Analysis of molecular mechanisms of cell division in Tetrahymena using cell-division-arrest mutants.

为了阐明动物细胞分裂的分子机制，我们利用四膜虫的细胞分裂停滞突变体CDAA和CDAC，分析了细胞分裂平面的确定、分裂沟的形成、皱纹的形成、收缩相关的收缩环微丝的解体和分裂沟收缩的钙调节机制。至于分裂沟收缩，通过对CDAC突变体的分析，认为突变的基因产物是一种能够与收缩环微丝捆绑在一起的肌动蛋白结合蛋白。我们最近发现了一种与四膜虫14 nm细丝相关的蛋白质，并通过基因分析确定它为EF-1α。我们正在研究EF-1α与CDAC基因产物的关系，因为四膜虫EF-1α具有肌动蛋白捆绑活性。在胞质分裂中，收缩环微丝的解体伴随着收缩环的收缩而发生。在这一点上，我们发现一个肌动蛋白聚合抑制因子，四膜虫的Profilin，与肌动蛋白细丝在分裂沟中共存，提示Profilin参与了收缩环微丝的形成和分解。关于分裂沟收缩对钙的调节，我们从四膜虫中克隆了三种钙结合蛋白(钙调素、TCBP-23和TCBP-25)的基因。我们现在正在仔细研究它们在胞质分裂中的生物学功能，方法是注射在大肠杆菌中产生的基因或修改后的基因产品。

To elucidate the molecular mechanisms of cell division in animal cells,by using Tetrahymena's cell-division-arrest mutants, cdaA and cdaC, we have analyzed mechanisms for division plane determination, division furrow formation, furrowing, contraction-related contractle ring microfilament disassembly, and for calcium-regulation of division furrow constriction.Concerning division plane determination, it was evident that cdaA mutant gene product,p85, plaied a crucial role in the determination and it served as polymerization nuclei of the contractile ring actin filaments. As for division furrow constriction, by the analysis of cdaC mutant, the mutant gene product is considered to be an actin-binding protein capable of bundling the contractile ring microfilaments. We recently found a protein which associates with Tetrahymena's 14-nm filaments and we identified it as EF-1alpha from its gene analysis. We are now investigating the relation between EF-1alpha and cdaC gene product, since Tetrahymena EF-1alpha has an actin-bundling activity. In cytokinesis, it has been proposed that contractile ring microfilament disassembly occurs accompanying with the contraction of contractile ring. In this regard, we found that an actin-polymerization-inhibitory factor, profilin of Tetrahymena, colocalized with actin filaments in the division furrow, suggesting the involvement of profilin in the formation and disassembly of contractile ring microfilaments. Concerning the calcium-regulation of division furrow constriction, we cloned the genes for 3 kinds of calcium-binding proteins (calmodulin, TCBP-23, and TCBP-25) from Tetrahymena. We are now scrutinized their biological functions in cytokinesis by injecting the gene or modified gene products yielded in E. coli.