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

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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 protozoan Tetrahymena.Tetrahymena cdaA mutant has a defect in the detemination 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 cDNA for p85. The data base analysis indicates that p85 is a new protein different from proteins known so far, but sharespartly homologous sequences with actin, EF-1alpha, Ca^<2+>/calmodulin-dependent protein kinase, suggesting that p85 is a multifunctional protein playing crucial roles in cell division.Tetrahymena cdaC mutant is relevant to the direct mechanism of division furrow constriction and the mutant gene product has been shown to be an F-actin bundling factor. In this regard, we proved that Tetrahymena EF-1alpha has remarkable F-actin bundling activity in a physiological condition. Moreover, we demonstrated that the F-actin bundling activity was clearly regulated by Ca^<2+>/calmodulin system.Concerning the Ca^<2+>-regulation in the constriction of contracatile ring by actomyosin system, we have investigated the functions in cell division of the threo kinds of Tetrahymena calmodulin family proteins, such as calmodulin, TCBP-25, TCBP-23, by purifying these proteins after expressing their genes in E.coli.