奈, 福田 佳代子, ハキミ・ハッサン , 麻田 正仁, 金子 修, 藤田 秋一   ヒトmalaria原虫Plasmodium falciparumでのラフト主成分の糖脂質GM3の局在  

    日本生化学会大会プログラム・講演要旨集  2022.11  (公社)日本生化学会

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    Language：Japanese  

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Works

  • 脂質を基盤とする情報伝達機序の解析

    2008

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  • 超微細レベルでの情報伝達機序解析

    2006

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Research Projects

  • Nanoscale Analysis of Lipid-Mediated Autophagy Regulation

    Grant number：25K09420  2025.4 - 2028.3

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

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    Grant amount：\4550000 （ Direct Cost: \3500000 、 Indirect Cost：\1050000 ）

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  • The development of a novel freeze-fracture replica labeling method for visualizing the hydrophobic part of the biological membrane

    Grant number：22K19252  2022.6 - 2024.3

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

    Fujita Akikazu

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    Grant amount：\6500000 （ Direct Cost: \5000000 、 Indirect Cost：\1500000 ）

    We thought that hydrophobic portion of phospholipids may exposes in the biological membrane of the replica thin membrane by etching. However, the specific labelings of alkyne oleate or alkyne arachidonate could not be detected in these replicas of the biological membranes. On the other hand, the labeling of the alkyne arachidonate was detected in the replica membrane of the mitochondrial membrane which was made by the normal QF-FRL method. These results suggest that the alkyne of arachidonate indicating the terminal end of fatty acid of arachidonate is exposed on the hydrophilic surface of the mitochondrial membrane.

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  • Cell biological study for the distribution and dynamic of lipid in the biological membrane with nano-scale analyzing techniques

    Grant number：20H03154  2020.4 - 2023.3

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

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    Grant amount：\17810000 （ Direct Cost: \13700000 、 Indirect Cost：\4110000 ）

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  • 生体膜での脂肪酸ナノスケール解析技術確立による不飽和脂肪酸生理機能発現機序の解明

    Grant number：19K22435  2019.6 - 2023.3

    日本学術振興会  科学研究費助成事業  挑戦的研究(萌芽)

    藤田 秋一, 正谷 達謄

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    Grant amount：\6500000 （ Direct Cost: \5000000 、 Indirect Cost：\1500000 ）

    申請者らは脂質の超微局在を明らかにすることが、膜脂質の機能を解明するために必須であると考え、そのための方法開発に注力してきた。その結果、急速凍結・凍結割断レプリカ標識法（QF-FRL: Quick Freezing & Freeze-fracture Labeling）によって膜脂質を特異的に標識することが可能であることを示し、生体膜脂質の二次元的分布をナノレベルで定量的に解明することに成功した。このQF-FRL法が識別するのは糖鎖、イノシトール環など、膜脂質の“親水性頭部”の違
    いであるのに対し、膜脂質の多様性のもう一つの要因である“疎水性尾部”の違いを明らかにすることは原理的に困難であった。本研究では、現在用いているQF-FRL法をさらに改良することにより、凍結レプリカ脂肪酸標識法を新たに改良し、生体膜における脂肪酸分布をナノレベルで解析することを可能にする。哺乳類培養細胞あるいは培養組織の培養液中にアルキン化不飽和脂肪酸を入れ細胞内に取り込ませ、そのアナログが本来の内在性脂質と同様の性質を持つことを確認し、そしてクリックケミストリー法により不飽和脂肪酸を標識することができた。標識方法を確立するために、まず蛍光標識法で行い光学顕微鏡を用いて解析し、最適な条件を確定した。具体的には、市販されているアルキン化オレイン酸（１価不飽和脂肪酸）を用いて培養細胞において蛍光標識することに成功した。アルキン化オレイン酸を哺乳類培養細胞に取り込ませた後、アルデヒドで固定しbiotin-azideと反応させることにより、蛍光標識を得ることができた。ラット膵臓の培養組織における不飽和脂肪酸の標識には、培養細胞と同様の方法を用い、外来性に投与した不飽和脂肪酸の蛍光標識の条件検討を行い、不飽和脂肪酸の標識ができる条件を見出すことができた。

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