High-resolution direct stochastic optical reconstruction microscopy of the human kinetochore chromatin

The kinetochore is a chromatin-protein complex on the chromosome centromere. Kinetochore attache chromosomes to the microtubules of the spindle apparatus, recognize attachment to microtubules, and regulate the progress of mitosis. The kinetochore protein complex is built on the periphery centromere chromatin. It seems that kinetochores react to forces of the spindle apparatus microtubules in their interior. Due to the limited resolving power of the optical microscope and limitations of electron microscopy, the spatial arrangement of kinetochore protein and chromatin complex is unresolved. In this thesis, the human periphery centromere chromatin assembled with CENP-A (kinetochore chromatin in further text), on which kinetochore complex is built, was imaged by high-resolution optical microscopy called direct Stochastic Optical Reconstruction Microscopy (dSTORM). Resolving power of less than 30 nm was achieved, and the architecture of more than 900 kinetochores in different sub-stages of interphase and mitosis was resolved. Kinetochore chromatin was formed in a rectangle between 250 and 400 nm long and between 150 and 270 nm wide. It was composed of parallel and orthogonal lines between 12 and 75 nm wide. The arrangement of kinetochore chromatin in mitosis was narrower and longer than in interphase. In interphase, subtle changes in the dimensions of kinetochore lines were measured. The mitotic toxin nocodazole disturbed kinetochore chromatin organization. The discovered change of arrangement of the kinetochore chromatin assembled with CENP-A during the cell cycle could be the physical mechanism of recognition of proper attachment and positioning of the chromosomes at the equatorial plate. Based on the discovered kinetochore structure and behavior in the cell cycle, a new shoo-lace model of assembly and function of kinetochore chromatin is suggested

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

During metaphase, forces on kinetochores are exerted by k fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser- cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non- kinetochore microtubules, which we term ‘bridging fibre’, bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape