Updated on 2024/02/01

写真a

 
TAKEI Kohji
 
Organization
Faculty of Medicine, Dentistry and Pharmaceutical Sciences Professor
Position
Professor
External link

Degree

  • 歯学博士 ( 東京歯科大学 )

Research Interests

  • 細胞内小胞輸送

  • 細胞生物

  • cytoskeleton

  • membrane traffic

  • cell biology

  • 細胞骨格

Research Areas

  • Life Science / Cell biology

Education

  • Tokyo Dental College   歯学研究科   病理学

    - 1989

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    Country: Japan

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  • Tokyo Dental College    

    - 1989

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  • Tokyo Dental College    

    - 1985

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  • Tokyo Dental College   歯学部   歯学

    - 1985

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    Country: Japan

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Research History

  • - 岡山大学医歯薬学総合研究科 教授

    2005

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  • - Professor,Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,Okayama University

    2005

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  • 岡山大学医歯学総合研究科 未設定

    2001 - 2005

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  • Research Specialist II,Howard Hughes Medical Institute

    1995 - 1999

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  • ハワードヒューズ 医学財団 リサーチスペシャリスト

    1995 - 1999

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  • Associate Research Scientist,Yale University, School of Medicine

    1993 - 1999

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  • エール大学医学部 アソシエートリサーチサイエンティスト

    1993 - 1999

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  • ハワードヒューズ 医学財団 ポストドクトラルアソシエート

    1992 - 1994

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  • Postdoctoral Associate,Howard Hughes Medical Institute

    1992 - 1994

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  • postdoctoral fellow,Yale Unversity, School of Medicine

    1988 - 1993

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  • エール大学医学部 ポストドクトラルフェロー

    1988 - 1993

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Professional Memberships

 

Papers

  • Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I. Reviewed International journal

    Hiromichi Okuma, Yumiko Saijo-Hamano, Hiroshi Yamada, Aalaa Alrahman Sherif, Emi Hashizaki, Naoki Sakai, Takaaki Kato, Tsuyoshi Imasaki, Satoshi Kikkawa, Eriko Nitta, Miwa Sasai, Tadashi Abe, Fuminori Sugihara, Yoshimasa Maniwa, Hidetaka Kosako, Kohji Takei, Daron M Standley, Masahiro Yamamoto, Ryo Nitta

    Genes to cells : devoted to molecular & cellular mechanisms   2023.11

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

    Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

    DOI: 10.1111/gtc.13080

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  • Dynamin: molecular scissors for membrane fission

    Tetsuya Takeda, Hiroshi Yamada, Kohji Takei

    Plasma Membrane Shaping   77 - 90   2023

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    Publishing type:Part of collection (book)   Publisher:Elsevier  

    DOI: 10.1016/b978-0-323-89911-6.00023-6

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  • Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation Reviewed

    Hiroshi Yamada, Tadashi Abe, Hikaru Nagaoka, Eizo Takashima, Ryo Nitta, Masahiro Yamamoto, Kohji Takei

    Frontiers in Cellular and Infection Microbiology   12   2022.9

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    Publishing type:Research paper (scientific journal)   Publisher:Frontiers Media SA  

    Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty “effector” IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction in vitro, and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted in vitro using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.

    DOI: 10.3389/fcimb.2022.992198

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  • Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells Reviewed

    Haymar Wint, Jianzhen Li, Tadashi Abe, Hiroshi Yamada, Yasutomo Nasu, Masami Watanabe, Kohji Takei, Tetsuya Takeda

    2022.8

  • Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model. Reviewed International journal

    Tetsuya Hori, Kohgaku Eguchi, Han-Ying Wang, Tomohiro Miyasaka, Laurent Guillaud, Zacharie Taoufiq, Satyajit Mahapatra, Hiroshi Yamada, Kohji Takei, Tomoyuki Takahashi

    eLife   11   2022.4

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    Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

    DOI: 10.7554/eLife.73542

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  • The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes. Reviewed International journal

    Eriko Hamasaki, Natsuki Wakita, Hiroki Yasuoka, Hikaru Nagaoka, Masayuki Morita, Eizo Takashima, Takayuki Uchihashi, Tetsuya Takeda, Tadashi Abe, Ji-Won Lee, Tadahiro Iimura, Moin A Saleem, Naohisa Ogo, Akira Asai, Akihiro Narita, Kohji Takei, Hiroshi Yamada

    Frontiers in cell and developmental biology   10   884509 - 884509   2022

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    Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

    DOI: 10.3389/fcell.2022.884509

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  • JRAB/MICAL-L2 undergoes liquid–liquid phase separation to form tubular recycling endosomes Reviewed

    Ayuko Sakane, Taka-aki Yano, Takayuki Uchihashi, Kazuki Horikawa, Yusuke Hara, Issei Imoto, Shusaku Kurisu, Hiroshi Yamada, Kohji Takei, Takuya Sasaki

    Communications Biology   4 ( 1 )   2021.12

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    <title>Abstract</title>Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

    DOI: 10.1038/s42003-021-02080-7

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    Other Link: http://www.nature.com/articles/s42003-021-02080-7

  • Imaging-based evaluation of pathogenicity by novel DNM2 variants associated with centronuclear myopathy. Reviewed International journal

    Kenshiro Fujise, Mariko Okubo, Tadashi Abe, Hiroshi Yamada, Kohji Takei, Ichizo Nishino, Tetsuya Takeda, Satoru Noguchi

    Human mutation   43 ( 2 )   169 - 179   2021.11

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    A centronuclear myopathy (CNM) is a group of inherited congenital diseases showing clinically progressive muscle weakness associated with the presence of centralized myonuclei, diagnosed by genetic testing and muscle biopsy. The gene encoding dynamin 2, DNM2, has been identified as a causative gene for an autosomal dominant form of CNM. However, the information of a DNM2 variant alone is not always sufficient to gain a definitive diagnosis as the pathogenicity of many gene variants is currently unknown. In this study, we identified five novel DNM2 variants in our cohort. To establish the pathogenicity of these variants without using clinicopathological information, we used a simple in cellulo imaging-based assay for T-tubule-like structures to provide quantitative data that enable objective determination of pathogenicity by novel DNM2 variants. With this assay, we demonstrated that the phenotypes induced by mutant dynamin 2 in cellulo are well correlated with biochemical gain-of-function features of mutant dynamin 2 as well as the clinicopathological phenotypes of each patient. Our approach of combining an in cellulo assay with clinical information of the patients also explains the course of a disease progression by the pathogenesis of each variant in DNM2-associated CNM.

    DOI: 10.1002/humu.24307

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  • Dynamin 2 and BAR domain protein pacsin 2 cooperatively regulate formation and maturation of podosomes

    Jianzhen Li, Kenshiro Fujise, Haymar Wint, Yosuke Senju, Shiro Suetsugu, Hiroshi Yamada, Kohji Takei, Tetsuya Takeda

    Biochemical and Biophysical Research Communications   571   145 - 151   2021.9

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    Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.bbrc.2021.07.041

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  • Mutant BIN1-Dynamin 2 complexes dysregulate membrane remodeling in the pathogenesis of centronuclear myopathy. Reviewed International journal

    Kenshiro Fujise, Mariko Okubo, Tadashi Abe, Hiroshi Yamada, Ichizo Nishino, Satoru Noguchi, Kohji Takei, Tetsuya Takeda

    The Journal of biological chemistry   2020.11

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    Membrane remodeling is required for dynamic cellular processes such as cell division, polarization and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis, and how mutations in these molecules cause CNM to develop.Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related centronuclear myopathy.

    DOI: 10.1074/jbc.RA120.015184

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  • Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes

    The Mon La, Hiromi Tachibana, Shun‐Ai Li, Tadashi Abe, Sayaka Seiriki, Hikaru Nagaoka, Eizo Takashima, Tetsuya Takeda, Daisuke Ogawa, Shin‐ichi Makino, Katsuhiko Asanuma, Masami Watanabe, Xuefei Tian, Shuta Ishibe, Ayuko Sakane, Takuya Sasaki, Jun Wada, Kohji Takei, Hiroshi Yamada

    The FASEB Journal   34 ( 12 )   16449 - 16463   2020.10

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    Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1096/fj.202001240rr

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.202001240RR

  • Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic β-cell Line. Reviewed

    The Mon La, Hiroshi Yamada, Sayaka Seiriki, Shun-Ai Li, Kenshiro Fujise, Natsuho Katsumi, Tadashi Abe, Masami Watanabe, Kohji Takei

    Cell structure and function   45 ( 2 )   121 - 130   2020.8

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    The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic β-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in β-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse β-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic β-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.

    DOI: 10.1247/csf.20020

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  • [Corrigendum]Phosphorylation of cortactin by cyclin-dependent kinase 5 modulates actin bundling by the dynamin 1-cortactin ring-like complex and formation of filopodia and lamellipodia in NG108-15 glioma-derived cells. International journal

    Tadashi Abe, The Mon La, Yuuzi Miyagaki, Eri Oya, Fan-Yan Wei, Kento Sumida, Kenshiro Fujise, Tetsuya Takeda, Kazuhito Tomizawa, Kohji Takei, Hiroshi Yamada

    International journal of oncology   56 ( 3 )   859 - 859   2020.3

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    Subsequently to the publication of the above article, the authors have realized that the second‑listed author, The Mon La, had not been properly credited as one of the co‑writers of the paper. Therefore, the Authors' Contributions of the Declarations section of the article should have read as follows: Authors' contributions HY, KTa and TML designed the research and wrote the paper. HY, TA, YM, EO and TT performed mutant protein construction, protein purification and actin bundling experiments. TA and YM performed electron microscopy. EO, TML, KS and KF performed immunofluorescent microscopy, cell migration assay and analyzed data. FYW and KTo identified phosphorylation sites by MALDI‑MS. All authors read and approved the final manuscript. The authors apologize to the readership of the Journal for the misinformation in this regard, and for any inconvenience caused. [the original article was published in International Journal of Oncology 54: 550‑558, 2019; DOI: 10.3892/ijo.2018.4663].

    DOI: 10.3892/ijo.2020.4962

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  • Initial phospholipid-dependent Irgb6 targeting to Toxoplasma gondii vacuoles mediates host defense. Reviewed International journal

    Youngae Lee, Hiroshi Yamada, Ariel Pradipta, Ji Su Ma, Masaaki Okamoto, Hikaru Nagaoka, Eizo Takashima, Daron M Standley, Miwa Sasai, Kohji Takei, Masahiro Yamamoto

    Life science alliance   3 ( 1 )   2020.1

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    Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals by ingestion. The organism enters host cells and resides in the cytoplasm in a membrane-bound parasitophorous vacuole (PV). Inducing an interferon response enables IFN-γ-inducible immunity-related GTPase (IRG protein) to accumulate on the PV and to restrict parasite growth. However, little is known about the mechanisms by which IRG proteins recognize and destroy T. gondii PV. We characterized the role of IRG protein Irgb6 in the cell-autonomous response against T. gondii, which involves vacuole ubiquitination and breakdown. We show that Irgb6 is capable of binding a specific phospholipid on the PV membrane. Furthermore, the absence of Irgb6 causes reduced targeting of other effector IRG proteins to the PV. This suggests that Irgb6 has a role as a pioneer in the process by which multiple IRG proteins access the PV. Irgb6-deficient mice are highly susceptible to infection by a strain of T. gondii avirulent in wild-type mice.

    DOI: 10.26508/lsa.201900549

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  • ATP6AP2 variant impairs CNS development and neuronal survival to cause fulminant neurodegeneration. Reviewed International journal

    Takuo Hirose, Alfredo Cabrera-Socorro, David Chitayat, Thomas Lemonnier, Olivier Féraud, Carmen Cifuentes-Diaz, Nicolas Gervasi, Cedric Mombereau, Tanay Ghosh, Loredana Stoica, Jeanne d'Arc Al Bacha, Hiroshi Yamada, Marcel A Lauterbach, Marc Guillon, Kiriko Kaneko, Joy W Norris, Komudi Siriwardena, Susan Blasér, Jérémie Teillon, Roberto Mendoza-Londono, Marion Russeau, Julien Hadoux, Sadayoshi Ito, Pierre Corvol, Maria G Matheus, Kenton R Holden, Kohji Takei, Valentina Emiliani, Annelise Bennaceur-Griscelli, Charles E Schwartz, Genevieve Nguyen, Matthias Groszer

    The Journal of clinical investigation   129 ( 5 )   2145 - 2162   2019.4

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    Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.

    DOI: 10.1172/JCI79990

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  • Phosphorylation of cortactin by cyclin-dependent kinase 5 modulates actin bundling by the dynamin 1-cortactin ring-like complex and formation of filopodia and lamellipodia in NG108-15 glioma-derived cells. Reviewed

    Abe T, La TM, Miyagaki Y, Oya E, Wei FY, Sumida K, Fujise K, Takeda T, Tomizawa K, Takei K, Yamada H

    International journal of oncology   54 ( 2 )   550 - 558   2019.2

  • A Novel Membrane Fission Mechanism by Dynamin Complex: Clusterase Model

    TAKEI Kohji, YAMADA Hiroshi, TAKEDA Tetsuya

    Seibutsu Butsuri   59 ( 5 )   255 - 261   2019

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    Language:Japanese   Publisher:The Biophysical Society of Japan General Incorporated Association  

    Dynamin GTPase, an essential endocytotic protein, helically polymerizes at the neck of endocytic pits, and mechanically sever the membrane upon GTP hydrolysis. However, it is not known exactly how the dynamin disconnect the membrane. To clarify the mechanisms we analyzed structural changes of dynamin complexes during membrane fission using electron microscopy and high-speed atomic force microscopy (HS-AFM). Surprisingly, the dynamin ring complexes were clustered upon GTP hydrolysis and membrane constriction occurred at uncoated regions between the clusters, suggesting a novel mode of action of dynamin. In this commentary, we illustrate dynamin’s membrane fission models proposed thus far, and our novel “clusterase” model.

    DOI: 10.2142/biophys.59.255

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    Other Link: http://id.ndl.go.jp/bib/030043645

  • Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis. Reviewed International journal

    Takeda T, Kozai T, Yang H, Ishikuro D, Seyama K, Kumagai Y, Abe T, Yamada H, Uchihashi T, Ando T, Takei K

    eLife   7   2018.1

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    Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

    DOI: 10.7554/eLife.30246

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  • Dynamin2 GTPase contributes to invadopodia formation in invasive bladder cancer cells. International journal

    Yubai Zhang, Maya Nolan, Hiroshi Yamada, Masami Watanabe, Yasutomo Nasu, Kohji Takei, Tetsuya Takeda

    Biochemical and biophysical research communications   480 ( 3 )   409 - 414   2016.11

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    Cancer cell invasion is mediated by actin-based membrane protrusions termed invadopodia. Invadopodia consist of "core" F-actin bundles associated with adhesive and proteolytic machineries promoting cell invasion by degrading extracellular matrix (ECM). Formation of the F-actin core in invadopodia is regulated by various actin-binding proteins including Arp2/3 complex and cortactin. Dynamin GTPase localizes to the invadopodia and is implicated in cancer cell invasion, but its precise role at the invadopodia remained elusive. In this study, we examined the roles of dynamin at the invadopodia of bladder cancer cells. Although all three dynamin isoforms (dynamin1, 2 and 3) are expressed in human bladder cancer cell line T24, only dynamin2 localizes to the invadopodia. Inhibition of dynamin2 function, using either RNA interference (RNAi) or the dynamin specific inhibitor Dynasore, caused defects in invadopodia formation and suppressed invasive activity of T24 bladder cancer cells. Structure-function analysis using dynamin2 deletion fragments identified the proline/arginine-rich domain (PRD) of dynamin2 as indispensable for invadopodia formation and invasiveness of T24 cells. Thus, dynamin2 contributes to bladder cancer invasion by controlling invadopodia formation in bladder cancer cells and may prove a valuable therapeutic target.

    DOI: 10.1016/j.bbrc.2016.10.063

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  • Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling Reviewed

    Yuji Henmi, Natsuko Oe, Nozomu Kono, Tomohiko Taguchi, Kohji Takei, Kenji Tanabe

    Experimental Cell Research   342 ( 1 )   1 - 10   2016.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier {BV}  

    DOI: 10.1016/j.yexcr.2016.02.011

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  • Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization

    Keiichiro Hayashi, Hiroyuki Michiue, Hiroshi Yamada, Katsuyoshi Takata, Hiroki Nakayama, Fan-Yan Wei, Atsushi Fujimura, Hiroshi Tazawa, Akira Asai, Naohisa Ogo, Hiroyuki Miyachi, Tei-ichi Nishiki, Kazuhito Tomizawa, Kohji Takei, Hideki Matsui

    Scientific Reports   6 ( 1 )   2016.3

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1038/srep23372

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    Other Link: http://www.nature.com/articles/srep23372

  • Inhibition of SGLT2 alleviates diabetic nephropathy by suppressing high glucose-induced oxidative stress in type 1 diabetic mice Reviewed International journal

    Hatanaka T, Ogawa D, Tachibana H, Eguchi J, Inoue T, Yamada H, Takei K, Makino H, Wada J

    Pharmacol Res Perspect   4 ( 4 )   e00239   2016

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    It is unclear whether the improvement in diabetic nephropathy by sodium glucose cotransporter 2 (SGLT2) inhibitors is caused by a direct effect on SGLT2 or by the improvement in hyperglycemia. Here, we investigated the effect of dapagliflozin on early-stage diabetic nephropathy using a mouse model of type 1 diabetes and murine proximal tubular epithelial cells. Eight-week-old Akita mice were treated with dapagliflozin or insulin for 12 weeks. Body weight, urinary albumin excretion, blood pressure, as well as levels of blood glucose and hemoglobin A1c were measured. Expansion of the mesangial matrix, interstitial fibrosis, and macrophage infiltration in kidneys were evaluated by histology. Oxidative stress and apoptosis were evaluated in kidneys and cultured proximal tubular epithelial cells. Compared with nontreated mice, dapagliflozin and insulin decreased blood glucose and hemoglobin A1c levels equally. Urine volume and water intake increased significantly in the dapagliflozin-treated group compared with those in the insulin-treated group, but there were no differences in body weight or blood pressure between the two groups. Macrophage infiltration and fibrosis in renal interstitium improved significantly in the dapagliflozin group compared with the insulin group. Oxidative stress was attenuated by dapagliflozin, and suppression occurred in a dose-dependent manner. RNAi knockdown of SGLT2 resulted in reduced oxidative stress. Dapagliflozin ameliorates diabetic nephropathy by suppressing hyperglycemia-induced oxidative stress in a manner independent of hyperglycemia improvement in Akita mice. Our findings suggest that dapagliflozin may be a novel therapeutic approach for the treatment of diabetic nephropathy.

    DOI: 10.1002/prp2.239

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  • Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice Reviewed

    Naoto Terami, Daisuke Ogawa, Hiromi Tachibana, Takashi Hatanaka, Jun Wada, Atsuko Nakatsuka, Jun Eguchi, Chikage Sato Horiguchi, Naoko Nishii, Hiroshi Yamada, Kohji Takei, Hirofumi Makino

    PLOS ONE   9 ( 6 )   e100777   2014.6

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    Inhibition of sodium glucose cotransporter 2 (SGLT2) has been reported as a new therapeutic strategy for treating diabetes. However, the effect of SGLT2 inhibitors on the kidney is unknown. In addition, whether SGLT2 inhibitors have an anti-inflammatory or antioxidative stress effect is still unclear. In this study, to resolve these issues, we evaluated the effects of the SGLT2 inhibitor, dapagliflozin, using a mouse model of type 2 diabetes and cultured proximal tubular epithelial (mProx24) cells. Male db/db mice were administered 0.1 or 1.0 mg/kg of dapagliflozin for 12 weeks. Body weight, blood pressure, blood glucose, hemoglobin A1c, albuminuria and creatinine clearance were measured. Mesangial matrix accumulation and interstitial fibrosis in the kidney and pancreatic beta-cell mass were evaluated by histological analysis. Furthermore, gene expression of inflammatory mediators, such as osteopontin, monocyte chemoattractant protein-1 and transforming growth factor-beta, was evaluated by quantitative reverse transcriptase-PCR. In addition, oxidative stress was evaluated by dihydroethidium and NADPH oxidase 4 staining. Administration of 0.1 or 1.0 mg/kg of dapagliflozin ameliorated hyperglycemia, beta-cell damage and albuminuria in db/db mice. Serum creatinine, creatinine clearance and blood pressure were not affected by administration of dapagliflozin, but glomerular mesangial expansion and interstitial fibrosis were suppressed in a dose-dependent manner. Dapagliflozin treatment markedly decreased macrophage infiltration and the gene expression of inflammation and oxidative stress in the kidney of db/db mice. Moreover, dapagliflozin suppressed the high-glucose-induced gene expression of inflammatory cytokines and oxidative stress in cultured mProx24 cells. These data suggest that dapagliflozin ameliorates diabetic nephropathy by improving hyperglycemia along with inhibiting inflammation and oxidative stress.

    DOI: 10.1371/journal.pone.0100777

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  • N '[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide is a dynamin GTPase inhibitor that suppresses cancer cell migration and invasion by inhibiting actin polymerization Reviewed

    Hiroshi Yamada, Tadashi Abe, Shun-Ai Li, Shota Tago, Peng Huang, Masami Watanabe, Satoru Ikeda, Naohisa Ogo, Akira Asai, Kohji Takei

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   443 ( 2 )   511 - 517   2014.1

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    Dynasore, a specific dynamin GTPase inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments. In search for novel dynamin inhibitors that suppress actin dynamics more efficiently, dynasore analogues were screened. N'-14-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide (DBHA) markedly reduced in vitro actin polymerization, and dose-dependently inhibited phosphatidylserine-stimulated dynamin GTPase activity. DBHA significantly suppressed both the recruitment of dynamin 2 to the leading edge in U2OS cells and ruffle formation in H1299 cells. Furthermore, DBHA suppressed both the migration and invasion of H1299 cells by approximately 70%. Furthermore, intratumoral DBHA delivery significantly repressed tumor growth. DBHA was much less cytotoxic than dynasore. These results strongly suggest that DBHA inhibits dynamin-dependent actin polymerization by altering the interactions between dynamin and lipid membranes. DBHA and its derivative may be potential candidates for potent anti-cancer drugs. (C) 2013 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2013.11.118

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  • Metallothionein deficiency exacerbates diabetic nephropathy in streptozotocin-induced diabetic mice Reviewed

    Tachibana H, Ogawa D, Sogawa N, Asanuma M, Miyazaki I, Terami N, Hatanaka T, Horiguchi C. S, Nakatsuka A, Eguchi J, Wada J, Yamada H, Takei K, Makino H

    Am J Physiol Renal Physiol   306 ( 1 )   F105 - 15   2014

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    Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT−/−) and MT+/+ mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT−/− mice compared with diabetic MT+/+ mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT−/− mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.

    DOI: 10.1152/ajprenal.00034.2013

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  • Cancer stem cell-like characteristics of a CD133+ subpopulation in the J82 human bladder cancer cell line. Reviewed

    Peng Huang, Masami Watanabe, Haruki Kaku, Hideo Ueki, Hirofumi Noguchi, Morito Sugimoto, Takeshi Hirata, Hiroshi Yamada, Kohji Takei, Shaobo Zheng, Kai Xu, Yasutomo Nasu, Yasuyuki Fujii, Chunxiao Liu, Hiromi Kumon

    Molecular and Clinical Oncology   180 - 184   2013.1

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  • REIC/Dkk-3-encoding adenoviral vector as a potentially effective therapeutic agent for bladder cancer. Reviewed

    Hirata T, Watanabe M, Kaku H, Kobayashi Y, Yamada H, Sakaguchi M, Takei K, Huh NH, Nasu Y, Kumon H

    International journal of oncology   41   559 - 564   2012.8

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  • A novel gene expression system for detecting viable bladder cancer cells Reviewed

    Hideo Ueki, Masami Watanabe, Haruki Kaku, Peng Huang, Shun-Ai Li, Kazuhiko Ochiai, Takeshi Hirata, Hirofumi Noguchi, Hiroshi Yamada, Kohji Takei, Yasutomo Nasu, Yuji Kashiwakura, Hiromi Kumon

    INTERNATIONAL JOURNAL OF ONCOLOGY   41 ( 1 )   135 - 140   2012.7

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    A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasm id with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.

    DOI: 10.3892/ijo.2012.1417

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  • Etiological Role of Dynamin in Charcot-Marie-Tooth Disease Reviewed

    Takei Kohji, Tanabe Kenji

    Peripheral Neuropathy – Advances in Diagnostic and Therapeutic Approaches   ( 55 )   3 - 9   2012

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  • The clathrin assembly protein PICALM is required for erythroid maturation and transferrin internalization in mice. Reviewed

    WATANABE Toshio, Mai Suzuki, Hirokazu Tanaka, Akira Tanimura, Kenji Tanabe, Natsuko Oe, Shinya Rai, Syunsuke Kon, Manabu Fukumoto, Kohji Takei, Takaya Abe, Itaru Matsumura, Yuzuru Kanakura, Toshio Watanabe

    PLoS ONE   10.1371/journal.pone.0031854   2012

  • Dynamin 2 in charcot-marie-tooth disease. Reviewed

    Tanabe Kenji, Takei Kohji

    Acta medica Okayama   66 ( 3 )   183 - 190   2012

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    DOI: 10.18926/AMO/48557

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  • Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments. Reviewed

    Ohashi Emiko, Tanabe Kenji, Henmi Yuji, Mesaki Kumi, Kobayashi Yuka, Takei Kohji

    PLoS ONE   6 ( 5 )   e19942   2011

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  • Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells. Reviewed

    Henmi Yuji, Tanabe Kenji, Takei Kohji

    PLoS ONE   6 ( 12 )   e28603   2011

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  • Dynamin 2 associates with microtubules at mitosis and regulates cell cycle progression. Reviewed

    Ishida Nobuhisa, Nakamura Yuichi, Tanabe Kenji, Li Shun-Ai, Takei Kohji

    Cell Structure and Function   36 ( 2 )   145 - 154   2011

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    Dynamin, a ~100 kDa large GTPase, is known as a key player for membrane traffic. Recent evidence shows that dynamin also regulates the dynamic instability of microtubules by a mechanism independent of membrane traffic. As microtubules are highly dynamic during mitosis, we investigated whether the regulation of microtubules by dynamin is essential for cell cycle progression. Dynamin 2 intensely localized at the mitotic spindle, and the localization depended on its proline-rich domain (PRD), which is required for microtubule association. The deletion of PRD resulted in the impairment of cytokinesis, whereby the mutant had less effect on endocytosis. Interestingly, dominant-negative dynamin (K44A), which blocks membrane traffic but has no effect on microtubules, also blocked cytokinesis. On the other hand, the deletion of the middle domain, which binds to γ-tubulin, impaired the entry into mitosis. As both deletion mutants had no significant effect on endocytosis, dynamin 2 may participate in cell cycle progression by regulating the microtubules. These data suggest that dynamin may play a key role for cell cycle progression by two distinct pathways, membrane traffic and cytoskeleton.<br>

    DOI: 10.1247/csf.10016

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  • Fission of tubular endosomes triggers endosomal acidification and movement. Reviewed

    Mesaki Kumi, Tanabe Kenji, Obayashi Masanori, Oe Natsuko, Takei Kohji

    PLoS ONE   6 ( 5 )   e19764   2011

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  • Receptor sorting and actin dynamics at early endosomes. Reviewed

    Tanabe Kenji, Ohashi Emiko, Henmi Yuji, Takei Kohji

    Communicative & Integrative Biology   4 ( 6 )   742   2011

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  • Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development. International journal

    Kai Zhang, Masami Watanabe, Yuji Kashiwakura, Shun-Ai Li, Kohei Edamura, Peng Huang, Ken Yamaguchi, Yasutomo Nasu, Yasuyuki Kobayashi, Masakiyo Sakaguchi, Kazuhiko Ochiai, Hiroshi Yamada, Kohji Takei, Hideo Ueki, Nam-Ho Huh, Ming Li, Haruki Kaku, Yanqun Na, Hiromi Kumon

    International journal of oncology   37 ( 6 )   1495 - 501   2010.12

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    The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells. In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions. We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types. We also examined the tissue distribution pattern in a variety of normal mouse organs. Ubiquitous REIC/Dkk-3 protein expression was observed in the organs. The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells. The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles. In cancer cells stably transfected with REIC/Dkk-3, the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy. Because REIC/Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium. Statistically significant acinar growth was observed in the culture condition with 10 µg/ml REIC/Dkk-3 protein, implicating the soluble form in prostatic acinar development. Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner.

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  • Use of liposomes to study vesicular transport. Reviewed International journal

    Kohji Takei, Hiroshi Yamada, Tadashi Abe

    Methods in molecular biology (Clifton, N.J.)   606   531 - 42   2010

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    Liposomes have been utilized for variety of membrane transport studies including clathrin-mediated endocytosis. Here we introduce clathrin-coated structures that are generated on large unilamellar liposomes by incubating with clathrin coat proteins. Large unilamellar liposomes are also used to reconstitute vesicle formation in a cell-free system, and the vesicle formation can be quantified by using dynamic light scattering (DLS). Furthermore, phagocytosis assay using liposome-conjugated styrene beads is demonstrated.

    DOI: 10.1007/978-1-60761-447-0_36

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  • Dynasore, a dynamin inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments. Reviewed

    Yamada H, Abe T, Li SA, Masuoka Y, Isoda M, Watanabe M, Nasu Y, Kumon H, Asai A, Takei K

    Biochem Biophys Res Commun   2009.12

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    DOI: 10.1016/j.bbrc.2009.10.105

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  • Dynamic Interaction of Amphiphysin with N-WASP Regulates Actin Assembly Reviewed

    Hiroshi Yamada, Sergi Padilla-Parra, Sun-Joo Park, Toshiki Itoh, Mathilde Chaineau, Ilaria Monaldi, Ottavio Cremona, Fabio Benfenati, Pietro De Camilli, Maite Coppey-Moisan, Marc Tramier, Thierry Galli, Kohji Takei

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 49 )   34244 - 34256   2009.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidicliposome-triggered, N-WASP dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.

    DOI: 10.1074/jbc.M109.064204

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  • Dynamin 2 is required for actin assembly in phagocytosis in Sertoli cells Reviewed

    OTSUKA A

    Biochemical and Biophysical Research Communications   378   478 - 482   2009.1

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  • Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis. Reviewed

    Liang Shuang, Wei Fan-Yan, Wu Yu-Mei, Tanabe Kenji, Abe Tadashi, Oda Yoshiya, Yoshida Yumi, Yamada Hiroshi, Matsui Hideki, Tomizawa Kazuhito, Takei Kohji

    Journal of neurochemistry   102 ( 5 )   1466   2007

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  • Norepinephrine triggers Ca2+-dependent exocytosis of 5-hydroxytryptamine from rat pinealocytes in culture. Reviewed

    Yamada H, Hayashi M, Uehara S, Kinoshita M, Muroyama A, Watanabe M, Takei K, Moriyama Y

    Journal of neurochemistry   81 ( 3 )   533 - 540   2002.5

  • Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals. Reviewed

    Nemoto Y, Wenk MR, Watanabe M, Daniell L, Murakami T, Ringstad N, Yamada H, Takei K, De Camilli P

    J Biol Chem.   276 ( 44 )   41133 - 41142   2001.9

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    DOI: 10.1074/jbc.M106404200

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  • ラット精巣におけるグルタミン酸受容体の発現と局在性 Reviewed

    石尾 将吾, 林 美都子, 森本 理代, 本江 誠, 桑木 孝明, 渡部 昌美, 山田 浩司, 竹居 孝二, 森山 芳則

    生化学   73 ( 8 )   1017 - 1017   2001.8

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Books

  • Use of liposomes to study vesicular transport

    Humana Press  2010 

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  • Methods in Molecular Biology “Liposomes”

    Humana Press (USA), Springer Publishing Group,  2009 

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  • 生体の科学55

    医学書院,東京  2004 

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  • 文部科学省科学研究費特定領域研究C「ゲノム」2000年度報告書

    2001 

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  • 平成12?13年度科学研究費補助金(基盤研究(B)(2))研究成果報告書

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MISC

  • 細胞操作と定量イメージングで知る細胞骨格ダイナミズム 細胞の形態形成における膜-細胞骨格の動的相互作用

    竹田 哲也, 藤瀬 賢志郎, 山田 浩司, 竹居 孝二

    日本細胞生物学会大会講演要旨集   72回   W3 - 5   2020.6

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  • 腎糸球体ポドサイトにおけるアンフィファイジン1の機能

    山田 浩司, The Mon La, 竹田 哲也, 阿部 匡史, 淺沼 克彦, 竹居 孝二

    日本生化学会大会プログラム・講演要旨集   92回   [3P - 138]   2019.9

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  • 腎糸球体ポドサイトにおけるダイナミンイソフォームの局在と機能

    阿部 匡史, The Mon La, 橘 洋美, 竹田 哲也, 竹居 孝二, 山田 浩司

    日本生化学会大会プログラム・講演要旨集   92回   [3P - 139]   2019.9

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  • 筋細胞膜リモデリングにおけるメカニカルストレス応答の解析

    藤瀬 賢志郎, 山田 浩司, 竹居 孝二, 竹田 哲也

    日本筋学会学術集会プログラム・抄録集   5回   107 - 107   2019.8

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  • A Novel Membrane Fission Mechanism by Dynamin Complex: Clusterase Model

    竹居孝二, 山田浩司, 竹田哲也

    生物物理(Web)   59 ( 5 )   2019

  • メカノエンザイム・ダイナミンGTPaseによるアクチン線維束化機構の解析

    山田 浩司, 阿部 匡史, 竹田 哲也, 高島 英造, 森田 将之, 竹居 孝二

    日本生物工学会大会講演要旨集   平成30年度   122 - 122   2018.8

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  • 膜リモデリングおよびリン脂質代謝異常に起因する先天性ミオパチー発症機序の解明

    藤瀬 賢志郎, 背山 佳穂, 山下 恭加, 山田 浩司, 戸井 基道, 竹居 孝二, 竹田 哲也

    日本筋学会学術集会プログラム・抄録集   4回   110 - 110   2018.8

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  • メカノエンザイム・ダイナミンGTPaseによるアクチン線維束化機構の解析

    山田 浩司, 阿部 匡史, 竹田 哲也, 高島 英造, 森田 将之, 竹居 孝二

    日本生物工学会大会講演要旨集   平成30年度   122 - 122   2018.8

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  • ダイナミン2のシャルコー・マリー・トゥース病の原因変異は腎ポドサイトのアクチン再構成を阻害する

    和木田 夏輝, La The Mon, 隅田 健斗, 竹田 哲也, 阿部 匡史, 竹居 孝二, 山田 浩司

    生命科学系学会合同年次大会   2017年度   [2P - 1089]   2017.12

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  • ダイナミン-コルタクチンらせん状複合体の解析 機械的なアクチン線維束形成とアクチン脱重合保護作用

    阿部 匡史, 山田 浩司, 竹田 哲也, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    生命科学系学会合同年次大会   2017年度   [2P - 0320]   2017.12

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  • エンドサイトーシス生物学の新展開 ダイナミンによる膜切断過程の動態イメージング

    竹田 哲也, 小財 稔矢, 楊 恵然, 石黒 大輝, 背山 佳穂, 熊谷 祐介, 阿部 匡史, 山田 浩司, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    生命科学系学会合同年次大会   2017年度   [4AW17 - 2]   2017.12

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  • GTP加水分解に共役したダイナミン依存的膜切断機構の高速原子間力顕微鏡解析

    竹田 哲也, 石黒 大輝, 楊 恵然, 小財 稔矢, 背山 佳穂, 熊谷 祐介, 山田 浩司, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    日本細胞生物学会大会講演要旨集   69回   72 - 72   2017.5

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  • Actin bundling by dynamin 2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells International journal

    Hiroshi Yamada, Tetsuya Takeda, Hiroyuki Michiue, Tadashi Abe, Kohji Takei

    INTERNATIONAL JOURNAL OF ONCOLOGY   49 ( 3 )   877 - 886   2016.9

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    The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.

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  • Expression of a dynamin 2 mutant associated with Charcot-Marie-Tooth disease leads to aberrant actin dynamics and lamellipodia formation

    Hiroshi Yamada, Kinue Kobayashi, Yubai Zhang, Tetsuya Takeda, Kohji Takei

    NEUROSCIENCE LETTERS   628   179 - 185   2016.8

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    Specific mutations in dynamin 2 are linked to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy. However, the effects of these mutations on dynamin function, particularly in relation to the regulation of the actin cytoskeleton remain unclear. Here, selected CMT-associated dynamin mutants were expressed to examine their role in the pathogenesis of CMT in U2OS cells. Ectopic expression of the dynamin CMT mutants 555 Delta 3 and K562E caused an approximately 50% decrease in serum stimulation dependent lamellipodia formation; however, only K562E caused aberrations in the actin cytoskeleton. Immunofluorescence analysis showed that the K562E mutation resulted in the disappearance of radially aligned actin bundles and the simultaneous appearance of F-actin clusters. Live-cell imaging analyses showed F-actin polymers of decreased length assembled into immobile clusters in K562E-expressing cells. The K562E dynamin mutant colocalized with the F-actin clusters, whereas its colocalization with clathrin-coated pit marker proteins was decreased. Essentially the same results were obtained using another cell line, HeLa and NG108-15 cells. The present study is the first to show the association of dynamin CMT mutations with aberrant actin dynamics and lamellipodia, which may contribute to defective endocytosis and myelination in Schwann cells in CMT. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.

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  • 腎糸球体ポドサイトにおけるダイナミンアイソフォームの局在と機能

    橘 洋美, 竹田 哲也, 山田 浩司, 小川 大輔, 竹居 孝二

    日本細胞生物学会大会講演要旨集   68回   98 - 98   2016.5

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  • Antiobesity Action of ACAM by Modulating the Dynamics of Cell Adhesion and Actin Polymerization in Adipocytes

    Kazutoshi Murakami, Jun Eguchi, Kazuyuki Hida, Atsuko Nakatsuka, Akihiro Katayama, Miwa Sakurai, Haruki Choshi, Masumi Furutani, Daisuke Ogawa, Kohji Takei, Fumio Otsuka, Jun Wada

    DIABETES   65 ( 5 )   1255 - 1267   2016.5

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    Coxsackie virus and adenovirus receptor-like membrane protein (CLMP) was identified as the tight junction-associated transmembrane protein of epithelial cells with homophilic binding activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM), and it is upregulated in mature adipocytes in rodents and humans with obesity. Here, we present that aP2 promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is abundantly expressed on plasma membrane of mature adipocytes and associated with formation of phalloidin-positive polymerized form of cortical actin (F-actin). By electron microscopy, the structure of zonula adherens with an intercellular space of approximate to 10-20 nm was observed with strict parallelism of the adjoining cell membranes over distances of 1-20 m, where ACAM and -actin are abundantly expressed. The formation of zonula adherens may increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin sensitivity.

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  • PtdIns4KII alpha generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes

    Yuji Henmi, Yoshiaki Morikawa, Natsuko Oe, Narumi Ikeda, Akikazu Fujita, Kohji Takei, Shane Minogue, Kenji Tanabe

    MOLECULAR BIOLOGY OF THE CELL   27 ( 6 )   990 - 1001   2016.3

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    Phosphatidylinositol 4-kinase II alpha (PtdIns4KII alpha) 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 PtdIns4KII alpha 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 PtdIns4KII alpha with small interfering RNA significantly reduced the amount of vesicular PtdIns(4) P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KII alpha 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 PtdIns4KII alpha. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KII alpha-depleted cells. Endosomal PtdIns(4,5)P-2 was also significantly reduced in PtdIns4KII alpha-depleted cells. These results show that PtdIns4KII alpha regulates receptor sorting at early endosomes through a PtdIns(4) P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P-2.

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  • 成長円錐におけるPKCαのコルタクチンリン酸化によるアクチン制御の可能性

    山田 浩司, 菊池 達也, 増本 年男, 魏 范研, 阿部 匡史, 竹田 哲也, 西木 禎一, 富澤 一仁, 渡部 昌実, 松井 秀樹, 竹居 孝二

    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   [1P1328] - [1P1328]   2015.12

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  • Possible role of cortactin phosphorylation by protein kinase C in actin-bundle formation at growth cone

    Hiroshi Yamada, Tatsuya Kikuchi, Toshio Masumoto, Fan-Yan Wei, Tadashi Abe, Tetsuya Takeda, Teiichi Nishiki, Kazuhito Tomizawa, Masami Watanabe, Hideki Matsui, Kohji Takei

    BIOLOGY OF THE CELL   107 ( 9 )   319 - 330   2015.9

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    Background Information. Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood.
    Results. Immunofluorescence microscopy revealed that protein kinase C (PKC) colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKC directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles.
    Conclusions. Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

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  • 腎糸球体ポドサイト分化におけるダイナミンGTPアーゼの役割

    橘洋美, 竹田哲也, 山田浩司, 小川大輔, 竹居孝二

    日本生化学会大会(Web)   88th   2015

  • Stabilization of actin bundles by a dynamin 1/cortactin ring complex is necessary for growth cone filopodia.

    Yamada, H, Abe, T, Satoh, A, Okazaki, N, Tago, S, Kobayashi, K, Yoshida, Y, Oda, Y, Watanabe, M, Tomizawa, K, Matsui, H, Takei, K

    Journal of Neuroscience   33 ( 10 )   4514 - 4526   2015

  • 肺がん細胞株における細胞運動を司るダイナミン2によるアクチン動態制御

    阿部匡史, 山田浩司, 竹田哲也, 竹居孝二

    日本生化学会大会(Web)   87th   2014

  • Dynaminを標的にした抗がん剤の開発

    岡崎奈奈, 阿部匡史, 有田美香子, 多湖翔太, 永井さや, 池田敏, 小郷尚久, 浅井章良, 山田浩司, 竹居孝二

    日本生化学会大会(Web)   85th   2012

  • 成長円錐糸状仮足形成におけるCortactinのPKCalphaによるリン酸化とアクチン制御の可能性

    山田 浩司, 勢井 麻梨, 阿部 匡史, 増本 年男, 菊池 達也, 富澤 一仁, 池田 敏, 松井 秀樹, 竹居 孝二

    日本生化学会大会プログラム・講演要旨集   84回   2P - 0345   2011.9

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  • ダイナミン・コルタクチン複合体はアクチン線維を束化し、この束化は成長円錐の糸状仮足形成に重要である

    山田 浩司, 阿部 匡史, 竹居 孝二

    日本薬学会年会要旨集   131年会 ( 3 )   152 - 152   2011.3

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  • Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

    山田浩司, PADILLA-PARRA Sergi, 朴宣奏, 朴宣奏, 伊藤俊樹, CHAINEAU Mathilde, CHAINEAU Mathilde, MONALDI Ilaria, CREMONA Ottavio, BENFENATI Fabio, CAMILLI Pietro De, COPPEY-MOISAN Maiete, TRAMIER Marc, GALLI Thierry, GALLI Thierry, 竹居孝二

    岡山医学会雑誌   123 ( 1 )   2011

  • ダイナミン/コルタクチン複合体によるアクチン細胞骨格制御機構

    竹居孝二, 山田浩司, 阿部匡

    生体膜と薬物の相互作用シンポジウム講演要旨集   33rd   2011

  • PKC phosphorylation of cortactin is implicated in the regulation of actin dynamics

    H. Yamada, T. Abe, T. Masumoto, M. Sei, T. Kikuchi, K. Tomizawa, S. Ikeda, H. Matsui, K. Takei

    MOLECULAR BIOLOGY OF THE CELL   22   2011

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  • ダイナミン1/コルタクチン複合体はアクチン線維束を形成し、成長円錐フィロポディア形成を支持する(Dynamin 1/cortactin complex mechanically bundles actin filaments and supports the formation of growth cone filopodia)

    竹居 孝二, 阿部 匡史, 川田 慎浩, 山田 浩司

    神経化学   49 ( 2-3 )   501 - 501   2010.8

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  • Dynamin 1/cortactin complex mechanically bundles actin filaments and supports the formation of growth cone filopodia

    Kohji Takei, Tadashi Abe, Yoshihiro Kawada, Hiroshi Yamada

    NEUROSCIENCE RESEARCH   68   E88 - E88   2010

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  • Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development.

    Zhang K, Watanabe M, Kashiwakura Y, Li SA, Edamura K, Huang P, Yamaguchi K, Nasu Y, Kobayashi Y, Sakaguchi M, Ochiai K, Yamada H, Takei K, Ueki H, Huh NH, Li M, Kaku H, Na Y, Kumon H

    Int J Oncol   37 ( 6 )   1495 - 1501   2010

  • The 2009 Okayama Medical Association Awards: Dynamic instability of microtubules regulated by Dynamin2 and Charcot-Marie-Tooth disease

    Journal of Okayama Medical Association   122 ( 2 )   113 - 117   2010

  • Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development

    Int. J. Oncol.   122   113 - 117   2010

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  • Dynamic Interaction of Amphiphysin with N-WASP Regulates Actin Assembly

    Hiroshi Yamada, Sergi Padilla-Parra, Sun-Joo Park, Toshiki Itoh, Mathilde Chaineau, Ilaria Monaldi, Ottavio Cremona, Fabio Benfenati, Pietro De Camilli, Maite Coppey-Moisan, Marc Tramier, Kohji Takei

    NEUROSCIENCE RESEARCH   68   E136 - E136   2010

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  • ダイナミン/コルタクチン複合体によるアクチン束化は糸状仮足形成と関連がある(Actin bundling by dynamin/cortactin complex is implicated in filopodia formation)

    山田 浩司, 阿部 匡史, 川田 慎浩, 竹居 孝二

    日本生化学会大会プログラム・講演要旨集   82回   3P - 372   2009.9

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  • Dynamic instability of microtubules requires dynamin 2 and is impaired in a Charcot-Marie-Tooth mutant

    Kenji Tanabe, Kohji Takei

    JOURNAL OF CELL BIOLOGY   185 ( 6 )   939 - 948   2009.6

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    Dynamin is a fission protein that participates in endocytic vesicle formation. Although dynamin was originally identified as a microtubule-binding protein, the physiological relevance of this function was unclear. Recently, mutations in the ubiquitously expressed dynamin 2 (dyn2) protein were found in patients with Charcot-Marie-Tooth (CMT) disease, which is an inherited peripheral neuropathy. In this study, we show that one of these mutations, 551 Delta 3, induces prominent decoration of microtubules with the mutant dyn2. Dyn2 was required for proper dynamic instability of microtubules, and this was impaired in cells expressing the 551 Delta 3 mutant, which showed a remarkable increase in microtubule acetylation, a marker of stable microtubules. Depletion of endogenous dyn2 with a small interfering RNA also resulted in the accumulation of stable microtubules. Furthermore, the formation of mature Golgi complexes, which depends on microtubule-dependent membrane transport, was impaired in both dyn2 knockdown cells and cells expressing the 551 Delta 3 mutant. Collectively, our results suggest that dyn2 regulates dynamic instability of microtubules, which is essential for organelle motility, and that this function may be impaired in CMT disease.

    DOI: 10.1083/jcb.200803153

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  • ダイナミン/コルタクチン複合体によるアクチン束形成の新しいメカニズム 糸状仮足形成における意義(Novel mechanism of actin bundle formation by dynamin/cortactin complex: its implication in filopodia formation)

    山田 浩司, 阿部 匡史, 吉田 祐実, 竹居 孝二

    日本細胞生物学会大会講演要旨集   61回   175 - 175   2009.5

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  • CaMキナーゼIα誘発性のDrp1リン酸化はミトコンドリア形態を制御する(CaM kinase Iα-induced phosphorylation of Drp1 regulates mitochondrial morphology)

    韓 小建, 陸 雲飛, 李 順愛, 貝塚 多久, 佐藤 泰史, 富澤 一仁, アンガス・シ・ネアン, 竹居 孝二, 松井 秀樹, 松下 正之

    日本生理学雑誌   71 ( 2 )   68 - 68   2009.2

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  • REGULATION OF MITOCHONDRIAL DYNAMICS AND MORPHOLOGY BY CHANNEL SPECIFIC CALCIUM SIGNALING

    Xiao-Jian Han, Yun-Fei Lu, Shun-Ai Li, Kazuhito Tomizawa, Kohji Takei, Masayuki Matsushita, Hideki Matsui

    JOURNAL OF PHYSIOLOGICAL SCIENCES   59   245 - 245   2009

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  • Dynamin 2 Cooperates with Amphiphysin 1 in Phagocytosis in Sertoli Cells

    Akira Nakanishi, Tadashi Abe, Masami Watanabe, Kohji Takei, Hiroshi Yamada

    ACTA MEDICA OKAYAMA   62 ( 6 )   385 - 391   2008.12

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    Testicular Sertoli cells highly express dynamin 2 and amphiphysin 1. Here we demonstrate that dynamin 2 is implicated in phosphatidylserine (PS)-dependent phagocytosis in Sertoli cells. Immunofluorescence and dual-live imaging revealed that dynamin 2 and amphiphysin 1 accumulate simultaneously at ruffles. These proteins are specifically bound in vitro. Over-expression of dominant negative dynamin 2 (K44A) inhibits liposome-uptake and leads to the mis-localization of amphiphysin 1. Thus, the cooperative function of dynamin 2 and amphiphysin 1 in PS-dependent phagocytosis is strongly suggested.

    DOI: 10.18926/AMO/30954

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  • CaM kinase l alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology

    Xiao-Jian Han, Yun-Fei Lu, Shun-Ai Li, Taku Kaitsuka, Yasufumi Sato, Kazuhito Tomizawa, Angus C. Nairn, Kohji Takei, Hideki Matsui, Masayuki Matsushita

    JOURNAL OF CELL BIOLOGY   182 ( 3 )   573 - 585   2008.8

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    Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca(2+) signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca(2+)/calmodulin-dependent protein kinase l alpha ( CaMKl alpha). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKl alpha is a widely expressed protein kinase, suggesting that Ca(2+) is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

    DOI: 10.1083/jcb.200802164

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  • ダイナミン1は、コルタクチンとともに、アクチンの動態に働く(Dynamin 1 participates in actin dynamics with cortactin)

    信崎 哲郎, 吉田 祐実, 阿部 匡史, 小田 吉哉, 富沢 一仁, 山田 浩司, 竹居 孝二

    日本細胞生物学会大会講演要旨集   60回   100 - 100   2008.6

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  • Regulation of clathrin-mediated endocytosis by p53

    Yoshie Endo, Atsumi Sugiyama, Shun-Ai Li, Kazuji Ohmori, Hirokazu Ohata, Yusuke Yoshida, Masabumi Shibuya, Kohji Takei, Masato Enari, Yoichi Taya

    GENES TO CELLS   13 ( 4 )   375 - 385   2008.4

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    The p53 gene encodes a multi-functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor-mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co-localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin-mediated endocytosis to control the correct signaling from EGFR.

    DOI: 10.1111/j.1365-2443.2008.01172.x

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  • SNX9 regulates tubular invagination of the plasma membrane through interaction with actin cytoskeleton and dynamin 2

    Narae Shin, Namhui Ahn, Belle Chang-Ileto, Joohyun Park, Kohji Takei, Sang-Gun Ahn, Soo-A Kim, Gilbert Di Paolo, Sunghoe Chang

    JOURNAL OF CELL SCIENCE   121 ( 8 )   1252 - 1263   2008.4

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    Dynamic membrane remodeling during intracellular trafficking is controlled by the intricate interplay between lipids and proteins. BAR domains are modules that participate in endocytic processes by binding and deforming the lipid bilayer. Sorting nexin 9 (SNX9), which functions in clathrin-mediated endocytosis, contains a BAR domain, however, the properties of this domain are not well understood. Here we show that SNX9 shares many properties with other BAR domain-containing proteins, such as amphiphysin and endophilin. SNX9 is able to deform the plasma membrane, as well as liposomes, into narrow tubules and recruit N-WASP and dynamin 2 to these tubules via its SH3 domain. SNX9-induced tubulation is antagonized by N-WASP and dynamin 2 while it is enhanced by perturbation of actin dynamics. However, SNX9 also has several unique properties. The tubulating activity requires the BAR and PX domains, as well as the low-complexity (LC) domain, which binds the Arp2/3 complex. SNX9 also binds to PtdIns(4)P-5-kinases via its PX domain and its tubulating activity is regulated by phosphoinositides. In addition, the kinase activity of PtdIns(4)P-5-kinases is stimulated by interaction with SNX9, suggesting a positive feedback interaction between SNX9 and PtdIns(4)P-5-kinases. These results suggest that SNX9 functions in the coordination of membrane remodeling and fission via interactions with actin-regulating proteins, endocytic proteins and PtdIns(4,5)P-2-metabolizing enzymes.

    DOI: 10.1242/jcs.016709

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  • Involvement of calcineurin in glutamate-induced mitochondrial dynamics in neurons

    Xiao-Jian Han, Yun-Fei Lu, Shun-Ai Li, Kazuhito Tomizawa, Kohji Takei, Masayuki Matsushita, Hideki Matsui

    NEUROSCIENCE RESEARCH   60 ( 1 )   114 - 119   2008.1

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    Alterations in the morphology and movement of mitochondria influence neuronal viability. However, the precise mechanisms of such alterations are unclear. In this study, we showed calcineurin was involved in the regulation of mitochondrial dynamics. Glutamate stimulation inhibited mitochondrial movement and decreased mitochondrial length in neurons. FK506 and cyclosporine A, calcineurin inhibitors, attenuated the effects of glutamate on mitochondrial dynamics. It was also found that glutamate treatment dephosphorylated, a proapoptotic protein, Bad and promoted its translocation to mitochondria in neurons via calcineurin. These results provide important new insights into intracellular signaling pathways that regulate mitochondrial dynamics and neuronal cell death. (c) 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

    DOI: 10.1016/j.neures.2007.09.012

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  • Amphiphysin 1 is important for actin polymerization During phagocytosis

    Hiroshi Yamada, Emiko Ohashi, Tadashi Abe, Norihiro Kusumi, Shun-AI Li, Yumi Yoshida, Masami Watanabe, Kazuhito Tomizawa, Yuji Kashiwakura, Hiromi Kumon, Hideki Matsui, Kohji Takei

    MOLECULAR BIOLOGY OF THE CELL   18 ( 11 )   4669 - 4680   2007.11

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    Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1(-/-) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate-induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.

    DOI: 10.1091/mbc.E07-04-0296

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  • cAMP-response element binding protein (CREB) regulates cyclosporine-A-mediated down-regulation of cathepsin B and L synthesis

    Kazuhiro Omori, Koji Naruishi, Tomoko Yamaguchi, Shun-Ai Li, Mayumi Yamaguchi-Morimoto, Kaori Matsuura, Hideo Arai, Kohji Takei, Shogo Takashiba

    CELL AND TISSUE RESEARCH   330 ( 1 )   75 - 82   2007.10

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    Cyclosporin A (CsA) is an immunosuppressant with severe side effects including gingival overgrowth. We have previously reported that CsA impairs the activity of the lysosomal enzymes cathepsin B and L in human gingival fibroblasts (HGFs). Here, we have examined the effects of CsA on the DNA-binding activity of the cyclic AMP response element-binding protein (CREB) and cell viability, and the effects of CREB on cathepsin B and L synthesis and activity in HGFs. We have confirmed that CsA down-regulates cathepsin B and L synthesis. Further, CsA has no effect on cell viability and dramatically impairs CREB-DNA binding activity. Importantly, the synthesis of cathepsin B and L is down-regulated, and their activity is also significantly impaired in HGFs transfected with plasmid expressing dominant-negative CREB. These results suggest that CREB is essential for the CsA-mediated down-regulation of cathepsin B and L synthesis in HGFs.

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  • Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation

    Yumei Wu, Shuang Liang, Yoshiya Oda, Iori Ohmori, Tei-Ichi Nishiki, Kohji Takei, Hideki Matsui, Kazuhito Tomizawa

    EMBO JOURNAL   26 ( 12 )   2981 - 2990   2007.6

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    Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca2+-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca2+ influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.

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  • Implication of amphiphysin 1 and dynamin 2 in tubulobulbar complex formation and spermatid release

    Norihiro Kusumi, Masami Watanabe, Hiroshi Yamada, Shun-Ai Li, Yuji Kashiwakura, Takashi Matsukawa, Atsushi Nagai, Yasutomo Nasu, Hiromi Kumon, Kohji Takei

    CELL STRUCTURE AND FUNCTION   32 ( 2 )   101 - 113   2007

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    Tubulobulbar complexes (TBCs) are composed of several tubular invaginations formed at the plasma membrane of testicular Sertoli cells. TBCs are transiently formed at the contact region with spermatids at spermatogenic stage VII in rat and mouse, and such TBC formation is prerequisite for spermatid release. Since the characteristic structure of TBCs suggests that the molecules implicated in endocytosis could be involved in TBC formation, we here investigated the localization and physiological roles of endocytic proteins, amphiphysin 1 and dynamin 2, at TBCs. We demonstrated by immunofluorescence that the endocytic proteins were concentrated at TBCs, where they colocalized with cytoskeletal proteins, such as actin and vinculin. Immunoelectron microscopy disclosed that both amphiphysin 1 and dynamin 2 were localized on TBC membrane. Next, we histologically examined the testis from amphiphysin 1 deficient {Amph(-/-)} mice. Morphometric analysis revealed that the number of TBCs was significantly reduced in Amph(-/-). The ratio of stage VIII seminiferous tubules was increased, and the ratio of stage IX was conversely decreased in Amph(-/-). Moreover, unreleased spermatids in stage VIII seminiferous tubules were increased in Amph(-/-), indicating that spermatid release and the following transition from stage VIII to IX was prolonged in Amph(-/-) mice. These results suggest that amphiphysin 1 and dynamin 2 are involved in TBC formation and spermatid release at Sertoli cells.

    DOI: 10.1247/csf.07024

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  • A human beta-cell line for transplantation therapy to control type 1 diabetes

    M Narushima, N Kobayashi, T Okitsu, Y Tanaka, SA Li, Y Chen, A Miki, K Tanaka, S Nakaji, K Takei, AS Gutierrez, JD Rivas-Carrillo, N Navarro-Alvarez, HS Jun, KA Westerman, H Noguchi, JRT Lakey, P Leboulch, N Tanaka, JW Yoon

    NATURE BIOTECHNOLOGY   23 ( 10 )   1274 - 1282   2005.10

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    A human pancreatic b-cell line that is functionally equivalent to primary b-cells has not been available. We established a reversibly immortalized human b-cell clone ( NAKT-15) by transfection of primary human b-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase ( hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed b-cell transcription factors ( Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

    DOI: 10.1038/nbt1145

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  • Analysis of the major cdk5 phosphorylation sites of amphiphysin 1

    Shuang Liang, Yumi Yoshida, Kazuhito Tomizawa, Tadashi Abe, Hiroshi Yamada, Hideki Matsui, Kohji Takei

    CELL STRUCTURE AND FUNCTION   30   70 - 70   2005.6

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  • Localization of dynamin2 during mitosis

    Nobuhisa Ishida, Shun-Ai Li, Yuuichi Nakamura, Hiroshi Yamada, Toshio Sugahara, Kohji Takei

    CELL STRUCTURE AND FUNCTION   30   58 - 58   2005.6

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  • Dynamin 2 is involved in PS-dependent phagocytosis in rat Sertoli cells

    O. Hiroshi Yamada, ShunAI Li, Norihiro Kusumi, Masami Watanabe, Yuji Kashiwakura, Kazuhito Tomizawa, Hiromi Kumon, Kohji Takei

    CELL STRUCTURE AND FUNCTION   30   73 - 73   2005.6

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  • Localization of amphiphysin1 and dynamin2 in tubulobulbar complexes (TBC) formation and sperm release at Sertoli cells

    Norihiro Kusumi, Masami Watanabe, Hiroshi Yamada, Shun-Ai Li, Yuji Kashiwakura, Atsushi Nagai, Yasutomo Nasu, Hiromi Kumon, Pietro De Camilli, Kohji Takei

    CELL STRUCTURE AND FUNCTION   30   71 - 71   2005.6

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  • Regulatory mechanisms of dynamin-dependent endocytosis

    K Takei, Y Yoshida, H Yamada

    JOURNAL OF BIOCHEMISTRY   137 ( 3 )   243 - 247   2005.3

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    Extensive studies on endocytosis in the last decade have resulted in identification of several key molecules that function in clathrin- and dynamin-dependent endocytosis. Most endocytic molecules contain multiple binding motifs that mediate protein-protein or protein-lipid interactions, which must be modulated spatially and temporally during endocytosis. Regulation of these interactions is the molecular basis of regulatory mechanisms involved in endocytosis. This review first describes current models of the mechanism of dynamin-dependent fission, then introduces several mechanisms that modulate dynamin GTPase activity and dynamin-dependent vesicle formation. Such mechanisms include regulation by inositol phospholipids, especially phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2], and their metabolism. It concludes by describing the regulation of dynamin 1 by its binding partner, amphiphysin 1, and regulation by cyclin-dependent kinase 5 (Cdk5)-dependent phosphorylation of dynamin 1 and amphiphysin 1. These mechanisms help endocytic molecules to function properly, and cooperatively regulate dynamin-dependent endocytosis.

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  • Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice

    SA Li, M Watanabe, H Yamada, A Nagai, M Kinuta, K Takei

    CELL STRUCTURE AND FUNCTION   29 ( 4 )   91 - 99   2004.12

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    Klotho mutant mouse (kl-/-), a mouse model for human aging. exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration. skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA. however. is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variery of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain. kidney. and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were dominantly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/- mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions. were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation. which may explain why mutation of klotho gene results in a variety of phenotypes.

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  • Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell

    Y Kashiwakura, M Watanabe, N Kusumi, K Sumiyoshi, Y Nasu, H Yamada, T Sawamura, H Kumon, K Takei, H Daida

    CIRCULATION   110 ( 21 )   3329 - 3334   2004.11

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    Background-On exposure to oxidized low-density lipoprotein (oxLDL), vascular cells generally undergo apoptosis, which is one of the major pathogenic factors of atherosclerosis. In this study, we examined the role of dynamin ( a crucial GTPase protein in endocytosis) in oxLDL-induced apoptosis of vascular smooth muscle cells (VSMC).
    Methods and Results-After oxLDL stimulation, dynamin-2 colocalized with LOX-1 around the cell surface, as well as oxLDL in the cytoplasm, suggesting that dynamin-2 was involved in scavenger receptor-mediated oxLDL endocytosis. Downregulation of dynamin-2 induced by dynamin-2 dominant negative plasmid (K44A) resulted in a decrease of oxLDL uptake and thereby in a reduction of apoptosis. These data demonstrated that dynamin-2 was involved in oxLDL-induced apoptosis via the oxLDL endocytotic pathway. On the other hand, dynamin-2 wild-type plasmid transfection promoted oxLDL-induced apoptosis without increasing oxLDL uptake. Interestingly, the p53 inhibitor pifithrin-alpha (PFT) significantly reduced apoptosis promoted by wild-type dynamin-2 (78% reduction compared with the PFT[-] condition). These results indicated that dynamin-2 enhanced oxLDL-induced apoptosis of VSMC by participating in the p53 pathway, probably as a signal transducer. Moreover, we demonstrated that, in advanced plaques of apolipoprotein E-/- mice, dynamin-2 expression was often enhanced in apoptotic VSMC, suggesting that dynamin-2 might participate in apoptosis of VSMC even in vivo.
    Conclusions - Our data demonstrated that dynamin-2 at least partially regulated oxLDL-induced apoptosis of VSMC by participating in 2 independent pathways: the oxLDL endocytotic pathway and the p53 pathway. These findings suggest that dynamin-2 may serve as a new research or therapeutic target in vascular disease.

    DOI: 10.1161/01.CIR.0000147828.86593.85

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  • Dynamin 2 is involved in PS-dependent formation of membrane ruffles and phagocytosis in rat sertoli cells

    H Yamada, S Li, M Watanabe, F Wei, N Kusumi, K Tomizawa, M McNiven, H Matsui, H Kumon, K Takei

    MOLECULAR BIOLOGY OF THE CELL   15   195A - 196A   2004.11

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  • The stimulatory action of amphiphysin on dynamin function is dependent on lipid bilayer curvature

    Y Yoshida, M Kinuta, T Abe, S Liang, K Araki, O Cremona, G Di Paolo, Y Moriyama, T Yasuda, P De Camilli, K Takei

    EMBO JOURNAL   23 ( 17 )   3483 - 3491   2004.9

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    Amphiphysin is a major dynamin-binding partner at the synapse; however, its function in fission is unclear. Incubation of large unilamellar liposomes with mice brain cytosol led to massive formation of small vesicles, whereas cytosol of amphiphysin 1 knockout mice was much less efficient in this reaction. Vesicle formation from large liposomes by purified dynamin was also strongly enhanced by amphiphysin. In the presence of liposomes, amphiphysin strongly affected dynamin GTPase activity and the recruitment of dynamin to the liposomes, but this activity was highly dependent on liposome size. Deletion from amphiphysin of its central proline-rich stretch dramatically potentiated its effect on dynamin, possibly by relieving an inhibitory intramolecular interaction. These results suggest a model in which maturation of endocytic pits correlates with the oligomerization of dynamin with either amphiphysin or other proteins with similar domain structure. Formation of these complexes is coupled to the activation of dynamin GTPase activity, thus explaining how deep invagination of the pit leads to fission.

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  • Dynamin及びAmphiphysinのリン酸化-脱リン酸化による小胞形成能変化

    絹田 正裕, 荒木 健太, 阿部 匡史, 梁 爽, 吉田 祐美, 山田 浩司, 永松 知洋, 富澤 一仁, 松井 秀樹, 保田 立二, 竹居 孝二

    生化学   76 ( 3 )   307 - 307   2004.3

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  • ダイナミンとエンドサイトーシス。

    竹居孝二

    蛋白質核酸酵素 増刊『細胞における蛋白質の一生』   49,938-941   2004

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  • Cophosphorylation of amphiphysin 1 and dynamin 1 by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

    K Tomizawa, S Sunada, YF Lu, Y Oda, M Kinuta, T Ohshima, T Saito, FY Wei, M Matsushita, ST Li, K Tsutsui, S Hisanaga, K Mikoshiba, K Takei, H Matsui

    JOURNAL OF CELL BIOLOGY   163 ( 4 )   813 - 824   2003.11

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    It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation-induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with P-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

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  • Formation of meso, N-diphenylprotoporphyrin IX by an aerobic reaction of phenylhydrazine with oxyhemoglobins

    A Nakanishi, K Kinuta, T Abe, K Araki, Y Yoshida, S Liang, SA Li, K Takei, M Kinuta

    ACTA MEDICA OKAYAMA   57 ( 5 )   249 - 256   2003.10

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    Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (H-1-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl- and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.

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  • Formation of S-[2-carboxy-1-(1H-imidazol-4-yl) ethyl]glutathione, a new metabolite of L-histidine, from cis-urocanic acid and glutathione by the action of glutathione S-transferase

    M Kinuta, K Kinuta, H Yamada, T Abe, Y Yoshida, K Araki, SA Li, A Otsuka, A Nakanishi, Y Moriyama, K Takei

    ELECTROPHORESIS   24 ( 18 )   3212 - 3218   2003.9

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    Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite Of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), An adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma-glutamyl transpeptidase resulted in the formation of N-{S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl}glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

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  • ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type I gamma

    M Krauss, M Kinuta, MR Wenk, P De Camilli, K Takei, Haucke, V

    JOURNAL OF CELL BIOLOGY   162 ( 1 )   113 - 124   2003.7

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    Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels Of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

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  • 小胞形成に対するdynamin及びamphiphysinのリン酸化-脱リン酸化の効果

    荒木 健太, 阿部 匡史, 福田 功, 富澤 一仁, 山田 浩司, 須田 城, 絹田 正裕, 竹居 孝二

    日本細胞生物学会大会講演要旨集   56回   52 - 52   2003.5

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  • Comprehensive research on brain. Analysis of neurodegenerative disease using variation of endocytosis in neural synapse as indicator.

    竹居孝二, 絹田正裕, 山田浩司

    脳研究の総合的推進に関する研究 平成13年度採択公募班員   2003

  • イノシトールリン脂質によるエンドサイトーシスの制御機構

    絹田正裕, 竹居孝二

    生化学   75,1449-1452   2003

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  • Effects of phosphorylation of dynamin and amphiphysin on vesicle formations from liposomes

    M Kinuta, K Tomizawa, T Abe, Fukuta, I, H Matsui, K Takei

    MOLECULAR BIOLOGY OF THE CELL   13   95A - 95A   2002.11

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  • ARF6によるPI(4)P5-kinaseの活性化機構

    福田 功, 絹田 正裕, 芝 直基, 重歳 正尚, 阿部 匡史, Volker Haucke, 竹居 孝二

    生化学   74 ( 8 )   895 - 895   2002.8

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  • Distribution of dynamins in testis and their possible relation to spermatogenesis

    A Kamitani, H Yamada, M Kinuta, M Watanabe, SA Li, T Matsukawa, M McNiven, H Kumon, K Takei

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   294 ( 2 )   261 - 267   2002.6

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    Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynainin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immuno reactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis. (C) 2002 Elsevier Science (USA). All rights reserved.

    DOI: 10.1016/S0006-291X(02)00470-9

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  • Arf6によるPI(4)P 5-kinaseの活性化機構

    芝 直基, 重歳 正尚, 絹田 正裕, 阿部 匡史, 福田 功, Haucke Volker, 竹居 孝二

    日本細胞生物学会大会講演要旨集   55回   51 - 51   2002.5

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  • Utilization of liposomes in vesicular transport studies

    M Kinuta, K Takei

    CELL STRUCTURE AND FUNCTION   27 ( 2 )   63 - 69   2002.4

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    Various coated vesicles are implicated in the intracellular transport between different compartments. In vitro reconstitution is a powerful experimental system to study molecular mechanisms involved in assembly of coat proteins from cytosol onto membranes as well as formation of coated vesicles. Liposomes have been recently utilized in the cell-free systems. In this review, we summarize studies on reconstitutions of coated vesicles or coated structures on liposomes. A novel method using dynamic tight scattering (DLS) to quantify vesicle formation from liposomes also is described. Our recent study on the role of phospholipids in vesicle formation, where the DSL assay is used in combination with lipid analysis, also is introduced.

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  • Cdk5/p35 regulates neurotransmitter release through phosphorylation and downregulation of P/Q-type voltage-dependent calcium channel activity

    K Tomizawa, J Ohta, M Matsushita, A Moriwaki, ST Li, K Takei, H Matsui

    JOURNAL OF NEUROSCIENCE   22 ( 7 )   2590 - 2597   2002.4

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    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/ threonine kinase with close structural homology to the mitotic Cdks. The complex of Cdk5 and p35, the neuron-specific regulatory subunit of Cdk5, plays important roles in brain development, such as neuronal migration and neurite outgrowth. Moreover, Cdk5 is thought to be involved in the promotion of neurodegeneration in Alzheimer's disease.
    Cdk5 is abundant in mature neurons; however, its physiological functions in the adult brain are unknown. Here we show that Cdk5/p35 regulates neurotransmitter release in the presynaptic terminal. Both Cdk5 and p35 were abundant in the synaptosomes. Roscovitine, a specific inhibitor of Cdk5 in neurons, induced neurotransmitter release from the synaptosomes in response to membrane depolarization and enhanced the EPSP slopes in rat hippocampal slices. The electrophysiological study using each specific inhibitor of the voltage-dependent calcium channels (VDCCs) and calcium imaging revealed that roscovitine enhanced Ca2+ influx from the P/Q-type VDCC. Moreover, Cdk5/p25 phosphorylated the intracellular loop connecting domains II and III (LII-III) between amino acid residues 724 and 981 of isoforms cloned from rat brain of the alpha(1A) subunit of P/Q-type Ca2+ channels. The phosphorylation inhibited the interaction of LII-III with SNAP-25 and synaptotagmin I, which were plasma membrane soluble N-ethylmaleimide- sensitive factor attachment protein (SNAP) receptor (SNARE) proteins and were required for efficient neurotransmitter release. These results strongly suggest that Cdk5/p35 inhibits neurotransmitter release through the phosphorylation of P/Q-type VDCC and downregulation of the channel activity.

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  • Expression of endocytic proteins in the testis, and their implication in spermatogenesis.

    A Kamitani, M Watanabe, H Iguchi, A Nagai, H Yamada, M Kinuta, K Takei, H Kumon

    JOURNAL OF UROLOGY   167 ( 4 )   319 - 319   2002.4

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  • Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

    M Kinuta, H Yamada, T Abe, M Watanabe, SA Li, A Kamitani, T Yasuda, T Matsukawa, H Kumon, K Takei

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   99 ( 5 )   2842 - 2847   2002.3

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    As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration Of L-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] but not of L-alpha-phosphatidylinositol 4-monophosphate or L-alpha-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P-2 to L-alpha-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P-2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5'-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P-2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

    DOI: 10.1073/pnas.261715599

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  • ラット培養セルトリ細胞におけるDynamin2,3の発現

    山田浩司, 紙谷章弘, 渡部昌実, 絹田正裕, 公文裕巳, 竹居孝二

    生化学   74 ( 8 )   2002

  • 脳及び精巣におけるKlothoタンパクの発現とKlotho変異マウスのエンドサイトーシス機能タンパクの発現変化

    李順愛, 絹田正裕, 紙谷章弘, 紙谷章弘, 山田浩司, 公文裕巳, 竹居孝二

    日本細胞生物学会大会講演要旨集   55th   2002

  • Norrpinephrine-evoked 5-hydroxytryptamine secretion is Ca2+ dependent and mediated through exocytosis in rat pinealocytes.

    Yamada H, Hayashi M, Kinoshita M, Uehara S, Watanabe M, Takei K, Moriyama Y

    J Neurochem   2002

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  • Generation of high curvature membranes mediated by direct endophilin bilayer interactions

    K Farsad, N Ringstad, K Takei, Floyd, SR, K Rose, P De Camilli

    JOURNAL OF CELL BIOLOGY   155 ( 2 )   193 - 200   2001.10

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    Language:English   Publisher:ROCKEFELLER UNIV PRESS  

    Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, AN. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev,V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.

    DOI: 10.1083/jcb.200107075

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  • Determination of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione, a novel metabolite of L-histidine, in tissue extracts from sunlight-irradiated rat by capillary electrophoresis

    M Kinuta, J Ohta, H Yamada, K Kinuta, T Abe, SA Li, A Otsuka, A Nakanishi, K Takei

    ELECTROPHORESIS   22 ( 16 )   3365 - 3370   2001.10

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    Exposure of the skin to sunlight results in an increase in the content of epidermal Urocanic acid, a key metabolite Of L-histidine, and some portions of the metabolite penetrate into the body fluid. S-[2-Carboxy-1 -(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of glutathione and urocanic acid, was proposed to be an origin of a urinary compound, S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). Various catabolites of Cys(CIE) were also isolated from human urine previously. However, no direct evidence to show the existence of GS(CIE) as a biological material had been found. By using capillary electrophoresis, the glutathione adduct has now been found in the extracts of rat tissues from the kidney, liver, skin and blood when the rat was kept under conditions of sunlight irradiation after the fur on the dorsal skin had been clipped. On the other hand, no or a trace of GS(CIE) was determined in rat tissue extracts when the animal was kept indoor in usual manner. The glutathione adduct was isolated from the kidney extract of the sunlight-irradiated rat using ion-exchangers and high-voltage paper electrophoresis, and determined by fast-atom-bombardment mass spectrometry. These results indicate that GS(CIE) formation actually occurs in the body and that the formation is accelerated by exposing the rat to sunlight irradiation. From these findings, we propose an alternative pathway of histidine metabolism which is initiated by the adduction of urocanic acid to glutathione to form GS(CIE) and terminates with the formation of the urinary compounds via Cys(CIE).

    DOI: 10.1002/1522-2683(200109)22:16<3365::AID-ELPS3365>3.0.CO;2-M

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  • Clathrin-mediated endocytosis: membrane factors pull the trigger

    K Takei, Haucke, V

    TRENDS IN CELL BIOLOGY   11 ( 9 )   385 - 391   2001.9

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    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:ELSEVIER SCIENCE LONDON  

    Clathrin-mediated endocytosis is a vesicular transport event involved in the internalization and recycling of receptors participating in signal transduction events and nutrient import as well as in the reformation of synaptic vesicles. Recent studies in vitro and in living cells have provided a number of new insights into the initial steps of clathrin-coated vesicle formation and the membrane factors involved in this process. The unexpected complexity of these interactions at the cytosol-membrane interface suggests that clathrin-coated vesicle assembly is a highly cooperative process occurring under tight regulatory control. In this review, we focus on the role of membrane proteins and lipids in the nucleation of clathrin-coated pits and provide a hypothetical model for the early steps in clathrin-mediated endocytosis.

    DOI: 10.1016/S0962-8924(01)02082-7

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  • Ptdlns(4,5)P2の分解とエンドサイトーシス

    山田浩司, 絹田正裕, 阿部匡史, 李順愛, 渡部昌美, 紙谷章弘, 公文裕巳, 竹居孝二

    神経化学   40 ( 2/3 )   2001

  • エンドサイトーシスにおけるPtdIns(4,5)P2の分解

    絹田正裕, 山田浩司, 阿部匡史, 李順愛, 渡部昌実, 紙谷章弘, 公文裕巳, 竹居孝二

    日本細胞生物学会大会講演要旨集   54th   2001

  • ラット培養セルトリ細胞におけるエンドサイトーシス関連蛋白質の発現

    紙谷章弘, 山田浩司, 渡部昌実, 絹田正裕, 公文裕巳, 竹居孝二

    日本細胞生物学会大会講演要旨集   54th   2001

  • ラット松果体細胞からのセロトニンの開口放出

    上原俊介, 山田浩司, 林美都子, 木下美香, 渡部昌美, 竹居孝二, 森山芳則

    生化学   73 ( 8 )   2001

  • Characterization of synaptojanin 2B isoforms expressed in brain and tests.

    Nemoto Y, Wenk M R, Watanabe M, Daniel L, Murakami T, Nemoto T, Ringstad N, Yamada H, Takei K, De Camilli P

    J Biol Chem   2001

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  • Interactions of dynamin and amphiphysin with liposomes

    K Takei, Slepnev, VI, P De Camilli

    REGULATORS AND EFFECTORS OF SMALL GTPASES, PT E   329   478 - 486   2001

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    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:ACADEMIC PRESS INC  

    DOI: 10.1016/S0076-6879(01)29109-5

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  • Vesicle formation and membrane lipid kinetics in model synaptic endocytosis using liposomes.

    M Kinuta, H Yamada, T Abe, M Watanabe, J Ohta, K Takei

    MOLECULAR BIOLOGY OF THE CELL   11   326A - 326A   2000.12

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:AMER SOC CELL BIOLOGY  

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  • 細胞膜のダイナミックス エンドサイトーシスにおける細胞膜脂質の動態

    竹居孝二, 絹田正裕, 阿部匡史, 山田浩司, 太田潤, 渡部昌実

    日本細胞生物学会大会講演要旨集   53rd   2000

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Presentations

  • Elucidation of cytoskeletal dynamics in glomerular podocyte

    Kohji Takei, Tadashi Abe, Tetsuya Takeda, Katsuyuki Tananbe, Hiroshi Yamada

    The 96th Annual Meeting of the Japanese Biochemical Society  2023.11.1 

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    Event date: 2023.10.31 - 2023.11.2

    Language:Japanese   Presentation type:Poster presentation  

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  • Recruitment of Irgb6 to the Membrane Directly Triggers Membrane Deformation

    Tadashi Abe, Hiroshi Yamada, Hikaru Nagaoka, Eizo Takashima, Ryo Nitta, Masahiro Yamamoto, Kohji Takei

    The 95th Annual Meeting of the Japanese Biochemical Society  2022.11.11 

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    Event date: 2022.11.9 - 2022.11.11

    Language:Japanese   Presentation type:Poster presentation  

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  • Allosteric changes of hecto-scale population of dynamin GTPases provide in dynamic regulation of membranes and cytoskeletons Invited

    Kohji Takei, Tadashi Abe, Tetsuya Takeda, Hiroshi Yamada

    The 60th Annual Meeting of the Biophysical Society of Japan  2022.9.29 

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    Event date: 2022.9.28 - 2022.9.30

    Language:English   Presentation type:Symposium, workshop panel (nominated)  

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  • Dynamin-dependent microtubule regulation and morphogenesis of glomerular podocyte

    Kohji Takei, Hiroshi Yamada

    The 74th Annual Meeting of the Japanese Society for Cell Biology  2022.6.30 

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    Event date: 2022.6.28 - 2022.6.30

    Language:English   Presentation type:Oral presentation (general)  

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  • 細胞操作と定量イメージングで知る細胞骨格ダイナミズム 細胞の形態形成における膜-細胞骨格の動的相互作用

    竹田 哲也, 藤瀬 賢志郎, 山田 浩司, 竹居 孝二

    日本細胞生物学会大会講演要旨集  2020.6  (一社)日本細胞生物学会

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    Event date: 2020.6

    Language:Japanese  

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  • 腎糸球体ポドサイトにおけるアンフィファイジン1の機能

    山田 浩司, The Mon La, 竹田 哲也, 阿部 匡史, 淺沼 克彦, 竹居 孝二

    日本生化学会大会プログラム・講演要旨集  2019.9  (公社)日本生化学会

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    Event date: 2019.9

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  • 腎糸球体ポドサイトにおけるダイナミンイソフォームの局在と機能

    阿部 匡史, The Mon La, 橘 洋美, 竹田 哲也, 竹居 孝二, 山田 浩司

    日本生化学会大会プログラム・講演要旨集  2019.9  (公社)日本生化学会

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  • 筋細胞膜リモデリングにおけるメカニカルストレス応答の解析

    藤瀬 賢志郎, 山田 浩司, 竹居 孝二, 竹田 哲也

    日本筋学会学術集会プログラム・抄録集  2019.8  日本筋学会

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  • 膜リモデリングおよびリン脂質代謝異常に起因する先天性ミオパチー発症機序の解明

    藤瀬 賢志郎, 背山 佳穂, 山下 恭加, 山田 浩司, 戸井 基道, 竹居 孝二, 竹田 哲也

    日本筋学会学術集会プログラム・抄録集  2018.8  日本筋学会

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  • メカノエンザイム・ダイナミンGTPaseによるアクチン線維束化機構の解析

    山田 浩司, 阿部 匡史, 竹田 哲也, 高島 英造, 森田 将之, 竹居 孝二

    日本生物工学会大会講演要旨集  2018.8  (公社)日本生物工学会

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  • ダイナミン-コルタクチンらせん状複合体の解析 機械的なアクチン線維束形成とアクチン脱重合保護作用

    阿部 匡史, 山田 浩司, 竹田 哲也, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    生命科学系学会合同年次大会  2017.12  生命科学系学会合同年次大会運営事務局

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    Event date: 2017.12

    Language:Japanese  

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  • エンドサイトーシス生物学の新展開 ダイナミンによる膜切断過程の動態イメージング

    竹田 哲也, 小財 稔矢, 楊 恵然, 石黒 大輝, 背山 佳穂, 熊谷 祐介, 阿部 匡史, 山田 浩司, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    生命科学系学会合同年次大会  2017.12  生命科学系学会合同年次大会運営事務局

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  • GTP加水分解に共役したダイナミン依存的膜切断機構の高速原子間力顕微鏡解析

    竹田 哲也, 石黒 大輝, 楊 恵然, 小財 稔矢, 背山 佳穂, 熊谷 祐介, 山田 浩司, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    日本細胞生物学会大会講演要旨集  2017.5  (一社)日本細胞生物学会

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  • ダイナミン1は微小管の配向を調節しTRPV2の細胞膜移行を制御する

    籔彩夏, 安岡宏樹, 片野坂友紀, 阿部匡史, 竹田哲也, 竹居孝二, 山田浩司

    第62回日本生化学会中国・四国支部例会  2021.9.11 

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  • ダイナミン2の自己重合と膜との相互作用は糸球体ポドサイトのアクチン制御に重要である

    濱崎英理子, 安岡宏樹, 和木田夏輝, 阿部匡史, 竹田哲也, 竹居孝二, 山田浩司

    第62回日本生化学会中国・四国支部例会  2021.9.11 

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  • 糸球体ポドサイトからのグルタミン酸の放出と開口放出関連タンパクの探索

    安岡宏樹, 西井尚子, 原田結加, 宮地孝明, 阿部匡史, 竹田哲也, 和田淳, 竹居孝二, 山田浩司

    第62回日本生化学会中国・四国支部例会  2021.9.11 

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  • Dynamin-Cortactin Ring Complex Physically Bundles Actin Filaments and Protects the Bundle from Depolarization

    IGER International Symposium on Now in actin study: Motor protein research reaching a new stage]  2016 

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  • 神経細胞成長におけるアクチンダイナミクスの制御(Regulation of actin dynamics in growing neurons)

    10th Cell Transplant Society Congress  2009 

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  • Mechanism of actin-regulation by dynamin/cortactin complex

    米国細胞生物学会/日本細胞生物学会/理化学研究所 発生・再生科学総合研究センター合同ミーティング  2009 

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Research Projects

  • Elucidation of morphogenesis, maintenance and stabilization, and maintenance mechanism of glomerular podocytes by microtubule bundles

    Grant number:21K19484  2021.07 - 2024.03

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

    竹居 孝二

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    Grant amount:\6370000 ( Direct expense: \4900000 、 Indirect expense:\1470000 )

    1)ダイナミン1の微小管制御機構解明のため、精製タンパクを用いて微小管束形成を蛍光顕微鏡観察した。微小管の束化は全長ダイナミンだけでなくダイナミン1のプレクストリン相同(PH)ドメインでも観られた。さらに、ダイナミン1のPHドメイン、プロリンリッチ(PR)ドメインに対応する合成ドデカペプチドを用いた結合阻害実験により、PHドメインに存在する微小管結合部位を特定した。
    2)ダイナミン1が微小管依存性の細胞内小胞輸送に及ぼす影響を細胞レベルで解析した。血清刺激によりラッフル形成させた肺癌H1299細胞をモデル細胞として、カルシウムチャンネルTRPV2のラッフル膜への輸送を蛍光免疫染色とカルシウムイオンの細胞内流入を指標に調べた。ダイナミン1をノックダウン細胞では微小管束が減少しTRPV2のラッフル膜への移行が減少したことから、ダイナミンに1による微小管制御は微小管依存性の細胞内小胞輸送に必要であると考えられた。
    3)ポドサイトは分化に伴い著しく伸展することから、アクチンのリモデリングと細胞膜ダイナミクスを連携させる機構が存在すると考えられる。この連携を担う分子としてダイナミン2の関与が予想されたので、野生型ダイナミン2および疾患変異K562Eを用いてポドサイトにおける機能を調べた。K562E発現ポドサイトではストレスファイバーが減少し、異常なアクチンクラスターが形成され、ストレスファイバー再形成能も低かった。精製タンパクを用いた解析では、K562Eのアクチン線維束化能は野生型より少なく、膜結合能、膜変形能、自己重合能は顕著に低かった。リポゾームと野生型ダイナミン2存在下では、アクチン線維は太い線維束を形成されたが、K562Eではその形成は少なかった。以上より、ダイナミン2の脂質膜に対する親和性と自己重合能がポドサイトにおけるアクチン束化に必要であることを明らかにした。

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  • Cooperative regulation of cytoskeleton and membrane dynamics by novel mechanism of dynamin

    Grant number:19H03225  2019.04 - 2022.03

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

    Takei Kohji

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    Grant amount:\17290000 ( Direct expense: \13300000 、 Indirect expense:\3990000 )

    We found that Charcot-Marie-Tooth disease-associated mutations of dynamin 2 cause aberrant stress fibers, showing that dynamin is required for the formation and stabilization of stress fibers. And we reconstituted in vitro the actin bundle formation by dynamin. We also found that dynamin 1 bundles microtubules, and showed that this bundling is necessary for the primary processes formation and stabilization of cell morphology of renal glomerular podocytes. The microtubule-binding site of dynamin 1 was identified. Furthermore, regarding the regulation of membrane dynamics, we demonstrated that dynamin 2 and BIN1, a BAR protein, cooperatively function in T-tubule formation and stabilization of skeletal muscle cells.

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  • Elucidation of the function of novel actin-binding factors in neutrophil phagocytosis and extracellular trap formation

    Grant number:19K07084  2019.04 - 2022.03

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

    ABE TADASHI

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    Grant amount:\4420000 ( Direct expense: \3400000 、 Indirect expense:\1020000 )

    Neutrophils play an essential role in the destruction of bacteria in innate immune system. In the process of killing pathogens, neutrophils drastically change their cell shape with accompanying the rearrangement of actin cytoskeleton. We found that expressing dynamin 2 K562E mutant, one of the pathogenic mutations in Charcot-Marie-Tooth disease in cells leads to aberrant actin clusters and stress fibers. The effect of this mutant on actin fibers was analyzed in vitro. Recombinant dynamin 2 K562E showed lower self-assembly ability and membrane binding ability than that of dynamin 2 wildtype. Although dynamin K562E directly bundled actin filaments, the formed bundles showed much less ability to bind to the lipid membranes as compared to dynamin 2 wildtype. In conclusion, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in neutrophil functions.

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  • Development of the anti-invasive drug for treatment of malignant glioma by drug-repositioning of anti-depressant.

    Grant number:16K10756  2016.04 - 2019.03

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

    ABE TADASHI

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    Grant amount:\4810000 ( Direct expense: \3700000 、 Indirect expense:\1110000 )

    For treatment of malignant glioma, highly invasive glioma cells become obstacle to surgical removal of primary tumor. To suppress the high invasive activity of glioma cells, we identified the novel anti-invasive drug, a fluvoxamine, by drug repositioning of anti-depressant. Screening for more potent anti-invasive drugs using fluvoxamine as a lead compound are currently in progress. Furthermore, we found that actin-bundling by dynamin-cortactin complex is required for glioma cell invation. Cortactin is phosphorylated by cyclin dependent kinase 5 (CDK5), and its phosphorylation negatively regulates glioma cell invasion.

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  • Molecular regulatory mechanism of podocyte, the filtration apparatus of glomeruli

    Grant number:15K15330  2015.04 - 2018.03

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

    Takei Kohji

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    Grant amount:\3640000 ( Direct expense: \2800000 、 Indirect expense:\840000 )

    Podocyte is a cell that forms a glomerular filter for protein filtration. For the formation and the function of the glomerular filter, the cytoskeleton of podocyte needs to be appropriately regulated. However, it is poorly understood how cytoskeletons are regulated in podocyte. In this study, we aimed to elucidate regulatory mechanisms involved in the cytoskeletal regulation using two types of podocyte cell lines, Mouse Podocyte (MPC), and Human Podocyte (HPC). We found that dynamin 1 regulates microtubules, whereas dynamin 2 regulates actin cytoskeleton. Furthermore, we found that expression of dynamin 2 mutation found in Charcot Marie Tooth disease, a neurodegenerative disease, leads to aberrant actin cytoskeleton. These results strongly suggest that dynamin plays an essential role in the regulation of cytoskeleton in podocyte.

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  • Development of antimalarial drugs targeting the membrane trafficking

    Grant number:26670201  2014.04 - 2017.03

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

    YAMADA Hiroshi, TAKEI Kohji

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    Grant amount:\3640000 ( Direct expense: \2800000 、 Indirect expense:\840000 )

    Malaria parasites live in erythrocyte by forming parasitophorous vacuole (PV) around the parasite, and by developing membrane trafficking system. In this study, we tried to examine whether or not malarial proteins could participate in these membrane remodeling. By microscopy, the candidate protein self-assembled under the low ionic strength conditions. Furthermore, the candidate protein deformed liposomes. The activity of membrane deformation by the protein was altered in the presence of GTP but not GTP gamma S, a non-hydrolyzable GTP analogue. We are now investigating the detail mechanism of membrane deformation by the protein using electron microscopy.

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  • シナプスにおけるエンドサイトーシス機能タンパク-膜リン脂質相互作用の解析

    Grant number:14380306  2002 - 2004

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    絹田 正裕, 竹居 孝二, 山田 浩司

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    Grant amount:\10700000 ( Direct expense: \10700000 )

    Amphiphysin 1によるDynamin GTPase活性の上昇
    Dynamin 1GTPアーゼ活性に対するAmphiphysin 1と膜脂質の影響を調べ、Amphiphysin 1が、大型リポゾームの存在下にDynamin 1のGTPアーゼ活性を上昇することを明らかにした。リポゾームにフォスファチジルセリン、フォスファチジルイノシトール2リン酸などの酸性リン脂質を含まれる場合、特にGTPアーゼ活性の増強効果が高かった。ミュータントAmphiphysin 1を用いた解析により、Dynamin 1GTPアーゼ活性の増強には膜脂質への結合を担うBARドメインと、dynaminとの結合ドメインであるSH3ドメインが必要でであることを明らかにした。また、クラスリン結合部位、AP2結合部位を含む中間部は、Dynamin 1GTPアーゼ活性に対して抑制的に働くことが示唆された。
    Amphiphysin 1によるDynamin 1と膜脂質の結合の増加
    リポゾームを用いた結合実験により、Amphiphysin 1がDynamin 1と膜脂質の結合を増加させることを明らかにした。この結合増加の一因は、Amphiphysin 1のBARドメインとSH3ドメインを介した間接的結合によるものであることを、ミュータントAmphiphysin 1を用いた解析により明らかにした。
    Amphiphysin 1とDynamin 1と脂質膜の結合増加
    ミュータントAmphiphysin 1を用いた解析により,Amphiphysin 1とDynamin 1のリング形成にはBARドメインとSH3ドメインが必要であることを明らかにした。また、リング形成とDynamin 1 GTPアーゼ活性の上昇が、Dynamin 1と膜結合性にある程度相関することを示唆した。
    以上の成果を論文にまとめ発表した。

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  • A Study on Regulatory Mechanisms of Endocytosis

    Grant number:14380336  2002 - 2004

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

    TAKEI Kohji, YAMADA Hiroshi

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    Grant amount:\11100000 ( Direct expense: \11100000 )

    Regarding to regulatory mechanisms of endocytosis, the followings were revealed.
    1.Regulation of endocytosis by Amphiphysin 1
    Dynamin-dependent endocytosis can be reconstituted in vitro by incubating large unilammelar liposomes with brain cytosol or dynamin in presence of GTP. Using amphipshyin knockout brain cytosol in this experimental system, it was clarified that amphiphysin 1 stimulates dynamin GTPase activity and thereby enhances dynamin-dependent vesicle formation. This effect required both BAR domain and SH3 domain of amphiphysin 1. Low membrane curvature of large liposomes was also requisite for the stimulatory effect of amphiphysin 1.
    2.Regulation of endocytosis cdk5-dependent phosphorylation
    Both dynamin 1 and amphiphysin 1 are phosphorylated by cyclin dependent kinase 5 (cdk5). Incubation of liposomes with phosphorylated dynain and phosphorylated amphiphysin 1 in presence of GTP resulted in few vesicle formation, whereas dephophorylated proteins massively generated vesicles. Thus, endocytosis is likely to be regulated by cdk5-dependent phosphorylation.
    3.Localization of dynamin 2 and dynamin3
    Distinct localization of dynamin 2 and dynamin3 in Sertoli cells was revealed suggesting different functions of these isoforms.

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  • AMPA型グルタミン酸受容体のエンドサイトーシスの分子機構

    Grant number:13041045  2001

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    竹居 孝二, 山田 浩司, 絹田 正裕

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    Grant amount:\4000000 ( Direct expense: \4000000 )

    1.膵島β細胞株MIN6のキャラクタライゼイション
    膵島β細胞にはAMPA受容体が発現していることが見い出されており、本研究ではAMPA受容体のエンドサイトーシスの機構を解明するためにβ細胞を用いることを提案した。
    まず特異的抗体を用いたWestern blot法により、MIN6におけるAMPA受容体の発現を明らかにした。次にMIN6におけるエンドサイトーシス機能タンパクの発現を調べた。その結果、クラスリン依存性エンドサイトーシスの機能タンパクであるアンフィファイジン、さらにアンフィファイジンのリン酸化酵素CDK5が高発現することがWestern blot法により見い出された。
    2.エンドサイトーシスのin vitro再構成系の確立
    AMPA受容体エンドサイトーシスの分子メカニズム解明のためには、エンドサイトーシスによる取り込み小胞の形成をin vitroで再現する実験系の確立が必要であると考えた。In vitroで人工脂質膜(リポソーム)を膜成分として、ATPおよびGTP存在下に細胞質と反応させることにより、大型(直径>1μm)のリポソームから直径100nm以下の小胞が多数形成される実験系を確立した。さらに、形成された小胞の大きさ、数、相対的質量を動的光拡散測定装置を用いて測定することにより、小胞形成の定量化を可能とした。この実験系はAMPA受容体の選択的取り込みを解析するための実験系として応用されうる。

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  • A Study on Molecular Mechanisms of Endocytosis

    Grant number:12480217  2000 - 2001

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

    TAKEI Kohji, YAMADA Hiroshi, KINUTA Masahiro

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    Grant amount:\14100000 ( Direct expense: \14100000 )

    Establishment of in vitro cell-free system: In order to elucidate molecular mechanisms involved in endocytosis, vesicle formation in endocytosis was reconstituted in vitro. Incubation of large liposomes, larger than 1 μm in diameter, with brain cytosol resulted in massive formation of small vesicles, smaller than 100 nm in diameter. The vesicle formation required both ATP and GTP. Vesicle formation was drastically reduced when liposomes were incubated with dynamin 1 -depleted cytosol, indicating that vesicle formation in this experimental system represents endocytic vesicle formation. The vesicle formed during the incubation can be analyzed quantitatively and qualitatively by dynamic light scattering.
    Functions and Kinetics of membrane lipids: Functions of membrane lipids were studied by analyzing vesicle formation from liposomes of with various compositions. Vesicle formation increased as phosphatidylinositol-4.5-bisphosphate (PIP_2) concentration in liposomes was increased. Furthermore, PIP_2 was degraded to phosphatidylinositol-4-bisphosphate, then to phosphatidylinositol. Next, PIP_2 synthesis during the reaction was analyzed by addition of neomycin, inhibitor for PIP_2 degradation, in the reaction mixture. PIP_2 synthesis was increased by active form of ADP-ribosylation factor 6 (Arf6). It was suggested that increase of membrane recruitment of AP2, clathrin adaptor protein, by Arf6 might attribute to the increase of PIP_2 synthesis.
    Kinetics of membrane lipids in culture cells: Degradation of PTP_2 synthesis upon endocytosis was examined in culture cells. Metabolically labeled HeLa cells were stimulated for endocytosis and the amount of PIP_2 was analyzed. Similar PIP_2 degradation as that observed in the cell-free system was observed.

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