Studies on the molecular mechanism of cell division using cell-division-arrest mutants in Tetrahymena

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For elucidation the molecular mechanism of cell division, we have carried out our studies using temperature-sensitive cell-division-arrest mutants in Tetrahymena. In this report, we describe the results concerning the roles of mutant gene products in cell division of cdaA and cdaC which we have persued extensively.1. Studies on cdaC : We previously showed that cdaC has a ts-defect in the structure (designated as LS) which binds contractile ring microfilaments. LS is thought to be the structure which transmits the force generated by the contraction of contractile ring to the surface layer and is inferred to be an actin-binding protein. However, the presence of actin in Tetrahymena has not so far been proved. In this study, we have succeeded in cloning and sequencing of Tetrahymena actin gene. We then synthesized a peptide deduced from the amino acid sequence of the predicted actin, prepared an antibody spedific for the peptide, and identified actin by immunoblotting. This Tetrahymena ac … More tin differs from ubiquitous actin in some properties, but has the same biological roles as other actin does. We also succeeded in isolating actin from Tetrahymena for the first time. The purified actin shows very unique biochemical properties. We are now trying to detect and purify actin-binding proteins to persue the mutant gene product of cdaC.2. Studies on cdaA : cdaA has a ts-defect in a determination of cell division plane. We have already identified the mutant gene product (p85) by 2-D gel electrophoresis and genetic analysis. In this study, we succeeded in isolating p85, and preparing its antibody. By using immunofluorescent antibody technique, p85 is shown to be localized in the presumptive division plane just before division. On the other hand, neither such a p85-deposit nor cell division occurred when the mutant cells were exposed to the restrictive temperature. The p85-deposit preceeded the formation of contractile ring microfilaments and both sites were closely related. Thus, we suggest that p85-deposit acts as nucleus of the formation of contractile ring microfilaments. Less