Book Description

Microtubule dynamics are regulated by a variety of proteins that bind to microtubule ends, and influence their polymerization properties. A number of these plus-end binding proteins have been identified and the interactions between these proteins are important for regulating microtubule dynamics. Stu2, Bik1, and Bim1 are three microtubule plus-end tracking proteins in Saccharomyces cerevisiae and each has been shown to influence microtubule assembly in vivo. I have found that Stu2, Bik1, and Bim1 interact both with themselves and each other in all pairwise combinations. Mapping of protein-protein interaction domains, competitive interaction assays, and physical characterization of protein complexes indicate that these proteins do not associate in a single complex but rather compete for binding to each other. Overall, these results suggest that the interactions among these proteins are dynamic and that different complexes of these proteins may perform distinct roles in the cell. In order to further characterize these proteins, I purified Bim1 and Bik1. I show that in vitro Bim1 and Bik1 form homodimers that can interact with each other to form a tetramer, indicating the Bim1-Bik1 interaction is direct. Using purified GFP tagged versions of these proteins, I observed Bim1 to localize directly to the microtubule plus-end, however, Bik1 required Bim1 for efficient plus-end localization. I also examined the effects of Bim1 and Bik1 on MT assembly and dynamics in vitro. I found that Bim1 has a stabilizing effect on microtubules resulting in an increase of polymer formation. This effect is primarily due to the suppression of microtubule catastrophes by Bim1, although Bim1 also increases microtubule growth rates and rescue frequencies. The ability of Bim1 to promote MT polymerization is likely due to a direct effect of Bim1 on MT plus-end structure as I found that Bim1 does not bind tubulin subunits. In contrast, Bik1 had a destabilizing effect on microtubules, increasing the frequency of catastrophes. This may be due to the formation of polymerization-incompetent tubulin oligomers as I observed Bik1 to promote oligomerization of tubulin subunits. Combining equal-molar amounts of Bim1 and Bik1 resulted in an effect on microtubule dynamics similar to Bim1 alone. These results indicate that Bim1 is able to suppress the catastrophe promoting activities of Bik1 and could be due to formation of a Bim1-Bik1 complex that disrupts Bik1-tubulin interactions. In agreement with this theory, Bik1 is known to bind both Bim1 and tubulin through the same domain. Overall, these results add to our knowledge of plus-end tracking proteins and the individual method each protein uses to influence MT dynamics.