Biggins, SusanBarrero, Daniel2024-09-092024-09-092024-09-092024Barrero_washington_0250E_27039.pdfhttps://hdl.handle.net/1773/52124Thesis (Ph.D.)--University of Washington, 2024In order to propagate, cells must be able to duplicate and faithfully segregate their genetic information. Eukaryotic chromosome segregation requires kinetochores, multimegadaltonprotein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from many complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple essential functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this we turned to a thermotolerant yeast, Kluyveromyces marxianus, in the hopes we would be able to purify kinetochores stable enough for structural studies. We were able to purify kinetochores from K. marxianus and study them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. We were also surprised to find two distinct classes of kinetochores, doublets and singlets. Suspecting that the doublets might indicate a regional centromere, we further interrogated their function and origin. We found that while the doublets could account for the high strength of its’ kinetochores, K. marxianus in fact utilized a point centromere much like Saccharomyces cerevisiae. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.application/pdfen-USCC BYelectron microscopyKinetochoremitosistomographyMolecular biologyBiophysicsMolecular and cellular biologyThesis