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水螅中的作用。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）。在此研究期间，我们成功克隆并测序了p85的eDNA。数据库分析表明，p85是一种不同于目前已知的frcin蛋白的新蛋白，但与Ca^<2+>/钙调素依赖性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Ca^<2+>依赖的方式与钙调蛋白结合。p85定位于压缩环微丝形成前的分形面。钙调蛋白也与p85在假定的分裂平面上共定位。这种p85与钙调蛋白复合物可能作为一个重要的起始信号在四膜虫的细胞分裂过程中发挥作用，因为钙调蛋白的特异性抑制剂VI完全抑制了赤道定位和分裂沟的形成。此外，我们发现ef - α和7lkDa纤维蛋白样蛋白两种肌动蛋白结合蛋白都是cdaC突变基因产物的候选蛋白，它们也定位于分裂沟中。特别地，ef - α的肌动蛋白捆绑活性被证明依赖于Ca^<2+>-钙调蛋白。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。在这方面，我们关注钙调蛋白在四膜虫细胞质分裂中的作用，因为钙调蛋白作为细胞质分裂的起始信号与p85一起起作用，钙调蛋白可以调节收缩环中肌球蛋白的活性，也可以调节收缩环肌动蛋白丝被ef - α捆绑。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