細胞質分裂におけるアクチン調節タンパク質の役割

• Project/Area Number: 10213201
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (B)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: University of Tsukuba
• Principal Investigator: 沼田 治 筑波大学, 生物科学系, 教授 (50189354)
• Co-Investigator(Kenkyū-buntansha): 阿部 洋志 千葉大学, 理学部・生物学科, 助教授 (00222662)
• Project Period (FY): 1998 – 2001
• Project Status: Completed (Fiscal Year 2001)
• Budget Amount: ¥75,100,000 (Direct Cost: ¥75,100,000); Fiscal Year 2001: ¥16,700,000 (Direct Cost: ¥16,700,000); Fiscal Year 2000: ¥17,100,000 (Direct Cost: ¥17,100,000); Fiscal Year 1999: ¥18,600,000 (Direct Cost: ¥18,600,000); Fiscal Year 1998: ¥22,700,000 (Direct Cost: ¥22,700,000)
• Keywords: テトラヒメナ / Ca^<2+> / カルモジュリン / アクチン / 収縮環 / コフィリン / プロフィリン / フィンブリン / ペプチド伸長因子-1α / 細胞質分裂 / 分裂溝 / アクチン繊維 / ミオシン / アフリカツメガエル / リン酸化脱リン酸化 / カルシウムイオン / フィブリン / リン酸化 / 脱リン酸化

Research Abstract

1.細胞分裂時におけるp85/カルモデュリン(CaM)とアクチンの相互作用の解明:テトラヒメナのp85はCa^<2+>/CaMとともに分裂面の決定に関わる。p85とF-アクチン、p85とG-アクチンの相互作用を共沈法および免沈法によって検討した。その結果、p85はF-アクチンに結合しなかったが、Ca^<2+>/CaMに依存してG-アクチンと結合することが分った。これはp85が分裂面にG-アクチンを集め、収縮環アクチン繊維の形成に重要な役割をしていることを示唆している。我々の成果をまとめて以下のようなモデルを提唱する。まず、Ca^<2+>/CaMとp85は分裂面の位置を決定し、さらにG-アクチンを分裂面に集める。プロフイリンがG-アクチンの重合を促進し、形成されたアクチン繊維をペプチド伸長因子1αやフィンブリンなどが束ね収縮環が完成するというものである。
2.コフィリン結合タンパク質CAPの機能の解明:コフィリンは、アクチン線維を切断し脱重合する活性を持つ。コフィリンに結合するCAPはアクチン分子と1対1の割合で複合体を形成し、CAPはアクチン、コフィリンと共に培養細胞のラッフル膜部位に共局在している。
(1)CAPはアクチンに対して添加量依存的に、重合阻害作用を示す。
(2)CAPはATP型アクチンに比べ、ADP型アクチンにより高い親和性を示す。
(3)CAPはアクチンのヌクレオチド交換反応を濃度依存的に促進する。
(4)CAPはアクチン繊維に結合しているコフィリンを引き離す作用を持つ。
以上のことから、コフィリンおよびCAPによりアクチン繊維のマイナス端からADP型G-アクチンが解離する。ADP型G-アクチン/コフィリン複合体にCAPが結合し、コフィリンをアクチンから引き離す。その結果、フリーのコフィリンの数が増加し、新たなアクチン線維の切断と脱重合が起こる。一方で、CAPはADP型アクチンのヌクレオチド交換を促進して、重合可能なアクチンを増加させる。つまり、CAPは重合阻害因子というよりは、相乗的にアクチンの重合を促進する因子である。

Research Products

• [Publications] Tetsuya Takeda: "Identification of Tetrahymena hsp60 as a 14-nm filament protein/citrate syntliase-binding protein and its possible involvement in the oral apparatus formation"Genes to Cells. 6巻. 139-149 (2001)
• [Publications] Hiroko Kojima: "Enzymatic form and cytoskeletal form of bifunctional Tetrahymena 49kDa protein is regulated by phosphorylation"Zoological Science. 19巻. 37-42 (2002)
• [Publications] Osamu Numata: "Determination of division plane and organization of contractile ring in Tetrahymena"Cell Structure and Function. 26巻. 559-567 (2001)
• [Publications] 久永真市: "細胞分裂と細胞運動"遺伝. 第56巻(印刷中). (2002)
• [Publications] T.Takahashi: "Functional involvement of Xenopus LIM-kinases in progression of oocyte maturation"Dev.Biol.. 229巻. 554-567 (2001)
• [Publications] Osamu Numata: "Tetrahymena elongation factor-1 a is localized with calmodulin in the division furrow."Journal of Biochemistry. 127巻. 51-56 (2000)
• [Publications] Atsushi Watanabe: "Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein."Journal of Biochemistry. 127巻. 85-94 (2000)
• [Publications] Osamu Numata: "Cytokinesis in Tetrahymena : Determination of division plane and organization of contractile ring."Microscopy Research and Technique. 49巻. 127-135 (2000)
• [Publications] Kenta Fujiu: "Reorganization of microtubules in the amitotically dividing macronucleus of Tetrahymena."Cell Motility and the Cytoskeleton. 46巻. 17-27 (2000)
• [Publications] Kohsuke Gonda: "Calmodulin and Ca^<2+>/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis."Cell Structure and Function. 25巻. 243-251 (2000)
• [Publications] Osamu Numata: "Tetrahymena calcium-binding proteins, TCBP-25 and TCBP-23.Methods in Cell Biology,62巻"Academic Press. 11 (2000)
• [Publications] Numata,O.,Kurasawa,H.,and Watanabe.Y.: "Tetrahymena elongation factor-1 α binds to Hsp70 family proteins."Zoological Science. 16. 279-284 (1999)
• [Publications] Fujiu,K.,andNumata,O.: "Localization of microtubules during macronuclear division in Tetrahymena and possible involvement of macronuclear microtubules in 'amitotical'chromatin distribution."Cell Structure and Function. 24. 401-404 (1999)
• [Publications] Gonda,K.,Nishibori,K.,Ohba,H.,Watanabe,A.,and Numata,O.: "Molecular cloning of the gene for p85 that regulates the initiation of Cytokinesis in Tetrahymena."Biochemical and Biophysical Research Communication. 246. 112-118 (1999)
• [Publications] Gonda,K.,Katoh,M.,Hanyu,K.,Watanabe,Y.,and Numata,O.: "Ca_<2+>/calmodulin and p85 cooperatively regulate an initiation of cytokinesis in Tetrahymena."Journal of Cell Science. 112. 3619-3626 (1999)
• [Publications] Wang,S.,Nakashima,S.,Numata,O.,Fujiu,K.,and Nozawa,Y.: "Molecular cloning and cell-cycle-dependent expression of the acetyl-CoA synthetase gene in Tetrahymena cells."Biochem J.. 343. 479-85 (1999)
• [Publications] Numata,O.,Kurasawa,H.,Gonda,K.,and Watanabe.Y.: "Tetrahymena elongation factor-1 α is localized with calmodulin in the division furrow."Journal of Biochemistry. 127. 51-56 (2000)
• [Publications] Watanabe,A.,Yonemura,I.,Gonda,K.,and Numata,O.: "Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein."Journal of Biochemistry. 127. 85-94 (2000)
• [Publications] Numata,O.,Gonda,K.,Watanabe,A.,and Kurasawa,Y.: "Cytokinesis in Tetrahymena:determination of division plane and organization of contractile ring."Microsco.Res.Tech.. (in press). (2000)
• [Publications] Numata,O.,Fujiu,K.,and Gonda,K.: "Macronuclear division and cytokinesis in Tetrahymena."Cell Biol.Int.. (in press). (2000)
• [Publications] Kusano,K.,Abe,H.and Obinata,T.: "Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin."Mol.Cell.Biochem.. 190. 133-141 (1999)
• [Publications] Tsubakimoto,K.,Matsumoto,K.,Abe,H.,Ishii,J.,Amano,M.,Kaibuchi,K.and Endo,T.: "Small GTPase RhoD suppresses cell migration and cytokinesis."Oncogene. 18. 2431-2440 (1999)
• [Publications] Okada,K.,Obinata,T.and Abe,H.: "XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1),which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins."J.Cell Sci.. 112. 1553-1565 (1999)
• [Publications] Numata,O.,Hanyu,K.,Takeda,T.,and Watanabe,Y.: "Tetrahymena calcium-binding proteins,TCBP-23 and TCBP25."In Tetrahymena thermophila,ed.by D.J.Asai and J.D.Forney,Methods in Cell Biology,62巻,Academic Press.. 11 (1999)
• [Publications] A.Watanabe: "The new Tetrahymena actin-binding proteins is localized in the division furrow" J.Biochem.123. 607-613 (1998)
• [Publications] S.Wang: "Molecular cloning and cell-cycle-dependent expression of a novel NIMA(never-in-mitosis in Aspergillus nidulans)-related protein kinase(TpNrk)in Tetrahymena cells" Biochem.J.334. 197-203 (1998)
• [Publications] O.Numata: "Tetrahymena elongation factor-1α binds to Hsp 70 family proteins(in press)" Zoological Science. (1999)
• [Publications] Y.Soen: "Generation of functional b-actinin(CapZ)in an E.coli expression system" J.Mus.Res.Cell.Motil.19. 639-646 (1998)
• [Publications] Y.Ohtsuka: "Functional characteristics and the complete primary structure of ascidian gelsolin" Biochim.Biophys.Acta.1383. 219-231 (1998)
• [Publications] K.Kusano: "Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin.(in press)" Mol.Cell.Biochem.(1999)
• [Publications] K.Tsubakimoto: "Small GTPase RhoD suppresses cell migration and cytokinesis." Oncogene. (in press). (1999)
• [Publications] O.Numata: "Tetrahymena calcium-binding proteins, TCBP-25 and TCBP-23 In TETRAHYMENA THERMOPHILA(D.J.Asai, J.D.Forney eds.)" Academic Press, San Diego(in press), (1999)