Chromosome segregation must occur with fidelity to maintain genome stability and prevent aneuploid disorders. During segregation, the kinetochore is assembled onto centromeric DNA and achieves the attachment of microtubule plus ends, which provide the force to physically segregate chromosomes to opposite poles of the cell. The kinetochore is a macromolecular protein/DNA machine composed of 8-9 biochemical complexes, the DNA-binding components of which define the inner kinetochore, and the microtubule-binding components of which define the outer kinetochore. The 3D protein architecture of the inner kinetochore in living cells remains poorly understood due to the resolution limits of live-cell imaging techniques. Here, we use pairwise, in vivo fluorescence microscopy to determine the position of kinetochore components relative to the centromere specific histone H3 variant, Cse4, with nanometer resolution. In addition, we complement kinetochore focus analysis with DNA dynamics simulations to propose a connection between chromatin dynamics and the observed anisotropy of the inner kinetochore proteins, Ame1 and Cse4, relative to the outer kinetochore proteins, Ndc80 and Nuf2.Bachelor of Scienc
