Using biophysical analysis to study the regulation of the budding yeast kinetochore

Thesis (Ph.D.)--University of Washington, 2019Kinetochores are large macromolecular machines that serve to physically segregate the genomic material of a cell during mitosis. To achieve this, the kinetochore must make strong attachments to dynamic microtubules under force for long periods of time, but also be able to correct erroneous attachments. These complicated tasks require exquisite regulation within the kinetochore. The flexible outer kinetochore complex Ndc80 is essential to all of these functions. Previous studies have primarily focused on posttranslational modifications that affect regulation of the complex. I sought to determine how additional kinetochore components might regulate the microtubule binding capacity of the Saccharomyces cerevisiae Ndc80 complex, as well as if the complex’s intrinsic flexibility (conformational changes) affects its function. I and a previous member of the lab initially found that the conserved MIND complex, a central kinetochore component, and the Ndc80 complex are connected by an extensive network of contacts: an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. However, MIND binds Ndc80 complex far from the microtubule-binding domain. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a tightly bent conformation. From these results, I hypothesized a previously unidentified allosteric mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex: the binding of MIND opposes the tightly bent (auto-inhibited) form of the Ndc80 complex, stabilizing the complex in a more open conformation which results in better microtubule binding. To more directly test this hypothesis, I developed a single molecule FRET assay to examine conformational changes of the complex while simultaneously monitoring its microtubule-binding activity. I uncovered a novel mechanism of regulation of the Ndc80 complex that is intrinsic to its structure; tight bending of the complex inhibits its microtubule binding. I showed that the Ndc80 complex can fluctuate between straight and bent forms, and that binding of the complex to microtubules selects for straightened forms. In addition, the kinetochore complex MIND enhances microtubule binding by opposing the auto-inhibited conformation of Ndc80 complex. I suggest that prior to its assembly at the kinetochore, the Ndc80 complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 complex in a straightened form for higher affinity microtubule binding