Mutations in α- and β-tubulin affect spindle formation in Chinese hamster ovary cells

Abstract

Two Chinese hamster ovary cell lines with mutated β-tubulins (Grs-2 and Cmd-4) and one that was a mutation in α-tubulin (Tax-1) are temperature sensitive for growth at 40.5°C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5°C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5°C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti-α-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered β-tubulins and the altered α-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.