Research Projects -
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ダイナミンによる細胞骨格制御機構とその破綻に関わる病態解明
2019.04 - 2020.03
愛媛大学 愛媛大学プロテオサイエンスセンター共同研究
Authorship：Principal investigator Grant type：Competitive
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Genome-wide expression of P. falciparum membrane proteins
Grant number：18K19455 2018.06 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
Takashima Eizo
Grant amount：\6240000 （ Direct expense: \4800000 、 Indirect expense：\1440000 ）
Membrane associated plasmodial proteins are usually difficult to predict using the present algorithms, and are not well expressed by under conventional wheat germ cell-free protein expression system (WGCFS) conditions. In this study, we aimed at expressing P. falciparum genes using WGCFS-liposome method for membrane protein production. As a result, we succeeded to express P. falciparum proteins which are not well expressed by usual conditions of WGCFS. Moreover, liposome encapsulated WGCFS successfully expressed some membrane proteins and the recombinant proteins were localized to the liposomal membrane. Based on these results, we conclude that WGCFS-liposome is a useful tool for the expression of membrane proteins and will facilitate production and functional analyses of P. falciparum membrane proteins.
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ダイナミンによる細胞骨格制御機構とその破綻に関わる病態解明
2018.04 - 2019.03
愛媛大学 愛媛大学プロテオサイエンスセンター共同研究
Authorship：Principal investigator Grant type：Competitive
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熱帯熱マラリア原虫ダイナミンホモログによる膜制御機構
2018.04 - 2019.03
神戸大学 神戸大学バイオシグナル総合研究センター共同利用研究
Authorship：Principal investigator Grant type：Competitive
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Analysis of membrane deforming proteins in Malaria parasite
Grant number：17K08808 2017.04 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Yamada Hiroshi
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
Malaria parasite live with erythrocyte and form parasitophorous vacuole (PV) around the parasite. And the parasite develops membrane trafficking system to get nutrients and transport many proteins. In this study, we tried to examine whether or not malarial proteins could participate in these membrane remodeling. We found that the candidate protein directly bound to liposomes and deformed them. The activity of membrane deformation by the protein was altered in the presence of GTP. We are now investigating the detailed mechanism of membrane deformation by the protein using electron microscopy and high speed atomic force microscopy.
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熱帯熱マラリア原虫ダイナミンホモログによる膜制御機構
2017.04 - 2018.03
神戸大学 神戸大学バイオシグナル総合研究センター共同利用研究
Authorship：Principal investigator Grant type：Competitive
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熱帯熱マラリア原虫ダイナミンホモログによる膜制御機構
2016.07 - 2017.03
神戸大学 神戸大学バイオシグナル総合研究センター共同利用研究
Authorship：Principal investigator Grant type：Competitive
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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) Grant-in-Aid for Scientific Research (C)
ABE TADASHI
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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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 Grant-in-Aid for Challenging Exploratory Research
YAMADA Hiroshi, TAKEI Kohji
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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The role of endocytosis in oateoarhthritis
Grant number：26670665 2014.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
HIROHATA Satoshi, YAMADA Hiroshi, OHTSUKI Takashi
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
The regulation of ADAMTS5 is crucial for osteoarthritis. However, little is known for its mechanism. We hypothesized that endocytosis may be involved in ADAMTS5 regulation. First, we found that endocytosis occurred in OUMS-27. We next examined endocytosis-related molecule, LRP-1 and RAP. The mRNA level of receptor-associated protein (RAP), antagonist of LRP-1, was not changed by IL-1 beta stimulation. It is interesting that the small-sized bands were found by Western blotting using anti-LRP-1 antibody after IL-1 beta stimulation. This is considered to be a short LRP-1 and the shedding of LRP-1 may be occurred by IL-1 beta stimulation.
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Multi-functionality of dynamin family and mechanism of integrated control
Grant number：23370089 2011.04 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
TAKEI Kohji, YAMADA Hiroshi, TANEBE Kenji
Grant amount：\18980000 （ Direct expense: \14600000 、 Indirect expense：\4380000 ）
We have clarified a novel regulatory mechanism of actin dynamics by dynamin/cortactin complex. The complex is ring-shaped and it changed the conformation upon dynamin GTP hydrolysis, from open ring to close ring. By the open-cloze motion, the complex bundled F-actins and stabilized the actin bundles. Furthermore, we identified N'-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide(DBHA)as a dynamin inhibitor that suppress dynamin-dependent actin regulation. DBHA inhibited recruitment of dynamin to the leading edge of migrating cancer cell line and ruffle formation. It also showed inhibitory effect in cell migration, invasion, and proliferation.
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Development of antimalarial drug targeting plasmodium falciparum dynamin
Grant number：23659213 2011 - 2013
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
TAKEI Kohji, YAMADA Hiroshi
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
By in vitro actin polymerization assay, we identified N'-(4-(diethylamino) benzylidene)-4-methoxybenzohydrazide (DBHA) as a dynamin inhibitor. Effects of DBHA on ruffle formation and cell migration were also examined, and the results on DBHA were published in a journal article.
Dynamin isoforms present in plasmodium falciparum, pfDyn1 and pfDyn2, were expressed in insect cells, and they were purified. Using these recombinant proteins, we demonstrated that both pfDyn1 and pfDyn2 have GTPase activity. It was also shown that pfDyn1 and pfDyn2 have an ability to deform lipid membranes. Furthermore, pfDyn2 inhibitor candidate molecules were determined by GTPase activity assay-based drug screening. -
Grant number：22240056 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
NARUSE Keiji, MOHRI Satoshi, NAKAMURA Kazufumi, TAKEI Kohji, YAMADA Hiroshi, IRIBE Gentaro, KATANOSAKA Yuki
Grant amount：\50570000 （ Direct expense: \38900000 、 Indirect expense：\11670000 ）
Physical and mechanical stimuli such as gravity, extension, and shearing stress are generated throughout a living body. It has been gradually revealed that these stimuli, which are transmitted via the mechanotransduction mechanisms of cells, are not simply detrimental stresses for living organisms, but rather are biological information essential to developmental processes and functional adaptation of organs. In this project, on the basis of the development of original loading systems for mechanical stress to cells and tissues, the cellular adaptive responses to the mechanical stimuli and their transduction mechanisms in a variety of mechanosensitive tissues are to be examined. Thus, the project aims toelucidate the molecular mechanisms and roles of mechanotransduction system, which is utilized as a basis of many physiological events. It can also contribute to develop therapeutic approach to cancer invasion, cardiac hypertrophy, and regeneration of neuronal circuit.
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The role of dynamin in actin dynamics: Development of anticancer drugs inhibiting dynamin function
Grant number：22501013 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
ABE Tadashi, YAMADA Hiroshi, WATANABE Masami, ASAI Akira
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
In search of more effective inhibitor for dynamin than dynasore, one hundred eighty one dynasore analogues were screened by in vitro actin polymerization assay. N'-(4-(diethylamino)benzylidene)-4-methoxybenzohydrazide (DBHA) was identified as a potent dynamin inhibitor. DBHA strongly inhibited the serum-stimulated ruffle formation, cell migration and invasion. Under these conditions, DBHA showed little toxicity for cultured cancer cell line. Furthermore, we clarified the function of dynamin/cortactin complex in the regulation of actin dynamics. The dynamin/cortactin complexes bundled several actin filaments and the bundling stabilized actin filaments. The actin bundling by dynamin/cortactin complex was necessary for formation of growth cone filopodia and cell migration. These findings might be crucial for developping anti cancer drugs.
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Grant number：22616004 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
YAMADA Hiroshi
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
The function of stretch-activated cation channel on actin dynamics was investigated. Calcium ion concentration in the ruffle membrane was increased during cell migration. Knockdown of the channel in cancer cell line resulted in marked reduction ofboth calcium influx via the channel and serum-stimulated ruffle formation, which were required for cell migration. These results suggest that stretch-activated cation channel in cancer cell is involved in cell migration possibly by regulating actin dynamics. Furthermore, we identified the function of dynamin/cortactin complex in the regulation of actin dynamics. The dynamin/cortactin complexes bundled several actin filaments and the bundling stabilized actin filaments. The actin bundling by dynamin/cortactin complex was necessary for formation of growth cone filopodia and cell migration. These findings might be crucial for the study for neuronal regeneration and cancer cell migration.
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Molecular mechanism of endocytosis that regulates membrane dynamics
Grant number：17370071 2005 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
TAKEI Kohji, YAMADA Hiroshi, LI Shun-ai, TANAE Kenji
Grant amount：\15280000 （ Direct expense: \14800000 、 Indirect expense：\480000 ）
In order to elucidate the role of endocytic proteins in membrane dynamics, localization and intracellular dynamics of dynamin and amphiphysin during phagocytosis were examined. For this purpose, testicular Sertoli cell phagocytosis was stimulated by phosphatidylserine containing liposomes, and intracellular localization and dynamics of the endocytic proteins were examined. By immunofluorescence and by live cell imaging, both dynamin2 and amphiphysin were concentrated at the leading edge of lamellipodia and ruffles. Ruffle formation, actin formation, and phagocytosis were markedly inhibited in Amphiphysin siRNA treated cells indicating that amphiphysin is essential for these processes. Furthermore, these effects in the amphiphysin 1-knocked down cells were rescued by co-overexpression of constitutive active Rac 1, suggesting that Rac 1 is involved in the process at the downstream of amphiphysin.
Next, effect of amphiphysin 1 on actin polymerization activity was examined in vitro. For this purpose, mouse testis cytosol supplemented with pyrene-conjugated actin, were subjected to quantitative actin in vitro polymerization assay. Actin polymerization was also observed under fluorescent microscopy using cytosol supplemented with rhodamine-conjugated actin. Actin assembly activity was considerably reduced in cytosol from amphiphysin 1 knock out mice, which can be recovered by adding back recombinant proteins.
Thus, amphiphysin 1, an endocytic protein, play a role in the regulation of actin dynamics, by which it stimulate in membrane dynamics and phagocytosis. -
エンドサイトーシスの分子機構:分子構造から細胞機能まで
Grant number：15079206 2003 - 2007
日本学術振興会 科学研究費助成事業 特定領域研究
竹居 孝二, 山田 浩司, 李 順愛, 田邊 賢司, 絹田 正裕
Grant amount：\106400000 （ Direct expense: \106400000 ）
1)ダイナミンによる微小管動態制御
エンドサイトーシスの機能タンパクであるダイナミンは、細胞骨格の一つである微小管に結合する遺伝子として同定されたが、その生理的意義は不明であった。そこで我々はRNAiによりダイナミン2の発現を抑制した細胞の微小管を形態的に観察した。その結果、ダイナミン2のノックダウンにより動的な微小管が減少し、微小管に沿った膜輸送の障害によりゴルジ体の分散が起こる事を見いだした。さらに、変性性末梢神経障害であるシャルコー・マリー・トゥース病の原因遺伝子として報告されたダイナミン変異体を発現させた細胞でも、同様の結果が観察され微小管の異常な蓄積が観察された。以上の結果からダイナミンが微小管のダイナミクスを制御している可能性が示唆された。
2)チューブ状エンドソームの切断と成熟機構
分子選別に機能する初期エンドソームでは、初期エンドソームからリサイクルに向かうチューブ状エンドうソームが形成切断され、リサイクルエンドソームに向かう。一方、残存したエンドソームは、分解に向かう物質を含んだ後期エンドソームへの成熟が行われる。この二つのプロセスがどのように連携しているのかは判っていなかった。我々は、阻害剤を用いてチュニブ状エンドソームの切断、後期エンドソームへの成熟を阻害し、これらのプロセスについて解析した。その結果、チュブ状エンドソームの切断にダイナミンが関与しており、チューブ切断がエンドソームの酸性化と移動などの成熟過程に必要である事を見出した。 -
エンドサイト-シス小胞形成のリアルタイム観察
Grant number：15657028 2003 - 2005
日本学術振興会 科学研究費助成事業 萌芽研究
竹居 孝二, 山田 浩司, 李 順愛, 絹田 正裕
Grant amount：\2900000 （ Direct expense: \2900000 ）
ファゴサイトーシスの分子機構解明のために、セルトリ細胞を用いてファゴサイトーシスのリアルタイム観察を行った。まず、セルトリ細胞株SerW3にフォスファチジルセリン(PS)を認識する受容体(SR-B1)が発現していることをウエスタンブロットにより確認し、細胞膜における局在のパターンを蛍光免疫染色により明らかにした。次に、SerW3細胞によるPS含有大型リポゾーム取込みを経時的、定量的に調べることにより、セルトリ細胞のファゴサイトーシスが、SR-B1を介してPS依存性に起こることを明らかにした。SerW3細胞に、PS含有リポゾームあるいはPSで被覆したスチレンビーズを貪食させ、その様子を超高速共焦点レーザー顕微鏡下でリアルタイム観察したところ、ファゴサイトーシスにおけるpseudo pod形成に先立って糸状突起やラッフルの形成など、F-アクチンの再編成による細胞膜の形態変化が著明に認められた。PS刺激からアクチン重合、ラッフル形成にいたる経路には、フォスファチジルイノシトール3リン酸キナーゼ、Rac、cdc42が関与することが阻害剤を用いた実験により示された。GFP-アンフィファイジン1を発現させたSerW3細胞をライブイメージングにより観察すると、ラッフル形成に伴ってアンフィファイジン1がラッフルに集積する様子が認められた。SerW3細胞のアンフィファイジン1をRNAiによりノックダウンするとラッフル形成が抑制されたことから、アンフィファイジン1がファゴサイトーシス初期におけるアクチンフィラメントの再編成に関与することが示唆された。
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Molecular mechanism of L-glutamate and GABA-mediated regulation of secretion of glucagon and insulin in islets of Langerhans
Grant number：15390026 2003 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
MORIYAMA Yoshinori, OTUSKA Masato, YAMADA Hiroshi
Grant amount：\13500000 （ Direct expense: \13500000 ）
Vesicular glutamate transporter (VGLUT) and vesicular GABA transporter (VGAT) play essential roles in the glutamatergic and GABAergic chemical transduction, respectively. VGLUT and VGAT are present not only in the neuronal synaptic vesicles but also in the glucagon-containing secretory granules of A cells of islets of Langerhans, and are responsible for vesicular storage of glutamate and GABA, respectively. This research aimed to reveal the molecular mechanism of the glutamate- and GABA-mediated regulation of secretion of glucagon and insulin in the islets. During three years, we have obtained the following important results.
(1)We have revealed topology of VGLUT2.
(2)We have established an in vitro assay system for VGLUT, and identified some essential amino acid residues for transport of glutamate as well as targeting.
(3)We have established an in vitro assay system for VGAT.
(4)We have observed decreased glutamatergic signaling in VGLUT1 KO mice.
(5)We have identified and characterized glutamate signaling pathway in intestinal L cells.
(6)We have identified and characterized a series of receptors for regulation of secretion of insulin and glucagon. -
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) Grant-in-Aid for Scientific Research (B)
TAKEI Kohji, YAMADA Hiroshi
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.