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During mitosis, the cell forms a spindle that equally segregates chromosomes into two daughter cells. Soon after nuclear envelope breakdown (NEB), kinetochores on chromosomes are captured by microtubules nucleated at the spindle pole. Some chromosomes immediately find themselves in the area between two spindle poles, yet approximately 7 out of 46 chromosomes, termed polar chromosomes, are positioned behind the pole at NEB in human cells. These polar chromosomes first approach the spindle pole with the help of kinetochore dynein and subsequently undergo CENP-E mediated sliding towards the equator [1, 2]. However, the mechanism of their passage across the polar region and its importance for faithful cell division remain unknown. Here we show that polar chromosomes are particularly prone to missegregation when the checkpoint is weakened. These chromosomes stall behind the pole and acquire speed similar to non- polar chromosomes once they reach the spindle body, indicating that the centrosome creates a physical barrier for chromosome movement in this area. To test this hypothesis, we removed one centrosome using centrinone, which indeed resolved the observed delay in the congression of polar chromosomes and suggested that they must have an additional and distinct mechanism of congression. Imaging of EB3, a microtubule plus end marker, revealed that astral microtubules attached to kinetochores of polar chromosomes pivot around the spindle pole and thereby transport polar chromosomes towards the spindle body. The angle that the kinetochores of these chromosomes form with the spindle axis changed during the period of rapid spindle elongation, indicating a role of centrosome separation in this process. By using different kinesin-5 (Eg5) inhibitors to perturb the sliding of antiparallel microtubules and consequently stop or reverse spindle elongation, we confirmed that pivoting occurs due to a hydrodynamic drag force created by centrosome movement. Stimulated emission depletion (STED) imaging of astral microtubules and Mad2, a marker for absent or immature end-on attachments, showed that during pivoting, both kinetochores of a polar chromosome typically attach to a single microtubule laterally, with more complex attachments observed less frequently. Altogether, we propose a model in which pivoting of microtubules around the spindle pole, driven by spindle elongation, promotes the movement of polar chromosomes towards the spindle body and consequently their proper congression to the spindle equator. [1] Barisic et al. Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces. Nat Cell Biol, 16(12): 1249-1256 (2014) [2] Kapoor et al. Chromosomes can congress to the metaphase plate before biorientation. Science, 311(5759): 388-391 (2006)

To ensure faithful mitosis, chromosomes located at different nuclear positions during nuclear envelope breakdown need to properly congress to the metaphase plate. The most unfavorably positioned chromosomes are polar chromosomes, which are located behind the spindle pole at nuclear envelope breakdown and make up approximately 7 out 46 chromosomes in human cells. These chromosomes first need to approach the spindle pole from the back and at a later point slide towards the metaphase plate in a motor- dependent manner. However, the mechanism of their passage across the polar region and its importance for faithful mitosis remain unknown. Here we show, by using live-cell confocal microscopy with high spatial and temporal resolution, that polar chromosomes are the last to reach the metaphase plate. They also stall behind the pole, indicating that the centrosome creates a physical barrier to their movement. To test this hypothesis, we removed one centrosome using centrinone, which indeed resolved the observed delay in the congression of polar chromosomes, suggesting the existence of an additional and distinct mechanism of congression in a polar region. By imaging the microtubule plus end marker EB3, we reveal that this mechanism is based on pivoting of astral microtubules with attached chromosomes around the spindle pole. The angle that the kinetochores of these chromosomes form with the spindle axis changed during the period of rapid spindle elongation, indicating a role of centrosome separation in this process. By using different kinesin-5 (Eg5) inhibitors to either stop or reverse spindle elongation, we confirmed that pivoting occurs due to a hydrodynamic drag force created by centrosome movement. Superresolution STED microscopy of astral microtubules and Mad2, a marker of unstable attachments, revealed that polar chromosomes are mostly laterally attached to one astral microtubule emanating from the pole behind which they are situated, with other more complex attachments observed less frequently. Finally, we show that polar chromosomes are prone to misalignment and cause aneuploidy in 62% of cases when the spindle checkpoint is weakened, highlighting their importance in faithful chromosome segregation. Altogether, we propose a model in which pivoting of microtubules around the spindle pole, driven by spindle elongation, promotes the movement of polar chromosomes towards the spindle body and consequently their proper congression to the spindle equator.

During mitosis, the cell forms a spindle that equally segregates chromosomes into two daughter cells. Soon after nuclear envelope breakdown, kinetochores on chromosomes are captured by microtubules nucleated at the spindle pole. Some chromosomes immediately find themselves in the area between two spindle poles, yet others positioned at the periphery first need to approach the spindle pole and subsequently travel to the equatorial plane. The question remains how these chromosomes located at the back of the spindle make their way across the centrosome and reach the spindle body, from where they can continue their congression towards the equator. By using confocal and STED live-cell microscopy of SPY- and SiR- tubulin stained RPE1 cells stably expressing CENP- A-GFP and Centrin1-GFP, we demonstrate that polar kinetochores, together with the microtubules they are attached to, pivot around the centrosome towards the spindle body. Pivoting was also evident for microtubules in RPE1 cells stably expressing EYFP-tubulin, where tubulin intensity of astral microtubules decreased over time, as bundles made their way towards the spindle center. The angle that the polar kinetochores form with the spindle axis coincided with rapid spindle elongation, indicating a mechanism in which spindle elongation creates a hydrodynamic drag force that brings kinetochores near the spindle body and enables efficient capture of these kinetochores by spindle body microtubules. This mechanism also ensured timely mitosis, as kinetochores that failed to pivot by the end of spindle elongation significantly delayed anaphase onset. Altogether, we propose a model in which pivoting of microtubules around the spindle pole, driven by spindle elongation, promotes the movement of peripheral chromosomes towards the spindle body and consequently their proper congression to the spindle equator.

During mitosis, the cell forms a spindle that equally segregates chromosomes into two daughter cells. Soon after nuclear envelope breakdown microtubules nucleated at the centrosomes of the spindle pole capture kinetochores, protein complexes on chromosomes. Some chromosomes immediately find themselves in the area between two spindle poles, yet others positioned at the periphery first need to approach the spindle pole in order to congress, or in other words, travel to the spindle equatorial plane. The question remains how these polar chromosomes located at the back of the spindle make their way across the spindle pole and reach the spindle body, from where they can continue their congression towards the equator. By using tubulin specific dyes in RPE1 cells stably expressing CENPA-GFP and Centrin1-GFP, markers for kinetochores and centrosomes, respectively, we demonstrate that polar chromosomes, together with the microtubules they are attached to, pivot around the centrosome towards the spindle body. The angle that the polar chromosomes form with the spindle axis changed faster during the period of rapid spindle elongation, indicating a mechanism in which spindle elongation creates a hydrodynamic drag force that brings kinetochores to the spindle body. This mechanism also ensured timely mitosis, as kinetochores that failed to pivot by the end of spindle elongation significantly delayed anaphase onset. Altogether, we propose a model in which pivoting of microtubules around the spindle pole, driven by spindle elongation, promotes the movement of peripheral chromosomes towards the spindle body and consequently their proper congression to the spindle equator.

During mitosis, the cell forms a spindle that equally segregates chromosomes into two daughter cells. Precise spatial organization of microtubule bundles and kinetochores is necessary for spindle function, yet it is unknown how unevenly distributed microtubules present during prometaphase organize into precise kinetochore and bridging fibers, important for positioning the kinetochores into the metaphase plate. By using live cell imaging of RPE1 cells stably expressing EYFP‐tubulin, we showed that in early prometaphase microtubules are organized in a few unevenly distributed and wide bundles. In contrast, late prometaphase and metaphase spindles are more ordered, consisting of numerous bundles that are similar in width and evenly spaced. Live cell imaging of HeLa cells stably expressing GFP‐tagged microtubule crosslinker PRC1 also revealed that in prometaphase, the spindle has a small number of unequally sized and spatially distributed PRC1‐ labeled overlap bundles, whose number increases either by forming de novo or exploiting the previously formed bundles. Depletion of PRC1 leads to different kinetochore movement during chromosome congression, which delays the formation of a stable metaphase plate. Thus, these results indicate a link between interpolar bundle formation and chromosome movements during congression in human cells.