Biophysical mechanisms of the kinetochore-microtubule interface and its regulation during mitosis

Thesis (Ph.D.)--University of Washington, 2014Chromosomes carry the genetic information that acts as a "blueprint" for every organism. When cells divide, replicated copies of the chromosomes are segregated into two new cells that are genetic copies of the original. This process is mediated by the mitotic spindle, a bipolar array of dynamic microtubules that connect to chromosomes and drive their segregation. Microtubules link to chromosomes via kinetochores, which assemble on the centromeres and present microtubule attachment sites. These kinetochore-microtubule linkages are tightly regulated to ensure accurate transmission of the genetic material during each division. In the budding yeast Saccharomyces cerevisiae, kinetochore-microtubule attachments are mediated by the Ndc80 and Dam1 complexes. Both are essential for viability, though their distinct contributions to kinetochore-microtubule coupling were previously unknown. We showed that these complexes interact directly to form robust linkages to dynamic microtubule ends. Furthermore, the interaction between these complexes can be disrupted by the mitotic regulatory kinase, Aurora B. During error correction, Aurora B detaches aberrant kinetochore-microtubule linkages, providing another chance to form correct attachments. We propose that Aurora B targets the interaction between the Ndc80 and Dam1 complexes during corrective detachment. In higher eukaryotes, error correction appears to depend additionally on modulating microtubule dynamics to promote microtubule disassembly. We showed that this effect is exerted through Aurora B regulation of the human Ndc80 complex. Kinetochore-microtubule linkages require the combined activity of many different kinetochore components. Moreover, these components are present in multiple copies, as ~20 Ndc80 complexes and ~30 Dam1 complexes act collectively at each kinetochore-microtubule interface. In vitro, Dam1 complexes associate together to form large oligomeric rings that encircle microtubules; how this oligomerization contributes to kinetochore function has remained unclear. We found that oligomerization of the Dam1 kinetochore complex is required for its ability to form microtubule attachments that are robust against tension in vitro and in vivo. In higher eukaryotes, the Ndc80 and Ska complexes are both reported to oligomerize on microtubules. Therefore, we propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis