Asymmetric Chromosome Oscillation during Mitosis and Protein Architecture of the Human Kinetochore Measured by K-SHREC (Kinetochore-Speckle High Resolution Co-Localization) Public Deposited

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  • March 19, 2019
  • Wan, Xiaohu
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
  • Mitotic chromosomes are known to oscillate during prometaphase and metaphase. This study demonstrated that kinetochores move faster in poleward (P) motion than in away-from-the-pole (AP) motion. P and AP motions also showed different position versus time curves, suggesting distinct mechanisms behind the phenomenon. Sister kinetochores oscillate with different phases relative to each other. The leading kinetochore usually switches first, from P to AP motion, followed by the trailing one switching from AP to P motion. Such asymmetry and phase lag produces oscillation in centromere stretch at twice the frequency of individual kinetochores. The leading kinetochore switches after sister chromosomes reach maximum centromere stretch, suggesting tension may trigger the kinetochore switching. To further investigate kinetochore dynamics, K-SHREC (Kinetochore-Speckle High Resolution Co-Localization) was developed to map the relative protein positions within kinetochores using two color fluorescent speckle microscopy, where centroids, orientations and geometries of fluorescent proteins were identified by asymmetric 3D Gaussian fitting in 3D image stacks. The accuracy of this method can reach +/-5nm. The relative positions of kinetochore proteins such as CenpA, Spc24, Spc25, Bub1, DC31, KNL1 and KNL3 to another kinetochore protein Hec1 were assessed in fixed Hela cells at metaphase. When centromeric tension is lost by taxol treatment, Ndc80 complex remains the same orientation and fully extended with 45nm-separation between the N-termini of Hec1 and Spc24. Most proteins moved about 30 nm closer to CENP-A, except Bub1. This result suggests that there is a tension-sensitive linkage between the KNL-1/Mis12 complex/Ndc80 complex (KMN) network of proteins in the core microtubule attachment site and the location of the majority of CENP-A within the peripheral centromere. The Ndc80 complex behaves like a stiff object, perhaps a thin rod. The relative positions between the end of kinetochore microtubule and the centroid of the fluorescent speckle of Hec1 were also measured by imaging GFP-tubulin. A 3D line scan method and an error function fitting algorithm were developed to identify the microtubule end position. Microtubule end stays closer to centromere with about 63nm distance from Hec1.
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  • Salmon, Edward D.
  • Open access

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