Molecular mechanisms of cytoplasmic division in Tetrahymena using cell-division-arrest mutants

使用细胞分裂停滞突变体研究四膜虫细胞质分裂的分子机制

• 批准号: 05454025
• 负责人: WATANABE Yoshio
• 金额: $ 3.9万
• 依托单位: Joubu University (1994-1995)University of Tsukuba (1993)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454025/
• 关键词: Tetrahymena; cytoplasmic division; cell-division-arrest mutant; p85 gene; F-actin bundling factor; EF-1alpha; Ca^<2+>; calmodulin; calmodulin family protein; p85; TCBP-25; TCBP-23; 分裂面決定因子; Ca^<2+>-結合蛋白質; テトラヒメナミオシン; small G-蛋白質

To elucidate the molecular mechanisms of cytoplasmic division in animal cells, we have mainly investigated the roles of mutant gene products of temperature-sensitive cell-division-arrest mutants (cda loci) in ciliated protozoan Tetrahymena.Tetrahymena cdaA mutant has a defect in the detemination factor of division plane. We demonstrated that the factor was responsible not only for division plane determination but also for the formation of contractile ring microfilaments as a polymerization nucleus. The mutant gene product has been shown to be a 85 kDa protein (designated as p85). In this research period. we succeeded in cloning and sequencing of cDNA for p85. The data base analysis indicates that p85 is a new protein different from proteins known so far, but sharespartly homologous sequences with actin, EF-1alpha, Ca^<2+>/calmodulin-dependent protein kinase, suggesting that p85 is a multifunctional protein playing crucial roles in cell division.Tetrahymena cdaC mutant is relevant to the direct mechanism of division furrow constriction and the mutant gene product has been shown to be an F-actin bundling factor. In this regard, we proved that Tetrahymena EF-1alpha has remarkable F-actin bundling activity in a physiological condition. Moreover, we demonstrated that the F-actin bundling activity was clearly regulated by Ca^<2+>/calmodulin system.Concerning the Ca^<2+>-regulation in the constriction of contracatile ring by actomyosin system, we have investigated the functions in cell division of the threo kinds of Tetrahymena calmodulin family proteins, such as calmodulin, TCBP-25, TCBP-23, by purifying these proteins after expressing their genes in E.coli.

为了阐明动物细胞中细胞质分裂的分子机制，我们主要研究了温度敏感的细胞 - 细胞 - 分割 - 数分突变突变（CDA loci）在纤毛介导的原生动物四氢烷中的突变基因产物。除法平面因素。我们证明了该因子不仅是分裂平面确定的原因，而且还导致收缩环微丝形成作为聚合核的原因。突变基因产物已显示为85 kDa蛋白（指定为p85）。在这个研究期间。我们成功地将p85的cDNA克隆和测序。数据库分析表明，p85是一种与迄今已知的蛋白不同的新蛋白四捕虫CDAC突变体在细胞分裂中至关重要的作用与分裂沟收缩的直接机理有关，并且已证明突变基因产物是F-肌动蛋白捆绑因子。在这方面，我们证明了四膜EF-1Alpha在生理状态下具有出色的F-肌动蛋白捆绑活性。此外，我们证明了F-肌动蛋白捆绑活性通过CA^<2+>/钙调蛋白系统明确调节。在Actomyosin System中调查CA^<2+> - 调节临界环的调节，我们已经研究了功能在三环甲钙化蛋白家族蛋白的细胞分裂中，例如钙调蛋白，TCBP-25，TCBP-23，通过在大肠杆菌中表达基因后净化这些蛋白质。

项目成果

沼田治: "テトラヒメナの多機能蛋白質" 蛋白質核酸酵素. 39. 106-118 (1994)

Osamu Numata：“四膜虫的多功能蛋白质”蛋白质核酸酶。39. 106-118 (1994)

Watanabe,Y.,Numata,O.,Katoh,M. & Kurasawa,Y.: "Cultivation of Tetrahymena pyriformis in Cell Biology:A Laboratory Handbook,ed.by J.E.Celis" Academic Press,San Diego(August,In Press.), (1994)

渡边，Y.，沼田，O.，加藤，M.

Watanabe,Y.: "Control of cell cycle" Europian Journal of Protistology. (印刷中). (1994)

Watanabe，Y.：“细胞周期的控制”欧洲原生学杂志（出版中）。

Watanabe, Y., Numata, O., Kurasawa, Y.& Katoh, M.: "Cultivation of Tetrahymena cells." In Cell Biology : A Laboratory Handbook, ed, by J.E.Celis, Academic Press, San Diego.Vol.I. 389-404 (1994)

渡边，Y.，沼田，O.，仓泽，Y.

Takagi, T., Iwasa, H., Yuasa, H., Shikama, K., Takemasa, T.& Watanabe, Y.: "Primary structure of Tetrahymena hemoglobins." Biochim.Biophys.Acta. 1173. 75-78 (1993)

高木，T.，岩佐，H.，汤浅，H.，鹿间，K.，竹正，T.