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阻滞突变体cdaA和cdaC，分析了分裂平面决定、分裂沟形成、划沟、收缩相关的收缩环微丝断裂的机制，以及钙调控分裂沟收缩的机制。在分裂面测定中，cdaA突变基因产物p85在测定中起着至关重要的作用，它作为收缩环肌动蛋白丝的聚合核。对于分裂沟收缩，通过对cdaC突变体的分析，认为突变基因产物是一种能够捆绑收缩环微丝的肌动蛋白结合蛋白。我们最近发现了一种与四膜虫的14nm细丝相关的蛋白质，并通过基因分析确定为EF-1alpha。我们现在正在研究EF-1alpha和cdaC基因产物之间的关系，因为四膜虫EF-1alpha具有肌动蛋白捆绑活性。在细胞分裂过程中，有人提出收缩环微丝的断裂是伴随着收缩环的收缩而发生的。在这方面，我们发现了一种肌动蛋白聚合抑制因子，四膜虫的profilin，在分裂沟中与肌动蛋白丝共定位，表明profilin参与了收缩环微丝的形成和分解。我们从四膜虫中克隆了3种钙结合蛋白（钙调蛋白、TCBP-23和TCBP-25）的基因，研究了钙对分裂沟收缩的调控作用。我们现在通过注射大肠杆菌中产生的基因或修饰的基因产物来仔细检查它们在细胞分裂中的生物学功能。

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.