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

为了阐明动物细胞胞质分裂的分子机制，我们主要研究了温敏性细胞分裂停滞突变体(CDA基因座)突变产物在纤毛虫四膜虫中的作用。我们证明，这一因素不仅是决定分裂平面的因素，也是形成收缩环状微丝作为聚合核心的原因。突变的基因产物被证明是一个85 kDa的蛋白质(标记为P85)。在本研究期间。成功克隆并测序了p85基因的全长序列。数据库分析表明，P85是一个新的蛋白质，与已知的蛋白质不同，但与肌动蛋白、EF-1α、钙离子和钙调素依赖的蛋白激酶有部分同源序列，表明P85是一种在细胞分裂中起关键作用的多功能蛋白质。四膜虫CDAC突变体与分裂沟收缩的直接机制有关，突变基因产物被证明是F-肌动蛋白结合因子。在这方面，我们证明了四膜虫EF-1α在生理条件下具有显著的F-肌动蛋白结合活性。此外，我们还证明了F-肌动蛋白结合活性明显受Ca~(2+)/钙调蛋白系统的调节。鉴于肌动球蛋白系统对收缩环中的Ca~(2+)~(2+)的调节，我们研究了四膜虫钙调蛋白家族的三种蛋白：钙调蛋白、TCBP-25、TCBP-23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.