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

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止突变体（cda位点）突变基因产物在纤毛原生动物四膜虫中的作用。四膜虫cdaA突变体在分裂面决定因子上存在缺陷。我们证明了该因子不仅决定了分裂面，而且还决定了作为聚合核的收缩环微丝的形成。突变基因产物已被证明是一个85 kDa的蛋白（命名为p85）。在本研究期间。我们成功克隆并测序了p85的cDNA。数据库分析表明，p85是一种不同于目前已知蛋白的新蛋白，但与actin、EF-1alpha、Ca^<2+>/calmodulin依赖性蛋白激酶有同源序列，提示p85是一种在细胞分裂中起重要作用的多功能蛋白。四膜虫cdaC突变体与分裂沟收缩的直接机制有关，突变基因产物已被证明是一种F-actin捆绑因子。为此，我们证明了四膜虫EF-1alpha在生理状态下具有显著的F-actin捆绑活性。此外，我们证明了F-actin的捆绑活性明显受到Ca^<2+>/calmodulin系统的调节。针对肌动球蛋白系统对钙调蛋白（Ca^<2+>-）在收缩环收缩中的调控作用，我们通过在大肠杆菌中表达三种四膜虫钙调蛋白家族蛋白（calmodulin, TCBP-25, TCBP-23）的基因纯化，研究了它们在细胞分裂中的功能。

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.