It remains unclear when sufficient antiplatelet effect is achieved after administration of a loading dose of clopidogrel in patients with acute ischemic stroke (AIS). This study aimed to evaluate the clopidogrel response in patients with AIS identified by the platelet function test (PFT).12 reaction unit (PRU) values measured using VerifyNow at baseline and at 6, 24, and 72 h after administration of a loading dose (300 mg) of clopidogrel were compared between patients with AIS and those of other cerebrovascular diseases (CVD). The prevalence of clopidogrel abnormal response and its associated factors were examined.P2Yp < 0.0001), and also in the AIS group (p = 0.0073). The PRU values were significantly higher in the AIS group than in the other CVD group . The prevalence of clopidogrel hypo-responder (PRU > 240 at 24 h after administration) was higher in the AIS group  and associated with the baseline PRU value but not with the cytochrome P450 2C19 genotype or clinical ischemic events.The PRU value was significantly reduced with time in the other CVD group (Residual platelet reactivity at 24 h after clopidogrel loading was substantially higher in patients with AIS than in patients with other CVD. In addition, most patients with AIS were judged to be hypo-responders on PFT. This should be carefully interpreted in patients with AIS because of poor specificity for predicting clinical ischemic events. Antiplatelet treatment with clopidogrel in combination with other antiplatelet agents such as aspirin is widely used for preventing recurrent ischemic stroke. Two major randomized control trials to evaluate an early initiation of combination antiplatelet therapy with clopidogrel and aspirin, as compared with aspirin alone, for patients with minor ischemic stroke (NIHSS score of ≤ 3) or transient ischemic attack have shown a reduction in the risk of recurrent stroke , 2. Patipost-hoc analysis for platelet reactivity assessed at only one point (12–24 h after clopidogrel administration), by which the intervention had been already finished.Individual responses to clopidogrel vary by CYP2C19 polymorphism , 7 or otThe optimum timing of the endovascular intervention requiring stenting for patients with AIS remains unclear because we do not know the duration for clopidogrel to sufficiently inhibit platelet reactivity.This study aimed to evaluate changes in platelet reactivity after clopidogrel loading to AIS patients and compare it with those in patients with other cerebrovascular diseases (CVD). The secondary aim of this study was to examine factors associated with PFT-identified abnormal clopidogrel reactivity.This study was approved by the relevant Ethics Committee and Institutional Review Board . The institutional review boards approved the exemption in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan. Written informed consent was obtained from all patients.Our protocol of early antithrombotic management for patients with AIS to prevent further progression or recurrence of ischemic stroke depends on the initial diagnosis of stroke etiology. For patients with cardioembolic or embolic stroke from undetermined sources (ESUS), anticoagulant therapy with continuous infusion of unfractionated heparin was the first-line treatment and then switched to oral anticoagulants the next day or after. If close examination later revealed the atherosclerotic cause in those patients, it was switched to antiplatelet therapy. For patients with atherosclerosis etiology defined as large-artery atherosclerosis or small-vessel occlusion (lacuna stroke) on TOAST criteria  or patien = 1) had thrombocytopenia (n = 1) and those who refused to participate in this study (n = 1) were excluded from this study; therefore, a total of 29 patients with AIS were included in the analysis  of clopidogrel on the day of onset. Patients who received oral anticoagulants  due to the patient's request, clopidogrel is started with a loading dose of 300 mg. In this study period, a total of 325 patients underwent elective endovascular treatment in our hospital. Among them, 27 patients received a loading dose of clopidogrel as periprocedural antiplatelet therapy for elective endovascular treatment and were included in the analysis .via a nasogastric tube for patients with consciousness disturbance or dysphagia.Written informed consent was obtained from all patients or their families. To confirm the adherence, a nurse checked oral administration or clopidogrel was administered by powder Clopidogrel reactivity was evaluated by the VerifyNow system , a point-of-care analysis of PFT, which is strongly associated with light transmittance aggregometry . Blood s12 reaction Unit (PRU), which represents platelets reactivity to clopidogrel, and the Aspirin reaction Unit (ARU), which represents platelets reactivity to aspirin, was measured at 4 points; baseline (before the administration of a loading dose of clopidogrel), and at 6, 24, and 72 h after the administration. The PFT-identified clopidogrel hypo-responder (CHypoR) was defined by a PRU > 240, and hyper-responder (CHyperR) was defined by a PRU <60 at 24 h after clopidogrel administration  variant , intermediate metabolizer , poor metabolizer  . The CYP2C19 gene has several polymorphysims , and *2  variant . The CYPalleles) .Data of variables included patient demographic and clinical characteristics such as age, sex, comorbidities, body mass index, concomitant medication use, and the CYP2C19 genotype were collected from hospital charts.PRU values at four points and the prevalence of CHypoR and CHyperR were compared between the groups. The associated factors for CHypoR and CHyperR were examined.Clinical outcomes were measured by clinical and radiological assessments during the patients' hospitalization (12–91 days). Radiological assessment by diffusion-weighted image (DWI) on magnetic resonance images (MRI) was performed 1–7 (median 2) days after administration of clopidogrel for the AIS group or after elective EVT for the other CVD group and when new neurological symptoms developed during hospitalization.Ischemic event was defined as a development of new ischemic lesions detected by DWI accompanied by any neurological symptom or enlarged primary ischemic area in the AIS group accompanied by an NIHSS score deterioration of ≥4 points. Bleeding event was defined based on the International Society on Thrombosis and Hemostasis's definition of major bleeding during hospitalization. The rates of these events in CHypoR or CHyperR were examined.Student's-t test. Time-dependent changes to the PRU values within the group were evaluated by a repeated measure ANOVA . The difference of PRU values between the two groups was analyzed using mixed-effect model, followed by a post-hoc analysis using the Bonferroni method. Univariate and multivariate logistic regression analyses were used to identify patient characteristics associated with PFT-identified clopidogrel abnormal response; p-values of <0.05 were considered statistically significant. All p-values were two-tailed. Factors identified as statistically significant in univariate analysis and factors with clinical relevance were included in multivariate analysis. All statistical analyses were conducted using JMP 13.0 .Categorical variables are presented as counts and percentages and compared using the chi-square test or Fisher's exact test. Continuous variables were expressed as mean ± standard deviation or median and interquartile range (IQR) and compared with the n = 23), small-vessel occlusion (n = 3), and arterial dissection (n = 3). Urgent EVT was performed in 13 patients. The median NIHSS in the AIS group was 5 (IQR 4–8) points, and the median time from onset was 8 (IQR 5.5–10) h. The details in the other CVD group were cerebral aneurysm (n = 10), intra-/extra-cranial artery stenosis or dissection (n = 17).Patient characteristics are shown in p = 0.042). The values of PRU and ARU were not different between the group . Significant reduction in PRU value with time was observed in the other CVD group , and also in the AIS group . The PRU values were significantly higher in the AIS group than in the other CVD group   . The PRU= 0.105) .p < 0.0001). CHyperR was rare (3.5%), observed in only one patient per group.The prevalence of CHypoR was higher in patients with AIS than in patients with other CVDs , vertebral artery stenosis (n = 1) and middle cerebral artery stenosis (n = 1). Their CYP2C19 genotype were PM , IM  and EM , and their PRU values at 24 h after the loading dose were 254, 355, 268, 362, respectively.Ischemic events were observed in five patients with ChypoR. Four of them were large-artery-atherosclerosis patients in the AIS group and the remaining one was a patient with intracranial artery stenosis who underwent PTA in the other CVD group. The patients with ischemic events in the AIS group included 3 females, and their etiology of stroke were artery-to-artery embolism from carotid artery stenosis (*1/*3). EVT was successfully performed 3 days after the loading dose, but she presented motor weakness on her upper limb the day after the procedure. Post-operative DWI revealed a new cerebral infarction.Remaining one patient with ischemic events in the other CVD group, a female patient in her 70s, was scheduled for an elective percutaneous transluminal angioplasty with stenting for symptomatic intracranial internal carotid artery stenosis. Her PRU value at 24 h after the loading dose was 249, and her CYP2C19 genotype was IM (*2/*2). The other two patients showed subcutaneous hematoma and required blood transfusion at the puncture site of cerebral angiography performed on the day of onset. Their etiology of AIS was large-artery-atherosclerosis, and the two patients immediately received loading doses of clopidogrel (300 mg) and aspirin (200 mg). PRU value at 24 h after loading dose was 48 in a male patient in his 60s with CYP2C19 genotype IM (*1/*2), and 355 in a male patient in his 80s with CYP2C19 genotype IM (*1/*2); this patient was the same as the second patient in ischemic events.Bleeding events were observed in three patients in the AIS group. Gastrointestinal bleeding which required urgent endoscopic clip and blood transfusion, was observed in a male patient in his 80s 20 days after onset of stroke due to artery-to-artery embolism from atherosclerotic carotid artery stenosis. His PRU value at 24 h after loading dose was 145, and CYP2C19 genotype was PM  were significantly associated with CHypoR  per 1 unit, 1.03; 95% confidence interval (CI), 1.01–1.96, h CHypoR ; howeverh CHypoR . MultivaPlatelet reactivity at 24 h after clopidogrel loading was significantly higher in patients with AIS than in other CVD patients. Although 79% of patients with AIS were judged as CHypoR, it was not associated with clinical events. The PFT-identified platelet reactivity to clopidogrel should be carefully interpreted in AIS patients with AIS.*2 or *3), associated with clopidogrel hypo-response, has been reported in >30% of the population, occurring at a rate higher than that observed in other populations  at 24 h to 35% (n = 11) at 72 h in this study supports this speculation.Campo et al. previously reported that, in patients undergoing a percutaneous coronary intervention, the rate of clopidogrel hypo-response varied over time. Post-clopidogrel PRU values at 1 month independently predicted ischemic and hemorrhagic events, suggesting that the rate of hypo-response might be overestimated in the early phase and that the PRU value may return to its normal range over time . The delN = 1,464), involving PFT evaluation with VerifyNow prior to flow diverter treatment, revealed that hypo-responders were at a 15% greater risk of thrombotic events and hyper-responders were at a 12% greater risk of hemorrhagic event than normal responders . The patients/participants provided their written informed consent to participate in this study.YEn contributed the conception and the design of the work, interpreted the data, drafted the original manuscript, reviewed all suggestions provided by all co-authors , approved the final version, agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, composed the final version of the manuscript, and assumed responsibility for final review. TI provided a final overview of the paper and revised a substantial portion of the manuscript. KS, DM, and HM provided a substantial contribution to the acquisition and statistical analysis of the data. YEg provided a substantial contribution to the interpretation of the provided data and contributed with substantial revisions of the original draft. All authors contributed to the article and approved the submitted version.This work was supported by JSPS KAKENHI Grant Number JP25861267.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


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. The mitotic spindle has a sophisticated architecture that enables it to accurately segregate chromosomes during cell division. It consists of three major classes of microtubules: kinetochore microtubules that form kinetochore fibers (k-fibers) connecting chromosomes to the spindle pole through kinetochores, midplane-crossing microtubules that form antiparallel arrays in the central part of the spindle, and astral microtubules that extend from the spindle poles towards the cell cortex . During o relative to the pre-existing microtubule   and confTo overcome the limitations of confocal microscopy and explore the relationship between different classes of microtubules within the crowded metaphase plate area, we performed stimulated emission depletion (STED) superresolution imaging  of microFigure 2—figure supplement 1A-CIn control cells, the vast majority of midplane-crossing microtubule bundles laterally attached to a pair of sister k-fibers and formed a bridging fiber between them, consistent with previous findings . These bThe appearance of disordered midplane-crossing microtubules in the metaphase plate area of spindles without augmin prompted us to investigate whether these microtubules affect mitotic fidelity. Augmin depletion has previously been linked to higher incidence of segregation errors , but theThe treatment with Mad2 siRNA resulted in a total of 1.5±0.2 segregation errors per cell . However, the effect was significantly more severe when Mad2 was codepleted with HAUS6, resulting in a total of 5.7±0.8 segregation errors per cell . TrackinAs misaligned chromosomes appeared equally frequently in Mad2 depletion and Mad2/HAUS6 codepletion, but differed in their ability to correctly segregate, we used them as a tool to isolate the role of augmin in resolving segregation errors during anaphase. Consistent with previous findings , both poWhereas lagging chromosomes have been previously observed following augmin depletion , their oTo further explore how this compromised spindle geometry affects lagging kinetochores, we measured their interkinetochore distance just before anaphase onset. The lagging kinetochore pairs in Mad2/HAUS6-codepleted cells had an interkinetochore distance of 0.89±0.03 µm, which was significantly smaller than the interkinetochore distance of 0.98±0.02 µm measured in error-free kinetochore pairs . This waAs sister k-fibers in the disorganized spindle region were sometimes diagonally positioned with respect to the pole-to-pole axis, we decided to test if this tilt is also connected to the appearance of segregation errors by measuring the angle that either lagging or error-free kinetochores form with the spindle axis just before anaphase onset. As for the interkinetochore distance following Mad2/HAUS6 codepletion, lagging kinetochore pairs were different from error-free kinetochore pairs, the tilt of which was 19.7±1.6° and 10.9±0.8°, respectively . In contAltogether, we propose that augmin ensures mitotic fidelity through the joint action of bridging and k-fibers. While augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled spindle geometry unfavorable for creating additional attachments, augmin-nucleated k-fibers produce robust force required to resolve any potentially appearing errors during anaphase .Ib) and k-fibers (Ik) which had no other microtubules in their immediate neighborhood, in a small square region between two kinetochores or at the pole-side of kinetochore, respectively . The accuracy of this calculation was additionally tested by measuring the intensity of astral microtubules, which presumably consist of single microtubules , and consistent with results obtained using visual inspection . The majThe compromised microtubule nucleation following augmin depletion led to the impairment of overall spindle geometry, creating a unique system where three main types of interactions between k-fibers and bridging fibers can be found within the same spindle: (1) sister k-fibers attached to bridging fibers, (2) sister k-fibers without a bridging fiber, and (3) solitary, long, interpolar bundles without associated kinetochores. This is in contrast with control cells, where the first group dominates and the other two groups are rarely found . To gainIn addition to spindle architecture, compromised microtubule nucleation following augmin depletion also affected spindle dynamics, as poleward flux in U2OS cells stably expressing CENP-A-GFP, mCherry-tubulin and photoactivatable-GFP-α-tubulin was significantly reduced , in agreOur finding that bridging fibers were more severely perturbed in the inner part of the spindle after augmin depletion prompted us to examine the spatial distribution of these midplane-crossing microtubules and their overlap regions throughout the spindle. We used protein regulator of cytokinesis 1 (PRC1) as a marker because it preferentially crosslinks overlap microtubules , thus prThe signal intensity of PRC1-GFP bundles in RPE1 cells was reduced by 55 ± 4% following augmin depletion . ConsistThe augmin-depleted cells showed a specific barrel-like distribution of the PRC1-GFP labeled bundles, with more overlap bundles being present around the perimeter of the spindle and fewer in the central part . HoweverDrosophila S2 cells and Xenopus egg extracts . Despite the spindles being wider in both cell lines, the diameter of the metaphase plate was not larger, as the spindles widened due to the long, curved bundles without kinetochores . While textracts . This diextracts . When boextracts , comparaextracts . AltogetInterestingly, the long curved bundles characteristic for augmin depletion  exhibiteThe interkinetochore distance, which is a readout of interkinetochore tension , decreasAlthough this result suggests a role of bridging fibers in regulating interkinetochore distance, this effect may be indirect and arise due to k-fibers, askinetochore pairs that lacked a bridging fiber typically had thinner k-fibers than those with a bridging fiber in augmin-depleted cells . Hence, In this paper, we propose a model of the metaphase spindle in which the bridging fiber, which laterally connects sister k-fibers, forms by augmin-based nucleation of microtubules along the existing microtubules. The newly nucleated microtubules in the bridging fiber create an antiparallel overlap in which the microtubules slide apart, generating a pushing force that the bridging fiber exerts on its k-fibers. In doing so, the bridging fiber works together with k-fibers to produce tension and maintain the appropriate orientation of sister kinetochores parallel to the spindle axis, thereby preventing merotelic attachments and ensuring mitotic fidelity .Our work shows that the depletion of the augmin complex by silencing the HAUS6 or HAUS8 subunits causes severe thinning of bridging fibers in metaphase spindles, combined with the appearance of wavy and disordered midplane-crossing microtubules. Thus, we conclude that the predominant nucleation of bridging microtubules by the augmin complex enables their directional bundling along the sister k-fibers, in agreement with previous observations on directionality of microtubule growth . K-fiberPrevious work showed that augmin depletion results in a decrease of interkinetochore distance , a readoPreprint). In addition to predisposing the kinetochore to merotelic attachments through impaired bridging fibers, thinning of k-fibers upon augmin depletion might ultimately be responsible for the inability to resolve merotelic attachments, as strong k-fibers are necessary to ensure proper segregation of kinetochores during anaphase ; human HeLa-Kyoto BAC cells stably expressing PRC1-GFP , courtesAll cell lines were cultured in flasks in Dulbecco’s Modified Eagle’s Medium with 1 g/L D-glucose, pyruvate and L-glutamine , supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum  and penicillin (100 IU/mL)/streptomycin (100 mg/mL) solution . For the selection of U2OS CENP-A-GFP mCherry-α-tubulin PA-GFP-α-tubulin, HeLa PRC1-GFP, HeLa CENP-A-GFP Centrin1-GFP and Hela GFP-α-tubulin cell lines, 50 μg/ml geneticin was added to the medium .2 in a Galaxy 170 R humidified incubator . They have also been regularly tested for mycoplasma contamination by examining the samples for extracellular DNA staining with SiR-DNA  and Hoechst 33342 dye  and have been confirmed to be mycoplasma free.All cells were kept at 37 °C and 5% CO2 in a humidified incubator for 5 min. After the incubation, trypsin was blocked by adding 2 ml of DMEM medium. For RNAi experiments, the cells were seeded to reach 60% confluence the next day and cultured on 35 mm uncoated dishes with 0.16–0.19 mm (1.5 coverglass) glass thickness  in 2 mL DMEM medium with previously described supplements. After one day of growth, cells were transfected with either targeting or non-targeting siRNA constructs which were diluted in OPTI-MEM medium  to a final concentration of 20 nM for HAUS6 and HAUS8 in medium with cells. HAUS6 and HAUS8 siRNA transfections were performed 48 hr prior to imaging and cotransfection with 100 nM Mad2 siRNA was additionally performed 24 hr prior to imaging using Lipofectamine RNAiMAX Reagent  according to the instructions provided by the manufacturer. After four hours of treatment, the medium was changed to the previously described DMEM medium. The constructs used were human HAUS6 siRNA , human HAUS8 siRNA , human Mad2 siRNA  and control siRNA . Mad2 was chosen instead of MPS1 inhibitors as it induces less segregation errors per cell in control cells, as well as to avoid a biased approach in which the inhibitor would likely be added at a different time point during an hours-long error correction process that occurs in augmin-depleted cells.At 80% confluence, DMEM medium was removed from the flask and cells were washed with 5 ml of PBS. Then, 1 ml 1% trypsin/EDTA  was added to the flask and cells were incubated at 37 °C and 5% COWhen microtubules were visualized during live cell-imaging in cell lines without stable expression of tubulin, staining was performed to a final concentration of 100 nM with a far-red silicon rhodamine (SiR)-tubulin-670 dye , 45 min to 2 hr prior to imaging. As for DNA, either SiR-DNA or SPY-555-DNA  were used to a final concentration of 100 nM, 20 min to 2 hr prior to imaging. In order to avoid dye efflux, a broad-spectrum efflux pump inhibitor verapamil  was added at a final concentration of 0.5 μM to RPE1 cells along with tubulin and/or DNA dyes. As an additional control for metaphase cells, MG-132  was added to the cells used for the measurement of spindle size for 30 min prior to imaging at a final concentration of 20 µM.AB_2169665), rabbit polyclonal HAUS6 , rabbit polyclonal HAUS8  and rat monoclonal tubulin . After the incubation with a primary antibody, cells were washed 3 times for 5 min with 1 ml of PBS and then incubated with 250 µl of secondary antibody for 45 min at a room temperature. Alexa Fluor 488 and 594  were used as secondary antibodies at a 1:1000 dilution for PRC1 staining, 1:500 dilution for HAUS6 and 1:250 for HAUS8 and tubulin staining. DAPI (1 µg/mL) was used for chromosome visualization.For confocal imaging, HeLa cells stably expressing PRC1-GFP were grown on glass-bottom dishes  and fixed by 2 ml of ice-cold methanol for 1 min at –20 °C. Following fixation, cells were washed three times for 5 min with 1 ml of PBS and permeabilized with 0.5% Triton-X-100 in water for 15 min at a room temperature. This step was repeated twice when tubulin staining was performed. To block unspecific binding, cells were incubated in 1 ml of blocking buffer ) for 1 hr at 4 °C. Cells were then washed three times for 5 min with 1 ml of PBS and incubated with 250 µl of primary antibody solution overnight at 4 °C. The primary antibodies used were as follows: rabbit polyclonal PRC1 , cell medium was removed, and cytoskeleton extraction buffer was added for 20 s to remove the components of the cytoplasm. Following extraction, cells were fixed in 3% paraformaldehyde and 0.1% glutaraldehyde solution for 10 min. To reduce the background fluorescence, quenching (100 mM glycine in PBS) and reduction (0.1% sodium borohydride in PBS) solution were added for 7 and 10 min, respectively. To prevent non-specific binding, cells were incubated in blocking/permeabilization buffer  for 2 hr at 4 °C. Microtubules were then stained using a rat anti-tubulin primary antibody solution  with a 4 °C overnight incubation. The next day, cells were washed with PBS three times for 5 min. After washing, a secondary antibody Alexa Fluor 594  was added and incubated for 1 hr at room temperature. Additionally, DAPI (1 µg/mL) was added and incubated for 15 min at room temperature to visualize chromosomes. Cells exposed to cold treatment were incubated with cold media on ice for 13 min prior to extraction of cytoplasmic components.AB_307275). Rabbit polyclonal HAUS8 antibody  resulted in no detectable bands under these conditions. Membranes were washed with 0.1% Tween 20 in PBS, incubated for 1 hr with anti-rabbit HRP-conjugated secondary antibodies  and visualized on the C-DiGit blot scanner  with WesternSure PREMIUM Chemiluminescent Substrate .RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP were grown on six-well plates (Greiner Bio-one) and subjected to HAUS6 or HAUS8 siRNA treatment as indicated before. Following transfection, cells were lysed in RIPA buffer  containing 1 x protease inhibitor , 1 x phosphatase inhibitor  and 1 mM PMSF by two cycles of freezing and thawing in liquid nitrogen. Protein extracts were mixed with 2 x Laemlli sample buffer  and heated at 95 °C for 10 min prior to SDS-PAGE. After protein transfer onto the nitrocellulose membrane  and blocking with blocking solution  for 1 hr, membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. The primary antibodies used were as follows: rabbit polyclonal HAUS6 , rabbit polyclonal GAPDH  with the 100 x/1.4NA UPLSAPO100x oil objective  and an avalanche photodiode (APD) detector. The 488 nm line was used for excitation in both cases, with the addition of the 561 nm line for excitation and the 775 nm laser line for depletion during STED superresolution imaging. Images were acquired using the Imspector software. The xy pixel size for fixed cells was 20 nm and 6 focal planes were acquired with 300 nm distance between planes were acquired. For confocal live-cell imaging of anaphase, the xy pixel size was 80 nm and 16 focal were acquired, with 1 µm distance between the planes and 30 s time intervals between different frames.2 in Okolab Cage Incubator . To excite Hoechst, GFP, mCherry or SiR fluorescence, a 405 nm, 488 nm, 561 nm or 647 nm laser lines were used, respectively. Opterra Dichroic and Barrier Filter Set 405/488/561/640 enabled the separation of excitation light from the emitted fluorescence. Images were acquired using Evolve 512 Delta Electron Multiplying Charge Coupled Device (EMCCD) Camera , with camera readout mode of 20 MHz. The xy pixel size was 83 nm. In all experiments where the whole spindle stack was imaged, z-stacks were acquired with unidirectional xyz scan mode at 37 focal planes and 0.5 μm distance between the planes. Photoactivation was performed perpendicular to pole-to-pole axis of metaphase spindles using a 405 nm laser diode  and a line pattern of 12 equally distributed points, with each point representing one laser hit. The interval between the points was 0.05ms and photoactivation area was set to 0.5 µm for each point. The interval between successive frames was set to 10 s and one central z-plane was imaged.Other confocal images and videos were acquired using a previously described microscope setup , consistSCR_003070). Raw images were used for quantification. In representative immunofluorescence images of augmin depletion (SCR_001622) was used for calculations and plotting and Rstudio  to transform the cells into an end-on view. Figures were assembled in Adobe Illustrator CS5 .In cells where HAUS6 or HAUS8 were silenced only bipolar metaphase spindles were imaged and analyzed, even though multipolar spindles were observed as reported previously . All imaepletion , all sigIb = Ib+bcg – Ibcg) and k-fiber intensities (Ik = Ik+bcg – Ibcg). 10 randomly positioned empty areas were taken into account while calculating background for STED imaging, whereas 2 empty areas just above and below bridging fiber were taken into account while calculating background for confocal imaging due to different nature of the acquired images. All measurements were performed on randomly selected bundles in single z-planes, after determining that no other microtubules were crossing the area of measurement. In STED images, the intensity of astral microtubules was additionally measured using the 25x25 pixel Square tool (ImageJ). Their background was measured using the same tool in the empty area between the two astral microtubules and it was subtracted from astral microtubule intensity (Ia=Ia+bcg-Ibcg).For the analysis based on small square regions which was performed on both STED and confocal images, tubulin intensity in bridging and k-fibers was measured using either a 25x25 or 5x5 pixel Square tool (ImageJ) on STED and confocal images, respectively. To measure bridging fibers, the square was positioned on the fiber spanning the area between two kinetochores. K-fibers were measured right next to the kinetochores. The average intensity of the two sister k-fibers was used for further analyses. The background was measured using the same tool at several empty areas within the spindle and its average was subtracted from bridging (Ib) was calculated as the mean value of intensities in the area 500 nm around the center of the bridging fiber. The final intensity of a k-fiber region (Ibk), which also includes the bridging fiber, was calculated as an average of two mean values of intensities in the area 500 nm around the distance of 1.5 μm away from the center of the bridging fiber. The intensity value of k-fibers alone (Ik) was then calculated as Ik = Ibk – Ib.On top of that, tubulin intensities of the bridging fiber and k-fiber region in confocal images were measured in a single z plane using the Segmented Line tool by drawing a 5 pixel line (ImageJ) along the contour of k-fibers and the corresponding bridging fiber. Background was measured in the same z plane by drawing the 5 pixel thick line along the length of the metaphase midzone, as this represents the background signal caused by the presence of neighboring microtubules. The minimum value of the midzone intensity profile was subtracted from the intensity profiles of bundle contours. The minimum value of the mean tubulin intensity profile was set as a distance of zero μm and was selected as the center of the bridging fiber. The final intensity of a bridging fiber  was calculated using the Pythagorean theorem, where a=projected distance of a middle point between two sister kinetochores in a particular z-plane from the long spindle axis, b=distance between the central z-plane and the z-plane of the kinetochore pair x 0.81 . Kinetochore pairs were defined as those in the inner or the outer part of the spindle if their distance to the pole-to-pole axis was smaller or larger than the average distance of all tracked kinetochore pairs to the pole-to-pole axis, respectively. Additionally, kinetochores with visible bridging fibers and kinetochores with no visible bridging fibers were separately tracked and analyzed in confocal images of RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP, where the presence of bridging fibers was determined by measuring intensity profiles of the tubulin signal between two kinetochores.Z-stacks of the mitotic spindles that were positioned horizontally were transformed into the end-on view using a home-written R script, based on the original version . Prior tThe number of bundles in HeLa and RPE1 PRC1-GFP cells was determined in an end-on view of the spindle by using sum intensity projections of 10 central z-planes covering 0.83 µm along the long spindle axis.Spindle length, width and metaphase plate diameter were measured on maximum intensity projections of the side view z-stack of spindles. Spindle length was determined as a distance between the two poles. The position of the poles was determined as either the center of Centrin1 signal or the outermost points of the tubulin or PRC1 signal at the spindle pole. Spindle width was measured as the distance between two lines parallel to the long axis of the spindle and encompassing the outermost PRC1- or tubulin-labeled bundles. Additionally, in RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP the metaphase plate diameter was measured as the distance between the outermost kinetochore pairs, whereas in HeLa PRC1-GFP it was measured as the distance between the outermost chromosome ends.Overlap length was measured on sum intensity projections of 2–4 z-planes covering the entire bundle of interest, using ImageJ Segmented line tool by drawing a pole to pole line along the contour of PRC1-GFP and acquiring an intensity profile. The overlap length was defined as the length of the base of the PRC1-GFP intensity peak .PRC1 intensity was measured in sum intensity projections of 10 central z-planes of the spindle. Total PRC1 signal in the cell was marked by using Polygon selection tool (ImageJ) and 5x5 Square tool was used to determine the background in the cytoplasm. The final intensity values were obtained using the following formula: PRC1 intensity = Integrated Density of the spindle – (Area of selected cell x mean fluorescence of background). Intensity profiles of PRC1-GFP were measured on the sum intensity projections of 10 central z-planes in an end-on view of the spindle by drawing a 50 pixel wide Straight line tool across the diameter of the spindle.The shape of spindles was determined in ImageJ using a Point tool. Ten points were distributed throughout the bundle, with the first and last point positioned at the spindle poles. The images were rotated to make the long spindle axis parallel to the x axis. In control cells, only the outermost bundle was tracked. In HAUS6 siRNA-treated cells, three different groups of outermost bundles were tracked: bundles with visible bridging fibers, bundles with no visible bridging fibers and curved bundles extending far from the metaphase plate. Shape and curvature were calculated using a home-written MatLab script by fitting a circle to the tracked points along the bundle. Contour lengths of the bundles were measured by calculating the cumulative distance between the first and the last point of the tracked bundle.For measuring the poleward flux rate, 10-pixel wide line was drawn from pole to pole along the bundle with photoactivation signal that lasted at least 5 time frames (40 s), using the Segmented Line tool in ImageJ. The position of photoactivated mark in each time frame was determined as the distance between the peaks of intensity profiles in photoactivation and SiR-tubulin channels for photoactivation mark and closer spindle pole, respectively. The analysis was performed on images processed with Gaussian Blur filter with Sigma set to 2 to improve the definition of the intensity profile peaks.Three types of segregation errors were analyzed in Mad2-depleted and Mad2/HAUS6-codepleted anaphase spindles: misaligned kinetochores, lagging kinetochores and other errors. Misaligned kinetochores were defined as those in which both kinetochores of the pair were situated outside the metaphase plate 30 s before anaphase onset. Lagging kinetochores were defined as those in which the CENP-A signal was visibly stretched and the kinetochore was situated in the central part of the spindle, outside the kinetochore mass that was moving towards the pole during anaphase. Finally, segregation errors classified as others included various kinetochores, precise classification of which was impossible using only CENP-A signal. These included kinetochores situated outside the moving kinetochore mass without stretched CENP-A signal, kinetochore pairs that remained completely unseparated for the whole duration of anaphase, as well as kinetochore pairs in which both kinetochores remained non-stretched and in the central part of the spindle despite the initial separation. Segregation errors were further divided based on their distance to the pole-to-pole axis into those in the inner and the outer part of the spindle. The distance of the kinetochore pair from the pole-to-pole axis was determined in 3D using a home written Matlab script, and the obtained value was then normalized to spindle half-width determined using maximum intensity projections in ImageJ. All kinetochores situated in the area less than 0.5 from the spindle axis 30 s before anaphase onset were defined as those in the inner part of the spindle and all kinetochore pairs in the area equal to or above the value of 0.5 were defined as kinetochores in the outer part of the spindle. All kinetochore pairs were manually tracked in time from just before anaphase onset until entering the daugther cell by using the Point tool in ImageJ. In images acquired using STED microscopy, merotelic attachments were defined as those in which one kinetochore forms attachments with microtubules from the opposite side of the spindle, with no visible microtubule signal just below or above the kinetochore.To measure anaphase A speed, the coordinates of kinetochores and poles were tracked in time using the Point tool in ImageJ. The speed was calculated from the time point when the distance between the kinetochore and its closer pole started gradually decreasing. The slope of a line equation obtained from the linear fitting of distances over time was determined for every kinetochore as the anaphase A speed. Anaphase B speed was calculated in a manner similar to anaphase A, but instead of kinetochores, the positions of poles were tracked in time with the first time frame determined as the frame when the distance between two poles started gradually increasing.Anaphase onset was determined in a frame just before the interkinetochore distance of the tracked kinetochore pair started gradually increasing. The coordinates of kinetochore pairs that ended up as errors and of error-free kinetochore pairs were tracked along with the coordinates of spindle poles. The angle that the kinetochores form with the long spindle axis was calculated using a home written Matlab script. This manuscript investigates how the protein augmin contributes to correct spindle architecture and chromosome segregation during cell division in cultured human cells. High-quality super-resolution imaging and functional studies provide new insight into the mechanism of proper chromosome attachment to the spindle microtubules. This is an important paper that will be of interest to researchers interested in cell biology and cell biophysics. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by  General Statements [optional]We thank the reviewers for providing thoughtful and constructive feedback on our manuscript. Motivated by their comments, we decided to perform a full revision with extensive new experiments, which led to important novel findings and thus a significant revision of the manuscript. The main changes lie in that we performed STED super resolution imaging of augmin depleted spindles, which allowed us to analyze the previously indistinguishable microtubule arrangements and erroneous attachments, as well as to quantify k-fibers and bridging fibers with unprecedented accuracy. Moreover, a key part of the revision is a functional approach where we tested how augmin depletion affects chromosome segregation fidelity, as suggested by Reviewer 3. Surprisingly, we found that without augmin, the lack of bridging fibers results in kinetochore tilt and, consequently, merotelic attachments in metaphase. Our STED images provide the first clear demonstration of such attachments, and these new findings offer a mechanistic explanation of how augmin-dependent microtubule branching promotes mitotic fidelity.The main conceptual advance of our work is that the augmin complex is crucial for the proper organization of midplane-crossing microtubules into uniformly arranged bridging fibers that connect two sister k-fibers and extend nearly-parallel to the spindle axis. We found that bridging fibers are often absent following augmin-depletion, and have a specific role in the maintenance of interkinetochore tension, as well as in promoting mitotic fidelity by preventing merotelic attachments. K-fibers were also impaired, but to a lesser extent, with their thinning amplifying the negative effect on mitotic fidelity by compromising resolution of merotelic attachments later in anaphase. Altogether, 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.We have now addressed the criticism of the reviewers by identifying the specific effects of bridging fibers upon augmin depletion and considerably enhanced the significance by exploring the effects on chromosome segregation fidelity, thereby transforming our paper from a descriptive to a mechanistic study. As such, the study is no longer of interest only to the mitotic field, but to a larger scientific community including cell biologists, molecular biologists and biophysicists interested in microtubules, mitotic and meiotic spindles, cell division, chromosome segregation, aneuploidy, cancer, and development, as well as scientists developing quantitative super resolution imaging protocols for imaging of cellular structures.Reviewer #1 ):In the manuscript "Augmin regulates kinetochore tension and spatial arrangement of spindle microtubules by nucleating bridging fibers", Manenica et al. explore the impact of augmin dependent microtubule nucleation on formation of a subset of spindle microtubules that bridge sister kinetochore fibers and investigate how this could affect the spindle forces and architecture. Using RNAi- and CRISPR-Cas9- based loss-of-function experimental approach, the authors propose that the bridging fibers are nucleated by augmin and that removal of augmin impairs proper spindle architecture, interkinetochore tension and microtubule poleward flux, specifically via its effect on the bridging fibers. Overall, the study is well designed and the manuscript well written. Expanding the knowledge on augmin contribution to the spindle functions and better understanding of the roles of bridging fibers would be important and of interest to cell biologists studying mitosis. Although this manuscript clearly shows that augmin depletion impairs the formation of bridging fibers (and other microtubules), the specific contribution of the bridging fibers to the augmin-dependent spindle functions is less clear.We thank the reviewer for pointing out the quality of our experiment design and writing. We also appreciate the criticism regarding the exact contribution of bridging fibers to the spindle architecture, interkinetochore tension and microtubule poleward flux. We addressed this in our revised manuscript, where we studied the specific effect of bridging fibers on each of these phenotypes.Using STED microscopy, we were now able to determine the specific effect of bridging fibers on spindle architecture by analyzing the previously indistinguishable microtubule arrangements within the crowded metaphase plate area and quantifying k-fibers and bridging fibers with unparalleled precision and accuracy. This type of analysis confirmed that augmin is vital for the organization of midplane-crossing microtubules into highly organized bridging fibers that connect sister kinetochore fibers and extend nearly-parallel to the spindle axis. Without augmin, proper bridging fibers were thin and often even absent, while the remaining midplane-crossing microtubules appear wavy and disorganized. K-fibers were also thinner, but to a lesser extent, as confirmed by two different types of analysis.We clarified the contribution of bridging fibers to the interkinetochore tension using different types of analysis, which revealed that augmin-nucleated bridging microtubules have a specific role in the maintenance of interkinetochore tension. Indeed, the severe impairment of bridging fibers had a stronger effect on the interkinetochore distance than thinning of k-fibers following augmin depletion, and the specific effect of bridging fibers was demonstrated for kinetochores with the same k-fiber intensity.As for poleward flux, a detailed analysis of poleward flux following augmin depletion, with a specific focus on the differences in bridging and k-fiber flux, is now published as a part of the recent Cell Reports paper from our lab . Thus, this is no longer a main point of our manuscript, which now shifted its focus to more striking consequences of augmin depletion, particularly microtubule arrangements within the metaphase plate and mitotic fidelity.The effect of augmin on mitotic fidelity, as suggested by Reviewer 3, was studied using sophisticated live-cell imaging and tracking protocols. We described the main results in the General Statement (see above), and dedicated an entire new section of the manuscript  to these exciting new results that provide a mechanistic explanation of how augmin-dependent microtubule nucleation prevents merotelic attachments.Major comments:1) Using cold treatment-induced microtubule destabilization, Zhu et al. (JCB 2008) showed that augmin depletion affected exclusively kinetochore microtubules. Since the bridging microtubules are usually not visible in the cold-treated spindles (due to being less stable/cold resistant compared to the k-fibers), it is unlikely that the observed effects were mainly associated with the bridging fibers. Thus, it would be important to further clarify the respective contribution of augmin to the formation of k-fibers and the bridging fibers. The cold-treatment experiment performed by Zhu et al. could be used in RPE1 and HeLa-PRC1-GFP cells to address the contribution of augmin nucleation to kinetochore- vs. bridging microtubules from another angle.Because of the above mentioned results by Zhu et al. it is difficult to grasp how augmin depletion could have a bigger effect on the bridging fibers than on the k-fibers, as concluded from the Figure 2C data. In fact, Figure 2A clearly shows a strong effect on k-fibers in spindles where the bridging fibers are reduced/missing.Also, Figure 1 D and E suggest that HAUS8 siRNA exclusively affected the bridging fibers, leaving the k-fibers intact, which is again against the data reported in Zhu et al. 2008 and in contrast with the representing image shown in Figure 1B. Even if the RNAi was less efficient compared to HAUS6 RNAi, as the authors proposed, this could still not explain the observed discrepancy.We thank the reviewer for this comment. To better address the contribution of augmin to either bridging or kinetochore microtubule nucleation, we repeated the experiments by using STED super-resolution microscopy on cells immunostained for tubulin, with or without HAUS6/8 depletion. STED imaging enabled a previously unattainable and exceptionally detailed visualization of k-fibers and bridging fibers , and subsequent analysis confirmed our initial result that augmin depletion has a larger effect on bridging fibers than on kfibers . Importantly, due to the increased precision of our measurements when using STED images, the new results for HAUS6 siRNA and HAUS8 siRNA were now similar, and showed that the lack of augmin affects k-fibers to an extent comparable to Zhu et al., 2008 . The data from the original manuscript, which were obtained by confocal microscopy and were therefore somewhat less precise due to the intrinsic limitations of the method, are now shown in Supplementary Figure S3.To independently test the effect of augmin depletion on k-fibers, we also performed experiments with cold treatment as suggested, imaged the cells by STED microscopy, and analyzed the images as in Zhu et al., 2008 . These experiments were done on RPE1 cells that stably express CENP-A-GFP. Cold treatment in HeLa-PRC1-GFP cells was no longer necessary, as STED imaging clearly showed the absence of bridging fibers in cold treated cells. The new results, that emphasise the effect on bridging fibers but no longer state that k-fibers are intact are described on page 12:“Quantification of STED images further revealed that HAUS6 depletion resulted in 68 ± 8% reduction of the bridging fiber signal intensity and 24 ± 6% reduction of the k-fiber signal intensity, with similar results obtained by HAUS8 depletion . These data indicate that augmin depletion affects not only k-fibers, but even more so bridging fibers. The contribution of augmin to the nucleation of k-fibers was independently tested by measuring their intensity in spindles exposed to cold treatment in which bridging fibers are removed . HAUS6 depletion resulted in a 37 ± 5% reduction of the k-fibers , which is consistent with a previous study  and comparable to values under non-cold conditions.”2) The authors showed that kinetochore pairs in the outer parts of Augmin-depleted spindles have larger inter-kinetochore distance compared to those in the inner parts of spindles. They indirectly related this to a predominant presence of the bridging fibers in the outer parts, concluding that augmin regulates inter-kinetochore tension via nucleation of the bridging fibers. A more direct way would be to show the eventual positive correlation between the inter-kinetochore distance and the bridging- and k- fibers intensity. Also, it would be nice to include the quantifications and correlation data for inter-kinetochore distance, distance from the spindle axis and the bridging- and k- fibers intensities for the control cells too.We analyzed the new data and included the correlations in the altered manuscript. We explained the correlation data for the interkinetochore distance and the distance from the spindle axis as follows: “… we noticed that the interkinetochore distance was smaller in the inner part of the spindle in augmin-depleted cells , where bridging fibers were most severely impaired . This was not the case in control cells, which showed no difference in interkinetochore distance between the inner and the outer part of the spindle .”We also included the correlation data for the interkinetochore distance and the bridging and kfibers intensity: “… we found that although the interkinetochore distance correlated both with bridging and k-fiber intensity after augmin depletion, the correlation with bridging fiber intensity was stronger . Such correlations were absent in control cells .”In addition, we performed yet another type of analysis where we divided kinetochore pairs into subsets with either comparable bridging fiber intensities and contrasting k-fiber intensities or comparable k-fiber intensities and contrasting bridging fiber intensities. We then compared the interkinetochore distance of kinetochore pairs within these subsets, as follows: "To explore a specific contribution of k-fibers to the interkinetochore tension, we divided the kinetochore pairs in augmin-depleted cells into two subsets, those with and without bridging fibers, and found that the interkinetochore distance did not correlate with k-fiber intensity within each group , which argues against the k-fiber intensity as a sole determinant of interkinetochore tension. In agreement with this, when we selected two subsets of kinetochore pairs with either very strong or very weak k-fiber intensity but with comparable bridging fiber intensities , we found no difference in the interkinetochore distance between these subsets . Finally, to examine a specific contribution of bridging fibers, we identified two subsets of kinetochore pairs with similar k-fiber intensity values, one of which had bridging fibers and the other which did not . We found that the interkinetochore distance was larger in the subset with bridging fibers than without , indicating a specific effect of bridging fibers on interkinetochore tension. Analysis of live-cell confocal images of RPE1 cells yielded similar results . Based on these data, we conclude that augmin has a significant role in regulating interkinetochore tension through the nucleation of bridging microtubules."In sum, in the altered manuscript we conclude that both bridging and k-fibers regulate the kinetochore tension, and that in augmin-depleted cells there is indeed a specific effect of bridging fibers on interkinetochore tension, which is larger than the effect of k-fibers.3) It is stated in the manuscript that the k-fibers without bridging fibers have shorter contour length compared to the k-fibers with bridging fibers, and that the curvature of k-fibers lacking the bridging fibers is drastically reduced. However, the data in Figure 5D and Table 1 show a slight effect on the contour length of the k-fibers lacking the bridging fibers compared to the ones containing the bridging fibers only in RPE1 siHAUS8 cells, while this effect seems to be missing in RPE1 HAUS8 KO cells, as well as in siHAUS6 in RPE1 and HeLa cells.Figure 2 shows that the kinetochore pairs without the bridging fibers are located closer to the spindle axis. Thus, it is not clear whether the effect on curvature observed in the augmin depleted cells is independent of the position of kinetochore pairs within the spindle, as the spindle axis-proximal pairs would anyway have a bigger radius compared to the more distant ones.As these analyses were previously performed on bundles stained with SiR-tubulin and using confocal microscopy, we have now determined their curvature on spindles immunostained for tubulin and imaged by STED microscopy, where the shape of these bundles can be determined more precisely both in control and HAUS6 depleted cells. In the revised manuscript, only those spindles were taken for further analysis , as this is no longer a major point in the revised manuscript which shifted its focus towards other more striking consequences of augmin depletion. The text was revised as follows: “Whereas the bundles without kinetochores in HAUS6 siRNA-treated cells had a significantly longer contour when compared to all other bundle types , k-fibers without bridging fibers in augmin-depleted cells had a significantly larger radius of curvature than any of the other bundle types in augmin-depleted or control cells . Taken together, the outer interpolar bundles without associated kinetochores are excessively long and make the spindle wider, whereas k-fibers lacking a bridging fiber are overly straight, ultimately resulting in a diamond-like shape of the spindle.”As for previous Figure 2, in all experiments regarding shape, we only analyzed the outermost bundles, so the potential effect of the position of kinetochore pairs within the spindle can be excluded. We explained that in the Methods section and highlighted it in the caption of the Supplementary Figure 3I: “In control cells, only the outermost bundle was tracked. In HAUS6 siRNA treated cells, three different groups of outermost bundles were tracked: bundles with visible bridging fibers, bundles with no visible bridging fibers and curved bundles extending far from the metaphase plate” and “Examples of each bundle type are shown in insets. From left to right: the outermost bundle in control cells, the outermost bundle with a bridging fiber, the outermost bundle without a bridging fiber and the outermost bundle without kinetochores in HAUS6-depleted cells”, respectively.4) The authors reported that augmin depletion impairs microtubule poleward flux and conclude that this happens exclusively due to the perturbation of bridging fibers. While the results from this and other studies clearly show that augmin depletion perturbs spindle microtubules in general, it is not clear whether this had a stronger effect on the bridging microtubules (see the comments in point 1). Thus, the impact of augmin depletion on kinetochore microtubules or other antiparallel microtubules within the spindle  cannot be ruled out as a potential cause of the impaired microtubule flux. Also, Steblyanko et al.  showed that PRC1 depletion hadffectt on microtubule poleward flux in metaphase cells. Since it has been previously shown by the authors of this manuscript that PRC1 depletion disrupts the formation of bridging fibers, it is unlikely that the bridging fibers are the main cause of the augmin depletion-mediated effect on the microtubule flux.We modified the text on poleward flux to include the contribution of both bridging and k-fibers. We also performed new experiments on U2OS cells and included references to the new work from our lab recently published in Cell Reports , which was able to distinguish between the effect of augmin depletion on bridging and k-fiber flux. The effect of augmin on poleward flux is now only a minor point in our revised manuscript which mainly focuses on microtubule arrangements and mitotic fidelity.However, as requested, we included a comment on PRC1 depletion: “Recent speckle microscopy experiments in RPE1 cells, which were able to separate the effect of augmin on poleward flux of bridging and k-fibers, revealed that both k-fibers and the remaining bridging fibers were significantly slowed down . Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells , supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules . We propose that augmin depletion results in slower flux of bridging fibers because the remaining bridging microtubules are likely nucleated at the poles, where microtubule depolymerization mechanisms might curb poleward flux speed . In contrast, PRC1 depletion does not affect the flux  even though it reduces bridging fibers , possibly because the remaining bridging microtubules are generated away from the poles via augmin and can thus flux freely.”Minor comments:1) Introduction: chromatin- and kinetochore- mediated generation of spindle microtubules are ignored when describing the origins of spindle microtubules in human somatic cells.We included the chromatin- and kinetochore-mediated generation of spindle microtubules in the Introduction. We revised the text as follows: “Spindle microtubules in human somatic cells are generated by several nucleation mechanisms, including centrosome-dependent and augmin dependent nucleation , with an addition of chromatin- and kinetochore-dependent nucleation as a third mechanism that contributes to the directional formation of k-fibers .”2) The authors proposed less efficient HAUS8 depletion as a potential reason of discrepancy between the siHAUS6 and siHAUS8 results. This should be shown by Western blot, like it is presented for the RNAi efficiency of siHAUS6.We agree with the reviewer that it would be best to include Western blot for the RNAi efficiency of siHAUS8. However, as we explained in the Methods section, commercially available HAUS8 antibodies resulted in no detectable bands in our hands, regardless of the modifications in the Western blot protocol. We explained this in the Methods section, as follows: “Rabbit polyclonal HAUS8 antibody  resulted in no detectable bands under these conditions”. For this reason, we performed immunocytochemistry to determine the efficiency of siHAUS8. The discrepancy was now also addressed as a part of our new super resolution STED analysis, which enabled increased precision and where depletion of HAUS6 and HAUS8 produced the same results.3) The measurements of total PRC1 intensities are mentioned in the manuscript text, but not shown in the figures.PRC1 measurements are now performed on both RPE1 and HeLa cells with corresponding graphs shown in Figure 4C and Supplementary Figure S4C.4) Supplementary Videos 3 and 4 are wrongly annotated as Supplementary Videos 1 and 2 in the text.As we have a new set of videos, this is no longer applicable.5) Given the spindle length phenotypes are opposite in HeLa and RPE1 cells, in order to be consistent with the other experiments it would be better to perform the PRC1 measurements in RPE1 cells .Drosophila S2 cells and Xenopus egg extracts . This difference in spindle length might be due to the overlaps remaining the same length after augmin depletion in RPE1 cells, while being longer and thereby able to push the spindle poles further apart in HeLa cells .”We have now performed size measurements in all three cell lines: RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP, RPE1 cells stably expressing PRC1-GFP and HeLa cells stably expressing PRC1-GFP treated with MG-132. These results are now shown in Supplementary Figure S4J-K. The phenotypes remained the same as in the original experiments. We revised the text to better explain the observed differences as follows: “While the spindles in RPE1 cells shortened following augmin depletion, those in HeLa cells were longer , consistent with previous observations on Please note that due to the extensive amount of new data regarding microtubule arrangements and segregation errors obtained from STED microscopy and live-cell imaging of kinetochores over time, size measurements are now only a small part of our revised story and are, as such, no longer presented in the main figure.6) Why are the microtubule flux rates for RPE1-PA-GFP-α-tubulin cells measured in this study largely different than the rates reported for the same cell line in Dudka et al., Nat Comms 2018 and Dudka et al., Curr Biol 2019? In order to better understand this difference and strengthen the microtubule flux data, it would be helpful to increase the experimental numbers to match the ones used in the mentioned studies.We performed photoactivation experiments on a higher number of U2OS cells stably expressing CENP-A GFP, mCherry-tubulin and PA-tubulin . U2OS cells with labelled kinetochores and tubulin were used to exclude the potential effects of SiR-tubulin on poleward flux, as well as to better determine the position of the metaphase plate. The results in control cells are comparable to the poleward flux measured in the same cell line .Similar to shape and size measurements, due to the extensive and exciting new data regarding microtubule arrangements and segregation errors and because poleward flux was studied into much more detail in a recent Cell Reports study from our lab, this is no longer a major point in our revised manuscript. Poleward flux, along with shape analysis, is now included in the Supplementary Figure 3, as a part of the section about spindle architecture and dynamics.7) The number of cells used per each experiment should be clearly stated.In all experiments included in the main figures, we have now performed 3 independent experiments with at least 10 cells each. The numbers are also clearly stated in the captions of figures for all experiments.Reviewer #1 (Significance (Required)):This study expands the analysis of augmin contribution to the spindle functions and focuses on its role in formation of the bridging fibers, which is of interest to cell biologists studying mitosis. It clearly shows that in addition to its effect on the k-fibers, augmin depletion also impairs the formation of bridging fibers. However, the exact contribution of the bridging fibers to the spindle functions affected by augmin depletion remains unclear.We thank the reviewer for the thoughtful comments and are convinced that our new experiments which include the never-before-seen arrangements of microtubules, along with functional analysis of augmin-depleted cells, demonstrated that bridging fibers have a specific k-fiber independent effect on kinetochore tension and a specific role in preventing merotelic attachments.Since our revised manuscript includes both the first quantitative analysis of the human spindle using STED microscopy, as well as new mechanisms by which augmin ensures mitotic fidelity, it is no longer of interest only to cell biologists studying mitosis but also to a wider scientific community, including cell biologists, molecular biologists and biophysicists interested in microtubules, mitotic and meiotic spindles, cell division, chromosome segregation, aneuploidy, cancer, and development, as well as scientists developing quantitative superresolution imaging protocols for imaging of cellular structures.Reviewer #2 ):Summary:The authors found that the microtubules in the bridging fibres of the mitotic spindle in a human cell line are predominantly supplied via augmin-dependent nucleation. On the other hand, the contribution of augmin to kinetochore fibre formation is ~40%. Augmin-depleted cells showed reduced inter-kinetochore tension and slower poleward flux of spindle microtubules, suggesting that bridging fibres play a role in these events. This study expands our knowledge on the role of augmin and augmin-mediated microtubules in animal somatic cells.We thank the reviewer for this accurate summary of our work. In addition to the findings nicely described here, we now have an entire new section on the role of augmin in mitotic fidelity, as suggested by Reviewer 3 and presented in the new Figure 2.Major comments:– Are the key conclusions convincing?Yes.– Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?In the current manuscript, the slower flux is attributed solely to the lack of bridging fibres in the augmin-depleted cells. This is an overinterpretation, as the augmin's role in the spindle is not limited to generating bridging fibres.We modified the text on poleward flux to include the contribution of both bridging and k-fibers. We also performed new experiments on U2OS cells and included references to the new work from our lab recently published in Cell Reports , which was able to distinguish between the effect of augmin depletion on bridging and k-fiber flux. Because poleward flux was analyzed into much more detail in this study, and because we produced surprising new data regarding microtubule arrangements and segregation errors, poleward flux is no longer a major point in our revised manuscript. It is now included in the Supplementary Figure 3 along with shape analysis, as a part of the section about spindle architecture and dynamics.The text about flux was modified as follows: “Recent speckle microscopy experiments in RPE1 cells, which were able to separate the effect of augmin on poleward flux of bridging and k-fibers, revealed that both k-fibers and the remaining bridging fibers were significantly slowed down . Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells , supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules . We propose that augmin depletion results in slower flux of bridging fibers because the remaining bridging microtubules are likely nucleated at the poles, where microtubule depolymerization mechanisms might curb poleward flux speed . In contrast, PRC1 depletion does not affect the flux  even though it reduces bridging fibers , possibly because the remaining bridging microtubules are generated away from the poles via augmin and can thus flux freely.”– Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.No.– Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.N/A– Are the data and the methods presented in such a way that they can be reproducedYes.– Are the experiments adequately replicated and statistical analysis adequate?Yes.Minor comments:– Specific experimental issues that are easily addressable.None.– Are prior studies referenced appropriately?Yes.– Are the text and figures clear and accurate?1) Page 15: "To determine the curvature of the bundles, we ….. with all other bundles types ." – I could not understand this sentence well, and would like to ask for a revision.The text has now been changed to: “To gain insight into the contribution of each of these functionally distinct microtubule bundles to the maintenance of spindle geometry, we traced the outermost bundles in HAUS6 siRNA treated RPE1 cells imaged using STED microscopy and fitted a circle to the bundle outline ”.2) The following words may be too strong:Page 20: whereas k-fiber microtubules are "mainly" nucleated in an augmin-independent manner .We revised this sentence on page 24 as follows: “K-fibers were also thinner, though to a lesser extent, indicating that they are largely nucleated in an augmin-independent manner, at the centrosome or kinetochores and chromosomes.”Page 21, bottom: "demonstrates".As we changed this section of the manuscript, this is no longer applicable.– Do you have suggestions that would help the authors improve the presentation of their data and conclusions?No.Reviewer #2 (Significance (Required)):– Describe the nature and significance of the advance  for the field.The presence of bridging fibres has been recognised for decades; however, until recently, little attention has been paid to this structure from a mechanistic and functional point of view. The Tolic lab has been shedding light on this structure for the past several years. The current study represents another step forward in the research of the origin and function of bridging fibres.– Place the work in the context of the existing literature .Augmin's critical contribution to microtubule nucleation in the human somatic spindle has been well documented, as cited by the authors. The current study is the first to show that augmin also contributes to bridging fibres. The >70% contribution may be more than expected, given that centrosomal microtubules frequently reach the spindle midzone.Reduced inter-kinetochore tension has also been documented, but previous studies attributed this exclusively to reduced number of kinetochore microtubules. The current study has revised this view.We thank the reviewer for correctly pointing out the main points of our original manuscript. These results are now strengthened by new experiments, and accompanied by STED microscopy imaging and a section about the role of augmin-nucleated microtubules in the maintenance of mitotic fidelity.– State what audience might be interested in and influenced by the reported findings. Spindle researchers.Since our revised manuscript includes both the first quantitative analysis of the human spindle using STED microscopy, as well as new mechanisms by which augmin ensures mitotic fidelity, it is no longer of interest only to spindle researchers but also to a wider scientific community, including cell biologists, molecular biologists and biophysicists interested in microtubules, mitotic and meiotic spindles, cell division, chromosome segregation, aneuploidy, cancer, and development, as well as scientists developing quantitative super resolution imaging protocols for imaging of cellular structures.– Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.This review is written by a researcher who is familiar with the literature of the mitotic spindle.We thank the reviewer for an accurate summary of our work and perceptive comments.Reviewer #3 ):In this study Manenica et study how the presence of the augmin complex affects the overall spindle architecture and the different types of spindle microtubules. The authors propose that depletion of augmin affects particularly bridging microtubules, leading to their disappearance on sister-kinetochores located in the central part of the metaphase plate.Overall the manuscript is well written, clear and supported by excellent explanatory schemes. The main conclusion of the manuscript, i.e. that augmin plays an essential role in the formation of bridging microtubules is generally well supported by the data. A number of other conclusions, however, are less well supported by the data and would benefit from a number of additional experiments, repetitions or analysis. Specifically:We thank the reviewer for complimenting the clarity of our manuscript, and the data supporting the main conclusion. Other conclusions, which the reviewer states were not equally well supported by the data, are now strengthened by a new set of experiments using STED microscopy, increased repetitions (at least 30 cells from 3 independent experiments) and multiple new analyses. In addition, our revised manuscript now also includes both the first quantitative analysis of the human spindle using STED microscopy, as well as an entire new section on the mechanisms by which augmin ensures mitotic fidelity, as thoughtfully suggested below.1) Throughout all the figures the authors use a t-test, which is fine when comparing two conditions, but not for multiple experimental conditions. The authors should instead use an ANOVA test or apply a Bonferroni correction. This can strongly affect the significance of some of the reported results.We agree and have performed either ANOVA with post-hoc Tukey test or Mann-Whitney U-test instead of t-test where appropriate throughout the whole manuscript. The statistical analyses that were used are clearly stated in the captions of the figures.2) Another general concern is that the authors rely throughout the manuscript on live cell imaging data from few cells (5-10). Live cell imaging data has the advantage to avoid fixation artifacts, but the low sample size is a major concern, as for every experiment the authors rely on 10 cells (and for inter-kinetochore distances on 5) in three independent experiments overall. This means that two our of three of those independent experiments are based on 3 cells only, which is too low, given that siRNA depletions are known to be variable in their efficiencies. With such low number of cells, there is always the danger of an unconscious selection bias, which can skew a result. Just to take an example the spindle length, structure and density for HAUS-8 depleted RPE1 cells looks very different in the examples show in Figure 1B, 2A, or 5B.It is therefore essential to work with a higher sample size, at minimum 10 cells per independent experiment.This is a valid critique, which we addressed by performing 3 independent experiments with at least 10 cells each for all of our new analyses included in the main figures.3) Throughout all experiments the authors use 100nm Sir-Tubulin, which in our hands already leads to substantial changes in microtubule dynamics, as it stabilizes spindle microtubules. I understand why the authors did this, as they wanted to also stain for weak bridging fibers, but tt would be important to validate some of the obtained results with an independent approach, for example fixed-cell imaging and tubulin staining, to rule out artifacts introduced by SiR-tubulin.We thank the reviewer for this suggestion. To validate our results, we performed tubulin immunostaining, as suggested. Moreover, we imaged the immunostained cells by using super resolution STED microscopy, which significantly increased the precision of our measurements, and included these results in the main figures . The data obtained from cells stained with SiR-tubulin and imaged using live-cell confocal microscopy are now shown in Supplementary figures.4) Figure 1: Given that the authors later report that augmin affects more strongly bridging fibers in the central part of the spindle, how were the values in terms of microtubule densities obtained in the experiments in Figure 1: only on the outer microtubules, or overall in the spindle?Values in Figure 1  were obtained overall in the spindle. We described the selection in the text as follows: “We measured tubulin signal intensity of randomly selected bridging (Ib) and k-fibers (Ik) which had no other microtubules in their immediate neighborhood…”5) Figure 2: the authors conclude that depletion of augmin has a much stronger effect on the bridging fibers located in the central part of the spindle. This is a very interesting result, but it begs the question as to the origin of this difference. If the authors analyze in control cells the interkinetochore distances and the density of the bridging fibers of the kinetochores located in the central part of the metaphase plate vs those located at the outer part of the plate, do they already see a difference? In other words, is the effect of augmin due to already weaker bridging fibers in the central part of the spindle, or is the depletion effect indeed specific for those bridging fibers located in the middle. This analysis should be possible with the existing data (+ a higher sample size)This was now analyzed in the cells imaged using STED microscopy with increased resolution and a higher sample size. From our new data, it is clear that the depletion effect is indeed specific for bridging fibers located in the middle as there was no significant difference between the interkinetochore distance in the inner and the outer part of the spindles in control cells . The same trend can also be seen for bridging fiber density . We modified the text as follows: “However, we noticed that the interkinetochore distance was smaller in the inner part of the spindle in augmin-depleted cells , where bridging fibers were most severely impaired . This was not the case in control cells, which showed no difference in interkinetochore distance between the inner and the outer part of the spindle .”6) Figure 4: the authors study spindle width, length and diameter of the metaphase plate in a small number of cells (10). One concern is that these values might change as cells progress from late prometaphase to anaphase onset (metaphase plate width decreases for example). Given the low number of cells the authors do not know if they are comparing cells at similar mitotic times. To circumvent this issues, they could: either arrest the cells with MG132 for 1 hour, to obtain an endpoint, or record these different values as cells progress through mitosis and thus be able to compare similar conditions.We agree with this suggestion, and we performed new experiments by arresting the cells with MG-132: “… in HeLa  and RPE1  cells stably expressing PRC1-GFP with and without MG-132 treatment .” We measured spindle width, length and diameter of the metaphase plate in arrested RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP, RPE1 cells stably expressing PRC1-GFP, and HeLa cells stably expressing PRC1-GFP. We treated the cells with MG-132 for 30 minutes, as this was in our hands enough to arrest the cells, without causing other changes, e.g., problems with spindle orientation that occur after 1 hour of treatment. The results are now part of the Supplementary Figure S4 and are obtained from three independent experiments with at least 10 cells per experiment.7) In the discussion the authors conclude that the longer bundles and the reduction in microtubule poleward flux is due to the absence of bridging microtubules. This is an over-interpretation as augmin could in theory affect these parameters independently of the bridging microtubules, longer bundles could be generally due to the reduced number of microtubules in the k-fibers and the bridging microtubules. A better control would be to affect bridging microtubules with an independent tool, such as PRC1 depletion, and to measure these paramenters in the same RPE1 cell line, since differences can arise from cell line to cell line as the authors also document in their study .We modified the Discussion based on new results, so these statements are now in the Results section. For the long, curved bundles, we modified the sentence as follows: “These bundles likely arose either due to PRC1 crosslinking excessively long astral microtubules that were now able to reach the spindle midzone or due to PRC1 activity combined with the excess of free tubulin present as a consequence of less tubulin being incorporated in bridging and k-fibers.”Regarding the reduced poleward flux following augmin depletion, we revised the text as follows: “Recent speckle microscopy experiments in RPE1 cells, which were able to separate the effect of augmin on poleward flux of bridging and k-fibers, revealed that both k-fibers and the remaining bridging fibers were significantly slowed down . Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells , supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules . We propose that augmin depletion results in slower flux of bridging fibers because the remaining bridging microtubules are likely nucleated at the poles, where microtubule depolymerization mechanisms might curb poleward flux speed . In contrast, PRC1 depletion does not affect the flux  even though it reduces bridging fibers , possibly because the remaining bridging microtubules are generated away from the poles via augmin and can thus flux freely.”However, due to the extensive and thrilling new data regarding microtubule arrangements and segregation errors and because poleward flux was studied into much more detail in a recent Cell Reports study from our lab , this is no longer a major point in our revised manuscript. Poleward flux, along with shape analysis, is now included in the Supplementary Figure 3, as a part of the section about spindle architecture and dynamics.Minor comment:– The reported flux rate for control-depleted cells is substantially higher than the flux rates normally reported for human cells. This could be due to the experimental conditions (slight changes in temperature), but at minimum the authors should comment on this.We performed photoactivation experiments on a higher number of U2OS cells stably expressing CENP-A GFP, mCherry-tubulin and PA-tubulin . U2OS cells with labelled kinetochores and tubulin were used to exclude the potential effects of SiR-tubulin on poleward flux, as well as to better determine the position of the metaphase plate. The results in control cells are comparable to the poleward flux measured in the same cell line .Please note that, as previously stated, this is no longer a major point in our revised manuscript and can be found in Supplementary Figure 3.Reviewer #3 (Significance (Required)):The significance of the study is that the authors performed a detailed description of the effects of augmin depletion on the spindle architecture, in particular bridging fibers. Nevertheless, many of the reported results are already known (and as cited by the authors): the reduction in interkinetochore distances or the change in spindle architecture. The 3 main novel results, is the fact that augmin affects more bridging microtubules, particularly in the central part of the spindle, and that it also affect poleward microtubule flux, which limits the impact of this study to a specialized mitotic spindle audience. Nevertheless, if the authors address the reviewers concerns, this could be a nice, descriptive study for the mitotic field.One way to expand the significance of this study would be to test how augmin depletion and the lack of bridging microtubules in the central part of the metaphase plate affects chromosome segregation. Does the specific absence of bridges in this part lead to more lagging chromosomes, chromosome segregation errors, or micronuclei amongst sister chromatids located in the central part of the spindle? Is there a differential anaphase A speed for those kinetochore vs those at the periphery that still are associated to bridging fibers? Such a functional approach could allow to highlight the most interesting aspect of this study, the spatial difference in the effects of augmin depletion. Such experiments would, however, not be part of a revision, but rather a substantial enhancement of the present study.Patrick MeraldiThis is a great idea that we truly appreciate! We performed new experiments to study lagging chromosomes and indeed found that they were more often located in the inner part of the spindle in HAUS6-depleted than in control cells, which is likely due to the specific impairment of bridging fibers in that area. We also found that lagging chromosomes typically had a lower interkinetochore distance and a higher kinetochore tilt just before the onset of anaphase, which we interpret as a signature of perturbed bridging fibers. Using STED microscopy, we were able to clearly see merotelic attachments at kinetochores lacking a bridging fiber in metaphase following augmin depletion. Thus, we propose that augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled microtubule arrangement unfavorable for creating additional attachments. On the other hand, augmin-nucleated k-fibers produce robust force required to resolve errors during anaphase. We dedicated an entire new section on pages 6-11 and a new Figure 2 to these exciting new results.We agree with the Reviewer 3 that these results substantially increase the significance of this study, which is now no longer descriptive nor of interest only to the mitotic field, but offers a new mechanism that safeguards mitotic fidelity, which makes it of interest to a wider scientific community. This includes cell biologists, molecular biologists and biophysicists interested in microtubules, mitotic and meiotic spindles, cell division, chromosome segregation, aneuploidy, cancer, and development, as well as scientists developing quantitative superresolution imaging protocols for imaging of cellular structures.We are thankful to the reviewer for raising such an interesting question, and motivating us to use a functional approach to study the mechanism by which the observed effects of augmin depletion compromise mitotic fidelity.