| During mitosis, kinetochores link centromeric DNA to spindle microtubules to facilitate accurate segregation of the replicated genome. Spindle poles also must form stable microtubule attachments to generate the opposing forces required for the depolymerization-coupled movement of chromosomes during anaphase. Kinetochores contain between 80-100 proteins, but the mechanism by which this macromolecular structure couples to microtubules remains unknown. Models invoking a kinetochore sleeve, a sliding ring and a fibrillar coupler have been proposed to explain how the kinetochore harnesses the energy released by a depolymerizing microtubule to power chromosome movements. In this dissertation we test two of these models through extensive study of two proteins thought to function as key kinetochore couplers.;Chapter 1 provides a general introduction to mitosis, kinetochores and microtubules. Chapter 2 tests whether xCep57R is a functional vertebrate homolog of Dam1, a budding yeast protein that forms a sliding ring on microtubules. Chapter 3 provides an extensive review of Ndc80 complex function, while Chapter 4 identifies a tripartite attachment point in the Hec1/Ndc80 subunit that allows the Ndc80 complex to couple to microtubules. Chapter 5 concludes the dissertation by providing data suggesting that the unstructured tail of Hec1/Ndc80 facilitates chromosome congression by packing Ndc80 complexes close together and enhancing kinetochore processivity on a depolymerizing microtubule. Chapter 5 finishes with a section detailing future directions for more precisely determining the coupling mechanism of the vertebrate Ndc80 complex. |
