Evolution of resistance to induced microtubule depolymerization in S.Cerevisiae

Microtubules, polymers of alpha- and beta-tubulin, are essential cellular components. When microtubule polymerization is hindered, cells are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we here asked how cells cope on the long term with microtubule-impairing conditions. Taking advantage of mutations in beta-tubulin, we allowed 24 clonal populations of yeast cells unable to properly polymerize microtubules to evolve in the lab for ~150 generations. At the end of the evolution experiment, cells had re-gained the ability to form microtubules, and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing allowed us to identify recurrently-mutated genes, in particular for tubulins and kinesins, as well as the pervasive duplication of chromosome VIII. Recapitulating these mutations and disomy of chromosome VIII prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not lead to loss of function, but rather restored microtubule functionality. Analysis of the temporal order of mutations leading to resistance in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by one, and only one, additional adaptive mutation in either tubulins or kinesins. Given that tubulins are highly conserved among eukaryotes, our results are potentially relevant for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.