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

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

Aurora Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors

Merotelic kinetochore orientation is a misattachment in which a single kinetochore binds microtubules from both spindle poles rather than just one and can produce anaphase lagging chromosomes, a major source of aneuploidy [1]. Merotelic kinetochore orientation occurs frequently in early mitosis, does not block chromosome alignment at the metaphase plate, and is not detected by the spindle checkpoint 2, 3, 4 and 5. However, microtubules to the incorrect pole are usually significantly reduced or eliminated before anaphase 3 and 6. We discovered that the frequency of lagging chromosomes in anaphase is very sensitive to partial inhibition of Aurora kinase activity by ZM447439 at a dose, 3 μM, that has little effect on histone phosphorylation, metaphase chromosome alignment, and cytokinesis in PtK1 cells. Partial Aurora kinase inhibition increased the frequency of merotelic kinetochores in late metaphase, and the fraction of microtubules to the incorrect pole. Measurements of fluorescence dissipation after photoactivation showed that kinetochore-microtubule turnover in prometaphase is substantially suppressed by partial Aurora kinase inhibition. Our results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotelic attachments are destabilized by being pulled toward the inner centromere

The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Constitutive Centromere Associated Network (CCAN) proteins, particularly CENP-C, CENP-T and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, like the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ~16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20-60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex

Dynamic bonds and polar ejection force distribution explain kinetochore oscillations in PtK1 cells

A computational model of kinetochore dynamics suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed position-dependence of metaphase chromosome behavior.Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments to spindle microtubules via their kinetochores, and multiple motor and nonmotor proteins cooperate to regulate their behavior. Depending on the system, sister chromatids may display either of two distinct behaviors, namely (1) the presence or (2) the absence of oscillations about the metaphase plate. Significantly, in PtK1 cells, in which chromosome behavior appears to be dependent on the position along the metaphase plate, both types of behavior are observed within the same spindle, but how and why these distinct behaviors are manifested is unclear. Here, we developed a new quantitative model to describe metaphase chromosome dynamics via kinetochore–microtubule interactions mediated by nonmotor viscoelastic linkages. Our model reproduces all the key features of metaphase sister kinetochore dynamics in PtK1 cells and suggests that differences in the distribution of polar ejection forces at the periphery and in the middle of PtK1 cell spindles underlie the observed dichotomy of chromosome behavior

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment