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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)

Background: During prometaphase all chromosomes must congress to the metaphase plate in order to satisfy the spindle assembly checkpoint (SAC) and initiate correct chromosome segregation. Contrary to healthy cells, human cancer cells often start mitosis via prometaphase pathway - without fully separating their centrosomes. This initial centrosome distance is believed to be an important determinant of chromosome segregation fidelity, as this pathway likely promotes the generation of syntelic attachments. However, the importance of chromosome position relative to centrosomes remains unknown. Aims: We set out to elucidate how chromosome positions relative to the position of centrosomes predict their speed of alignment and likelihood of mis-segregation. Methods: Using live-cell imaging and kinetochore tracking in three cellular systems with distinct fractions of peripherally positioned chromosomes at the onset of mitosis, we explore the paths of chromosome congression and the outcomes of their segregation. Results: We show that non-transformed RPE1 cells start mitosis with only a small fraction of polar chromosomes that are more prone to mis-segregate than non-polar chromosomes when SAC is weakened. Meanwhile, in the U2OS cancer cell line the fraction of polar chromosomes is higher, and they are prone to late alignment that predisposes them to mis-segregation. As the third system we use monastrol washout in RPE1 cells to increase the fraction of polar chromosomes before the spindle elongation, and we demonstrate that this leads to an increase in the proportion of late-aligning chromosomes after the washout, mimicking U2OS cells. By tracking congression of kinetochores during bipolarization, we notice that polar chromosomes take substantially longer to align to the metaphase plate, inducing long mitotic delays when compared to non-treated RPE1 cells. Interestingly, the majority of non-polar chromosomes reach the metaphase plate before spindle elongation is completed, contrary to polar chromosomes, which are often detained in the region between the pole and the emerging metaphase plate. The increase in the number of segregation errors following monastrol washout could not be rescued by depolymerizing all microtubules with additional nocodazole treatment to remove preformed syntelic attachments before the final washout and bipolarization. Moreover, in monopole, the angles of kinetochore pairs toward their respective poles that suggest syntelic orientation, cannot predict their future late alignment. These two results imply that formation of syntelic attachments that takes place in the monopole is not the primary cause of late alignment and segregation errors. Conclusions: We propose that unfavorable position of chromosomes behind the spindle pole at the start of spindle elongation increases the likelihood of their mis-segregation and that syntelic monooriented intermediates form during their delayed congression.

The human mitotic spindle is made of microtubules nucleated at centrosomes, at kinetochores, and from pre-existing microtubules by the augmin complex. However, it is unknown how the augmin- mediated nucleation affects distinct microtubule classes and thereby mitotic fidelity. Here we use superresolution microscopy to analyze the previously indistinguishable microtubule arrangements within the crowded metaphase plate area and demonstrate that augmin is vital for the formation of uniformly arranged parallel units consisting of sister kinetochore fibers connected by a bridging fiber. This ordered geometry helps both prevent and resolve merotelic attachments. Whereas augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled microtubule arrangement unfavorable for creating additional attachments, augmin-nucleated k-fibers produce robust force required to resolve errors during anaphase. STED microscopy revealed that bridging fibers were impaired twice as much as k-fibers following augmin depletion. The complete absence of bridging fibers from a significant portion of kinetochore pairs, especially in the inner part of the spindle, resulted in the specific reduction of the interkinetochore distance. Taken together, we propose a model where augmin promotes mitotic fidelity by generating assemblies consisting of bridging and kinetochore fibers that align sister kinetochores to face opposite poles, thereby preventing erroneous attachments.

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.

The mitotic spindle functions as a molecular micromachine that evenly distributes chromosomes into two daughter cells during cell division. A major mechanical element of the spindle are kinetochore fibers attached to sister kinetochores on each chromosome and laterally linked by a bundle of antiparallel microtubules called the bridging fiber. Spindle microtubules are mainly nucleated at the centrosome and on the lateral surface of existing microtubules by the augmin complex. However, it is unknown how the augmin‐ mediated nucleation affects functionally distinct microtubule bundles and thus the architecture and forces within the spindle. Here we show, by using siRNA depletion and CRISPR knock‐out of the augmin complex subunits HAUS6 and HAUS8 in human cells, that augmin is a major contributor to the nucleation of bridging microtubules. Augmin depletion resulted in a ∼70% reduction of the microtubule number in bridging fibers and ∼40% in kinetochore fibers, suggesting that the bridging microtubules are largely nucleated at the surface of present microtubules. In augmin‐depleted cells, the interkinetochore distance decreases preferentially for kinetochores that lack a bridging fiber, independently of the thickness of their k‐fibers, indicating that augmin affects forces on kinetochores largely via bridging fibers. Without augmin the number of bridging fibers decreases, with the remaining ones mostly confined to the spindle periphery with an increased overlap length. The reduced number of microtubules also results in a slower poleward flux. Our results demonstrate a critical role of augmin in the formation of bridging microtubules and proper architecture of the metaphase spindle, suggesting a model where sliding of augmin‐ nucleated bridging microtubules promotes poleward flux of k‐fibers and thus tension on kinetochores.

The mitotic spindle in human cells consists of microtubules nucleated at the centrosome, at kinetochores and chromosomes, and on the lateral surface of existing microtubules by the augmin complex. However, it is unknown how the augmin- mediated nucleation affects functionally distinct microtubule bundles, the interkinetochore tension and the mitotic fidelity. Here we show, by using stimulated emission depletion (STED) microscopy and siRNA depletion of HAUS6 or HAUS8 subunits of the augmin complex, that augmin is crucial for the formation of bridging fibers, which laterally link sister kinetochore fibers. Without augmin, the structure of bridging fibers was severely impaired, with 40% of bridging fibers being undetectable in contrast to only 4% in control cells. The number of microtubules within bridging fibers was reduced by 65% and in kinetochore fibers by 30%, suggesting that the bridging fibers, but not kinetochore fibers, are primarily nucleated on the surface of present microtubules. Moreover, bridging fibers had a kinetochore fiber- independent role in the regulation of interkinetochore tension, given that the interkinetochore distance of kinetochore pairs that lack a bridging fiber decreased independently of the thickness of their kinetochore fibers. In HAUS6-depleted cells, the number of lagging kinetochores increased four-fold compared to control cells following Mad2 codepletion. As the lagging kinetochores were typically stretched, which is a signature of merotelic attachment, we propose a role of augmin in the correction of erroneous kinetochore attachments. We also observed misaligned kinetochore pairs that jointly segregated into the same cell and led to aneuploidy, suggesting a role of augmin in chromosome congression. Altogether, we propose a model in which augmin promotes the tension on kinetochores and the mitotic fidelity largely by generating bridging microtubules from the lateral sides of kinetochore microtubules.

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.

N-glikom predstavlja jedinstvenu poveznicu između genskih predispozicija za razvoj pojedinih bolesti i okolišnih faktora kojima je organizam izložen te je stoga moguća njegova uporaba kao biomarkera u preventivnoj i personaliziranoj medicini. Jedan je od glavnih ciljeva moderne glikobiologije razviti brze i osjetljive metode za analizu N-glikana u tragovima. Razvijenom metodom za ukoncentriravanje N-glikana PGC ekstrakcijom na čvrstoj fazi moguće je pomoću HILIC-UPLCFLR kvantitativno odrediti 5x manje koncentracije N-glikana u odnosu na prijašnju metodu. Kako bi kromatografski profil bio prihvatljiv, tijekom analize potrebno je pripaziti na kompatibilnost reagensa s mobilnom fazom za kromatografsku analizu, ionsku jakost prilikom eluacije glikana s PGC-a i udio vode u eluatu glikana. Razvijena metoda ima potencijal korištenja u istraživanjima različitih stanja i bolesti kod kojih dolazi do promjene glikozilacijskog profila, a u kojima je potrebna veća osjetljivost zbog male količine glikana u uzorku. N-glycome represents a unique connection between the genetic predisposition for the development of certain diseases and the environmental exposure of the organism, making it a potential biomarker in preventive and personalised medicine. Consequently, the development of fast and sensitive methods for the analysis of trace N-glycans is one of the main goals in the field. By using the newly developed PGC SPE method for the enrichment of N-glycans, followed by HILICUPLC-FLR, it is possible to quantitatively measure 5x lower concentrations of N-glycans than with previous methods. During the analysis, it is important to adjust the compatibility of the reagents with the mobile phase used in chromatography, ionic strength for eluation of the N-glycans from PGC and the amount of water in the N-glycan eluate. The method can be used in research of different conditions and diseases which are known to have an altered glycosylation profile, as well as in molecular diagnostics.