The coIR4 and colR15 B-Tubulin Mutations in Chlamydomonas reinhardtii Confer Altered Sensitivities to Microtubule Inhibitors and Herbicides by Enhancing Microtubule Stability

The colR4 and co1R15 B2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The coIR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the B-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wildtype like in their growth characteristics, the coIR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated ot-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated ct-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant B-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability. I N both animal and plant cells microtubules comprise a major component of the cytoskeleton and are involved in several vital cellular functions including nuclear division, several forms of cell motility, transport of organelles, cell shape determination, and cytoplasmic rearrangement during the cell cycle (Snyder and Mclntosh, 1975; Dustin, 1984; Schliwa, 1984). Studies on microtubule function and assembly have been facilitated by the use of a wide variety of inhibitor drugs which block microtubule-dependent processes in cells by binding to the tubulin dimers or polymerized microtubules. The in vivo mechanism of action of most of the microtubule inhibitors identified to date is to depolymerize microtubules and to inhibit their assembly. Microtubule-depolymerizing drugs include the plant alkaloids such as colchicine, vinblastine, podophyUotoxin, and maytansine, the antifungal and antihelminthic benzimidazole derivatives, and several plant-specific antimitotic herbicides including the dinitroanilines trifluralin and oryzalin and the phosDr. Sehibler's present address is La Jolla Cancer Research Foundation, La Jolla, CA 92037. phoric amide herbicide amiprophos-methyl (for review see Morejohn and Fosket, 1986). Unique among the microtubule inhibitors is the taxane alkaloid, taxol, which inhibits microtubule-dependent processes by stabilizing microtubules and promoting microtubule polymerization (Schiff et al., 1979, Schiff and Horwitz, 1980, Parness and Horwitz, 1981). One approach to the study of microtubule function in which microtubule inhibitor drugs has been particularly useful has been in the isolation of mutations in the structural genes for aand/3-tubulin. Over the past 10 yr tubutin mutants have been isolated on the basis of altered sensitivity to the growth-inhibitory effects of microtubule inhibitors in a number of different organisms including mammalian ceils in culture and various fungi such as Aspergillus, Neurospora, Physarum, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (for a review see Cabral, 1989). In previous studies from this laboratory, two /3-tubulin mutants in the unicellular green alga Chlamydomonas reinhardtii were isolated on the basis of resistance to the growth © The Rockefeller University Press, 0021-9525/91/05/605/10 $2.00 The Journal of Cell Biology, Volume 113, Number 3, May 1991 605-614 605 on F ebuary 0, 2013 jcb.rress.org D ow nladed fom Published May 1, 1991