Molecular Architecture and Regulation of the Budding Yeast Kinetochore-Microtubule Interface

Thesis (Ph.D.)--University of Washington, 2018Equal partitioning of replicated chromosomes during mitosis is vital. The genetic information stored in chromosomes provide the template for cells to produce RNA and protein molecules, which carry out cellular functions. Failure to equally divide chromosomes between two daughter cells means a disturbance in this central dogma of biology. Chromosome division is mediated by a bipolar structure specific to mitosis, called the spindle. Cytoskeletal microtubules anchored at each pole grow towards the other pole, resulting in the separation of two poles. During this process, the sister chromatids (replicated pair of chromosomes connected to each other) bind to spindle microtubules. This is made possible by the kinetochore, which assembles on the centromeric DNA region of each sister chromatid. During metaphase, sister kinetochores establish bioriented attachments on dynamic spindle microtubules. In anaphase, microtubule depolymerization is coupled with the segregation of sister chromatids towards their respective poles. Thus, the main function of the kinetochore is to make strong and processive attachments to microtubules. In the budding yeast S. cerevisiae outer kinetochore, the essential Ndc80 and Dam1 complexes are the main microtubule binding components. I show that these two protein complexes have a tripartite interaction and disrupting any one interaction site results in chromosome segregation error. Through these three interactions, the elongated Ndc80 complex bridges two Dam1 complex rings. In addition, I detail the mechanisms by which the Aurora B kinase and Cyclin dependent kinase 1 (Cdk1) regulate the kinetochore-microtubule interface. The Aurora B kinase phosphorylates the Dam1 complex to disrupt each interaction site between the Dam1 and Ndc80 complexes. Further phosphorylation of the Dam1 complex results in the inhibition of both cooperative and single molecule microtubule-binding ability. In contrast, the Cdk1 phosphorylates the Dam1 complex to enhance the cooperative binding onto microtubules. Together, I describe how the Ndc80 and Dam1 complexes are organized at the kinetochore-microtubule interface and the intricate mechanisms regulating these interactions. The Spc105 and MIND complexes are also essential and conserved components of the outer kinetochore. I show for the first time a recombinant expression and purification method for the Spc105 complex. I detected coiled-coil interactions between the two proteins that compose the Spc105 complex, Spc105p and Kre28p. Purification of the Spc105 complex from budding yeast co-purifies both MIND and Ndc80 complexes. These three protein complexes represent the highly conserved KMN network. Future studies will further utilize these tools for investigating how the Spc105 complex is organized with other outer kinetochore components