Accurate control of microtubule length is fundamental to all cellular microtubule -based processes including mitosis, spindle orientation, axon guidance, and vesicular transport 3-6. The goal of this proposal is to define the mechanisms underlying microtubule length control and to characterize how these mechanisms contribute to the fidelity of mitosis. Building on work in prior funding periods, we will approach this problem a two levels. First, we will define the molecular mechanism of a major regulator of microtubule length, spindle size, and spindle geometry in eukaryotes, the kinesin 8 motor (Kip3 in budding yeast 5,7-9). The yeast kinesin 8, Kip3 walks processively to the microtubule plus end where it binds with high affinity and promotes microtubule disassembly 8-10. Because most microtubule dynamics regulation and length control occurs at the microtubule plus end, how microtubule-associated proteins (MAPs) and motors interact with the plus end is a central question in the field. The mechanism by which Kip3 binds the MT plus end and regulates MT dynamics will be determined using a combination of bulk biochemistry, structural studies, single molecule imaging, in vivo imaging and genetic analysis. Building on our recent finding that Kip3 has the ability to slide antiparallel microtubules apart, we will define how Kip3 contributes to spindle assembly and anaphase spindle elongation. These experiments will build on novel in vitro systems, including the ability to reconstitute a spindle elongation-like reaction on micropatterned glass substrates. The second line of experiments will employ in vitro microbial evolution as an unbiased approach to define how optimal MT length control and spindle function can emerge from the destabilizing effects of a genome doubling 2,11. We previously showed that tetraploidy in budding yeast causes a profound distortion in spindle geometry, which in turn results in a >200-fold increase in chromosome missegregation11. Genome doubling or tetraploidy is common during organismal evolution and during tumorigenesis 12-15.
The goal of this project is to define the mechanism of microtubule and spindle length control, a fundamental aspect of the maintenance of genome stability. Budding yeast will be used as a model system. The approaches include yeast molecular genetics, bulk biochemistry, single molecule imaging, electron microscopy, laboratory evolution of yeast, and genomics.