| Research Abstract |
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
Tetrahyrnena. 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
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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
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