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，我们主要研究了温度敏感型细胞分裂停滞突变体(CDA基因座)的突变基因产物在纤毛虫原生动物中的作用。四膜虫CDAA突变体在决定分裂平面的因子上存在缺陷。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，我们成功地克隆和测序了P85基因的EDNA。数据库分析表明，P85是一种新的蛋白，不同于已知的frins蛋白，但与钙调素依赖的蛋白激酶有部分同源序列，提示P85具有钙调素结合位点。事实上，P85以钙依赖的方式与钙调蛋白结合。P85已本地化到PRES…紧靠收缩环微丝形成前的平面图。钙调蛋白也与P85共定位于假定的分裂平面。由于钙调素的特异性抑制剂VI完全抑制了四膜虫的赤道定位和分裂沟的形成，因此我们成功地从四膜虫中分离纯化了肌球蛋白。此外，我们还发现了两种肌动蛋白捆绑蛋白EF-α和7lkDa纤毛蛋白样蛋白，这两种蛋白都是CDAC突变基因产物的候选者，它们也分布在分裂沟中。特别是EF-α的肌动蛋白结合活性被证明是钙依赖的。鉴于肌动球蛋白系统对收缩环的钙调节作用，我们研究了三种四膜虫钙调蛋白家族蛋白：钙调蛋白、TCBP-25、TCBP-23在细胞分裂中的功能。在这一点上，我们关注钙调蛋白在四膜虫细胞质分裂中的作用，因为钙调蛋白与P85一起作为胞质分裂的起始信号，而钙调蛋白可能调节收缩环上的肌球蛋白活性，还通过EF-α调节收缩环肌动蛋白细丝的捆绑。较少

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