Analysis of molecular mechanisms of cell division in Tetrahymena using cell-division-arrest mutants.

使用细胞分裂停滞突变体分析四膜虫细胞分裂的分子机制。

• 批准号: 02454020
• 负责人: WATANABE Yoshio
• 金额: $ 4.29万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454020/
• 关键词: Tetrahymena; cell division; cytokinesis; cell-division mutant; actin; profilin; EF-1alpha; calcium-binding protein; 分裂停止突然変異株; テトラヒメナ・アクチン; Ca^<2+>-結合蛋白質; 分裂停止突然変異; 収縮環; DNA複製; EFー1α; テトラヒメナ アクチン; テトラヒメナ プロフィリン; カルシウム 結合蛋白質; アンチセンス RNA /

To elucidate the molecular mechanisms of cell division in animal cells,by using Tetrahymena's cell-division-arrest mutants, cdaA and cdaC, we have analyzed mechanisms for division plane determination, division furrow formation, furrowing, contraction-related contractle ring microfilament disassembly, and for calcium-regulation of division furrow constriction.Concerning division plane determination, it was evident that cdaA mutant gene product,p85, plaied a crucial role in the determination and it served as polymerization nuclei of the contractile ring actin filaments. As for division furrow constriction, by the analysis of cdaC mutant, the mutant gene product is considered to be an actin-binding protein capable of bundling the contractile ring microfilaments. We recently found a protein which associates with Tetrahymena's 14-nm filaments and we identified it as EF-1alpha from its gene analysis. We are now investigating the relation between EF-1alpha and cdaC gene product, since Tetrahymena EF-1alpha has an actin-bundling activity. In cytokinesis, it has been proposed that contractile ring microfilament disassembly occurs accompanying with the contraction of contractile ring. In this regard, we found that an actin-polymerization-inhibitory factor, profilin of Tetrahymena, colocalized with actin filaments in the division furrow, suggesting the involvement of profilin in the formation and disassembly of contractile ring microfilaments. Concerning the calcium-regulation of division furrow constriction, we cloned the genes for 3 kinds of calcium-binding proteins (calmodulin, TCBP-23, and TCBP-25) from Tetrahymena. We are now scrutinized their biological functions in cytokinesis by injecting the gene or modified gene products yielded in E. coli.

为了阐明动物细胞分裂的分子机制，我们利用四膜虫细胞分裂停滞突变体cdaA和cdaC，分析了分裂平面决定、分裂沟形成、沟形成、收缩相关的收缩环微丝解体以及分裂沟收缩的钙调节机制。在此过程中起着关键作用，它是收缩环肌动蛋白丝的聚合核。对于分裂沟收缩，通过对cdaC突变体的分析，认为突变基因产物是一种肌动蛋白结合蛋白，能够使收缩环微丝成束。我们最近发现了一种与四膜虫14纳米细丝相关的蛋白质，并通过基因分析将其鉴定为EF-1 α。由于四膜虫EF-1 α具有肌动蛋白集束活性，我们现在正在研究EF-1 α和cdaC基因产物之间的关系。在胞质分裂中，已经提出收缩环微丝的解体伴随着收缩环的收缩而发生。在这方面，我们发现，肌动蛋白聚合抑制因子，profilin的四膜虫，共定位与肌动蛋白丝在分裂沟，这表明参与profilin的形成和拆卸的收缩环微丝。针对钙离子对分裂沟收缩的调控，我们克隆了四膜虫中3种钙离子结合蛋白（钙调素、TCBP-23和TCBP-25）的基因。我们现在通过注射在大肠杆菌中产生的基因或修饰的基因产物来仔细检查它们在胞质分裂中的生物学功能。杆菌

项目成果

Numata,O.,Tomiyoshi,T.,Kurasawa,Y.,Fu,Z-x., Takahashi,M.,Kosaka,T.,Chiba,J. & Watanabe,Y.: "Antibodies against Tetrahymena 14-nm filament-forming protein recognize the replication band in Euplotes" Exptl. Cell Res.193. 183-189 (1991)

沼田，O.，富吉，T.，仓泽，Y.，Fu，Z-x.，高桥，M.，小坂，T.，千叶，J.

Numata,O.,Takemasa,T.,Takagi,I.,Hirono,M.,Hirano,H.,Chiba,J. & Watanabe,Y.: "Tetrahymena 14-nm filament-forming protein has citrate synthase activity" Biochem. Biophys. Res. Commun.174. 1028-1034 (1991)

沼田，O.，武正，T.，高木，I.，广野，M.，平野，H.，千叶，J.

Takemasa,T.,Takagi,N.and Watanabe,Y.: "Calciumーbinding proteins from Tetrahymena.In Novel CalciumーBinding Proteins.Fundamentals and Clinical Implications.,ed.by C.W.Heizman,Chapter26" Springer Press, 481-495 (1991)

Takemasa, T.、Takagi, N. 和 Watanabe, Y.：“来自四膜虫的钙结合蛋白。新型钙结合蛋白。基本原理和临床意义。”，C.W. Heizman 编辑，第 26 章”Springer Press，481- 495 (1991)

Hirono,M.,Tanaka,R.,& Watanabe,Y.: "<Tetrahymena>___ー actin:Copolymerization with skeletal muscle actin and interactions with muscle actinーbinding proteins." J.Biochem.107. 32-36 (1990)

Hirono, M.、Tanaka, R. 和 Watanabe, Y.：“<四膜虫>___- 肌动蛋白：与骨骼肌肌动蛋白的共聚以及与肌肉肌动蛋白结合蛋白的相互作用。J.Biochem.107（1990 年）。 ）

Takemasa,T.,TAkagi,T.,Edamatsu,M. & Watanabe,Y.: "Calmodulin cDANs two species of Tetrahymena" Biochim. Biophys. Acta. 1132. 219-221 (1992)

武正，T.，高木，T.，毛松，M.