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

为了阐明动物细胞质分裂的分子机制，我们主要研究了温度敏感性细胞分裂停滞突变体（cda基因座）的突变基因产物在纤毛原生动物Tetrahyrnena中的作用。四膜虫cdaA突变体在分裂平面的决定因子上存在缺陷。我们证明，该因子不仅负责分裂平面的确定，但也为收缩环微丝作为聚合核的形成。突变基因产物已显示为85 kDa蛋白质（命名为p85）。在本研究期间，我们成功地克隆和测序了p85的eDNA。数据库分析表明，p85是一种新的蛋白质，不同于目前已知的frcin蛋白质，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与钙调蛋白的结合依赖于Ca^2+。p85定位于前列腺 ...更多信息 收缩环微丝形成前的假想分裂平面。钙调素也与p85共定位在假定的分裂平面。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，这种p85和钙调素复合物可能在四膜虫的胞质分裂中作为一种重要的起始信号发挥作用。它们都是cdaC突变基因产物的候选者，并且它们也定位于分裂沟中。本研究针对肌动球蛋白收缩环收缩过程中的Ca ^<2 +>调节，通过在大肠杆菌中表达钙调素、TCBP-25、TCBP-23三种四膜虫钙调素家族蛋白，并对其基因进行纯化，研究了它们在细胞分裂中的功能。在这方面，我们注意到钙调素在四膜虫细胞质分裂中的作用，因为钙调素的功能与p85作为胞质分裂的起始信号，钙调素可以调节收缩环中的肌球蛋白活性，并通过EF-α调节收缩环肌动蛋白丝的成束。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