Taxol-dependent Mutants of Chinese Hamster Ovary Cells with Alterations in aand /-Tubulin

Chinese hamster ovary cell mutants resistant to the microtubule stabilizing drug taxol were isolated in a single step. Of these 139 drug-resistant mutants, 59 exhibit an absolute requirement for taxol for normal growth and division, 13 have a partial requirement, and 69 grow normally without the drug. Twodimensional gel analysis of whole cell proteins reveal "extra" spots representing altered tubulins in 13 of the mutants. Six of these have an altered a-tubulin and seven have an altered ~-tubulin. Cells with an absolute dependence on taxol become large and multinucleated when deprived of the drug. In contrast, partially dependent cells exhibit some multinucleation, but most cells appear normal. In one mutant that has an absolute dependence on taxol, the cells appear to die more quickly and their nuclei do not increase in size or number. As previously found for another taxol-dependent mutant (Cabral, F., 1983, J. Cell. Biol., 97:22-29), the taxol dependence of the mutants described in this paper behaves recessively in somatic cell hybrids, and the cells are more susceptible to being killed by colcemid than are the wild-type parental cells. When compared with wild-type cells, taxol-dependent mutants have normal arrays of cytoplasmic microtubules but form much smaller mitotic spindles in the presence of taxol. When deprived of the drug, however, these mutants cannot complete assembly of the mitotic spindle apparatus, as judged by tubulin immunofluorescence. Thus, the defects leading to taxol dependence in these mutants with defined alterations in , and ~-tubulin appear to result from the cell's inability to form a functional mitotic spindle. Reversion analysis indicates that the properties of at least one atubulin mutant are conferred by the altered tubulin seen on two-dimensional gels. M ICROTUBULES are involved in many important cellular functions including mitosis, particle and organelle transport by saltatory motion, and secretion ( 11, 25). Much has been learned about microtubule assembly and function through the use of morphological techniques and specific microtubule-inhibiting drugs. Our understanding of microtubule assembly and organization has also been furthered by in vitro polymerization studies and by the ability to visualize arrays of these structures in whole cells with fluorescently labeled antibodies. Another approach that has been enormously successful in studying biological processes is the combination of genetics and biochemistry. This type of approach has only recently been used to study the cytoskeleton and should provide much useful information about the control of microtubule assembly and function, especially when combined with the morphological techniques available. Early attempts were made to select yeast and Chlamydomonas cells resistant to microtubule-inhibiting drugs (1, 15, 29). The specific defects responsible for the drug resistance phenotype, however, could not be demonstrated. Later investigations with the fungus Aspergillus proved more useful. Mutants resistant to benomyl were found to have alterations in ~-tubulin, and a revertant of one of these strains was found to have an altered a-tubulin (18, 26). Using these mutants, the authors were able to demonstrate that nuclear movement in that organism is microtubule dependent (19, 20). In mammalian cells, Ling and Thompson first isolated colchicineresistant mutants in Chinese hamster ovary (CHO) cells ~ (16). Although these mutants had defects in membrane permeability, later mutants isolated by the same group were shown to have altered colcemid binding affinity, and one had an alteration in c~-tubulin (14, 17). In our laboratory we have employed a similar approach, i.e., selecting cells resistant to microtubule-active drugs to obtain CHO mutants with distinct alterations in aand fl-tubulin (4, 6, 7, 9, 21, 22). Most microtubule inhibitors cause microtubules to break down, but one, taxol, stabilizes microtubules and promotes their assembly (23, 24). Among CHO cells resistant to taxol, a mutant was discovered that required the continuous presence of the drug for cell division and long term viability (3, 10). This mutant has no altered forms of c~or ~-tubulin detectable by two-dimensional gel analysis. The biochemical lesion responsible for the phenotype of this mutant is still unknown. We now report the isolation of many new taxol-requiring CHO cell lines, some of which carry altered forms of aand Abbreviations used in this paper. " CHO, Chinese hamster ovary; HAT, hypoxanthine-aminopterin-thymidine. © The Rockefeller U niversily Press, 0021-9525/86/04/1522/10 $1.00 The Journal of Cell Biology, Volume 102, April 1986 1522-1531 1522 on A ril 3, 2017 D ow nladed fom Published April 1, 1986