Davis, Trisha NHamilton, Grace Elizabeth2020-04-302020-04-302020Hamilton_washington_0250E_21196.pdfhttp://hdl.handle.net/1773/45444Thesis (Ph.D.)--University of Washington, 2020Equal partitioning of duplicated chromosomes between daughter cells is a microtubule- mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, tethers chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and initiates error correction mechanisms. But though the field has arrived at a “parts list” of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. I reconstituted functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self- assembling in vitro, tethering centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal that two inner kinetochore protein subcomplexes, Mif2 and OA, are independently capable of transmitting force from MIND to the centromeric nucleosome and suggest that these two pathways of outer kinetochore recruitment may be differentially regulated.application/pdfen-USCC BY-NCCentromereChromosome segregationKinetochoreMitosisOptical trapOptical tweezersBiochemistryBiological chemistryThesis