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: 08454272
• 负责人: WATANABE Yoshio
• 金额: $ 4.35万
• 依托单位: Jobu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08454272/
• 关键词: Tetrahymena; cytoplasmic division; cell-division-arrest mutant; p85 gene; F-actin bundling factor; EF-1alpha; Ca^<2+>; calmodulin; calmodulin family protein; アクチン繊維束化因子; 分裂開始シグナル; p85; p71; 分裂溝; アクチン束化因子; Ca^<2+>-結合蛋白質; ミオシン

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 protozoanTetrahyrnena. Tetrahymena cdaA mutant has a defect in the determination 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 eDNA for p85. The data base analysis indicates that p85 is a new protein different frcin proteins known so far, but shares partly homologous sequences with Ca^<2+>/calmodulin-dependent protein kinase, suggesting that p85 has calmodulin-binding sites. In fact, p85 binds to calmodulin in a Ca^<2+>-depenndent manner. p85 is localized to the pres … More umptive division plane just before contractile ring microfilament formation. Calmodulin also colocalized with p85 at the presumptive division plane. This p85 and calmodulin complex may play a role in cytokinesis of Tetrahymena as an important initiation signal, since VI, a specific inhibitor of calmodulin, completely inhibits the equatorial localization and division furrow formation,In this research period, we succeeded in purification of myosin from Tetrahymena.Furthermore, we found that two kinds of actin-bundling proteins, EF-alpha and 7lkDa fimbrin-like protein, both of them are candidates for cdaC mutant gene product, and they are also locarized in the division furrow. In especial, actin-bunndling activity of EF-alpha is proved to be Ca^<2+>-calmodulin dependent.Concerning the Ca^<2+>-regulation in the constriction of contracatile ring by actomyosin system, we have investigated the functions in cell division of the three kinds of Tetrahymena calmodulin family proteins, such as calmodulin, TCBP-25, TCBP-23, by purifying these proteins after expressing their genes in E.coli. In this regard, we pay the attention to the roles of calmodulin in the cytoplasmic division of Tetrahymena, since calmodulin functions with p85 as an initiation signal of cytokinesis, and calnodulin may regulates myosin activity in the contractile ring, and also regulates the bundling of contractile ring actin filaments by EF-alpha. Less

为了阐明动物细胞细胞质分裂的分子机制，我们主要研究了温度敏感细胞分裂停滞突变体（cda位点）的突变基因产物在纤毛原生动物四膜虫cdaA突变体中的作用，该突变体在决定因子上存在缺陷。我们证明该因子不仅负责划分平面的确定，而且还负责作为聚合核的收缩环微丝的形成。已被证明是一种85 kDa的蛋白质（命名为p85）。在本研究期间，我们成功地克隆并测序了p85的eDNA。数据库分析表明p85是一种与迄今为止已知的frcin蛋白质不同的新蛋白质。与Ca 2+ /钙调蛋白依赖性蛋白激酶部分同源的序列，表明p85具有钙调蛋白结合位点。事实上，p85在a中与钙调蛋白结合。 Ca^<2+> 依赖性方式。在收缩环微丝形成之前，p85 定位于预分裂面。该 p85 和钙调蛋白复合物可能在胞质分裂中发挥作用。四膜虫作为重要的起始信号，因为 VI 是钙调蛋白的特异性抑制剂，完全抑制赤道定位和分裂沟在本研究期间，我们成功地从四膜虫中纯化出了肌球蛋白。此外，我们还发现了两种肌动蛋白捆绑蛋白，EF-α和7lkDa fimbrin-like蛋白，它们都是cdaC突变基因产物的候选物，特别是，EF-α的肌动蛋白成束活性被证明是Ca^2+-钙调蛋白。针对肌动球蛋白系统对收缩环的Ca^2+-调节，我们研究了钙调蛋白、TCBP-25、TCBP-三种四膜虫钙调蛋白家族蛋白在细胞分裂中的功能。 23、通过在大肠杆菌中表达其基因后纯化这些蛋白质。在这方面，我们关注钙调蛋白在细胞质分裂中的作用。四膜虫，由于钙调蛋白与 p85 一起作为胞质分裂的起始信号发挥作用，并且钙调蛋白可能调节收缩环中的肌球蛋白活性，并且还通过 EF-α Less 调节收缩环肌动蛋白丝的成束。

项目成果

Kojima,H, Watanabe,Y.& Numata,O.: "The Dual Function of Tetrahymena citrate synthase are due to the polymorphism of its isoform" J.Biochem.122. 998-1003 (1997)

小岛，H，渡边，Y.

Watanabe,Y., Numata,O., Karasawa,Y.& Katoh,M.: "Cultivation of Tetrahymena" Celis's “Cell Biology : A Laboratory Hand Book" 2nd Ed.by Academic Press, 406-412 (1998)

Watanabe, Y.、Numata, O.、Karasawa, Y. 和 Katoh, M.：“四膜虫的培养”Celis 的“细胞生物学：实验室手册”第二版，学术出版社，406-412（1998 年）

Numata,O.,Yamazaki,A.& Watanabe,Y.: "Tetrahymena 14-nm filament protein (49KDa protein) and citrate synthase are expressed by the same gene." Europ.J.Protistol.33 suppl.1. 108-113 (1996)

沼田，O.，山崎，A.

Watanabe,A.,Kurasawa,Y.,Watanabe,Y.and Numata,O.: "The New Tetrahymena actin-binding protein is localized in the division furrow." The Journal of Biochemistry. 123巻. 406-412 (1998)

Watanabe, A.、Kurasawa, Y.、Watanabe, Y. 和 Numata, O.：“新的四膜虫肌动蛋白结合蛋白位于分裂沟中。”《生物化学杂志》123。406-412（1998 年） ）

Kanzawa, N., Numata, O., Watanabe, Y.and Maruyama, K.: "Identification and characterization of Tetrahymena myosin." Comp.Biochem.Physiol.115B. 574-551 (1996)

Kanzawa, N.、Numata, O.、Watanabe, Y. 和 Maruyama, K.：“四膜虫肌球蛋白的鉴定和表征。”