California Sea Grant College Program

The effects of structural changes in yeast b-tubulin on the dynamic properties of microtubules (MTs) were measured in Saccharomyces cerevisiae using real time digital microscopy. A galactose inducible GFP-TUB3 plasmid construct was transformed into yeast strains that were mutated at the putative colchicine binding site in the sole b-tubulin gene (TUB2). The dynamic properties of cytoplasmic MTs (cMTs) of two mutated strains and their respective wild-type strains were examined in vivo. CLC9 is a haploid strain in which TUB2 was mutated in a conserved residue (R318W), and is highly resistant to benomyl. In CLC8, a diploid benomyl sensitive strain, TUB2 is mutated in the sequence between amino acids 310-324 so that this region is identical to the porcine tubulin sequence. Several differences in specific dynamic parameters have been uncovered. For CLC9, all parameters were similar to wild-type except that the catastrophe frequency was higher and the mean attenuation duration lower. For CLC8, the shortening rate, catastrophe frequency, and the percent unrescued catastrophes, were higher than wild-type, while the mean attenuation duration was lower. Together, the cMTs in CLC8 are less stable than those in wild-type cells, which is reflected by the strain's longer doubling time. In the presence of increasing concentrations of benomyl, the three yeast strains sensitive to the drug exhibit greatly reduced MT dynamics while the super resistant strain showed little change. From 2 µg/ml to 3 µg/ml benomyl, MTs in the CLC8 strain transitioned from a highly dynamic state, to a completely attenuated state, whereas at 5 µg/mll MTs completly depolymerized. In contrast, MTs in the CLC9 strain at 10 µg/ml benomyl were as dynamic as MTs in untreated cells. These data support in vitro data demonstrating the stabilization of MT dynamics by low concentrations of antimitotic drugs but expand this fundamental property to a system in which the relationship between tubulin structure and dynamic function can be efficiently analyzed in vivo. This system will also be useful to elucidate how cellular factors modulate in vivo dynamics directly, and for identification of regions in the tubulin protein critical for modulating MT dynamics.