Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

Risteski, Patrik; Božan, Domagoj; Jagrić, Mihaela; Bosilj, Agneza; Pavin, Nenad; Tolić, Iva M.

doi:10.1016/j.celrep.2022.111169

Cited by 15 publications

(38 citation statements)

References 63 publications

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“…S3L-M), in agreement with findings in Indian Muntjac cells (Almeida et al, 2022). 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 (Risteski et al, 2022). Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells (Risteski et al, 2022), supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules (Brust-Mascher et al, 2009; Mitchison, 2005; Miyamoto et al, 2004).…”

Section: Resultsmentioning

confidence: 99%

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Manenica

Koprivec

Štimac

et al. 2020

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Manenica

Koprivec

Štimac

et al. 2020

Preprint

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“…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 ( Ganem et al, 2005 ). In contrast, PRC1 depletion does not affect the flux ( Risteski et al, 2022 ; Steblyanko et al, 2020 ) even though it reduces bridging fibers ( Kajtez et al, 2016 ; Polak et al, 2017 ), possibly because the remaining bridging microtubules are generated away from the poles via augmin and can thus flux freely. In sum, augmin ensures proper architecture and dynamics of the metaphase spindle largely through the nucleation of bridging fibers, which link sister k-fibers and ensure their proper shape and function.…”

Section: Resultsmentioning

confidence: 95%

“…In 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 ( Figure 3—figure supplement 1L-M ), in agreement with findings in Indian Muntjac cells ( Almeida et al, 2022 ). 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 ( Risteski et al, 2022 ). Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells ( Risteski et al, 2022 ), supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules ( Brust-Mascher et al, 2009 ; Mitchison, 2005 ; Miyamoto et al, 2004 ).…”

Section: Resultsmentioning

confidence: 99%

“…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 ( Risteski et al, 2022 ). Bridging fibers fluxed faster than k-fibers in control and augmin-depleted cells ( Risteski et al, 2022 ), supporting the model in which poleward flux is largely driven by sliding apart of antiparallel microtubules ( Brust-Mascher et al, 2009 ; Mitchison, 2005 ; Miyamoto et al, 2004 ). 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 ( Ganem et al, 2005 ).…”

Section: Resultsmentioning

confidence: 99%

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Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Štimac

Koprivec

Manenica

et al. 2022

eLife

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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.

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“…Variants were proposed by sliding bridging fibres along each other (Jagric et al, 2021). The motion of these fibres is transmitted to other microtubules, including kMT and sMTs, by cross-linking agents or motors like HSET KLP-15/16/17 , NuMA LIN-5 or PRC1 SPD-1 (Elting et al, 2014; Risteski et al, 2022; Steblyanko et al, 2020). Secondly, chromokinesin KIF4A KLP-19 congresses the chromosome arms and generates a reaction force that pushes the kMTs away from the chromosomes (Steblyanko et al, 2020; Wandke et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Kinetochore microtubules flux poleward along fixed centrosome-anchored microtubules during the metaphase ofC. elegansone-cell embryo

Soler

Chesneau

Bouvrais

et al. 2022

Preprint

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The microtubule array, assembled into the mitotic spindle, polymerises from the centrosomes and the chromosomes in many organisms. Their plus ends alternate between growing and shrinking. This dynamic instability plays a key role in pulling on the kinetochores to check the spindle assembly and correct the errors in chromosome attachments. In addition, the minus ends at centrosomes can undergo depolymerisation coordinated with the polymerisation of the plus ends at the kinetochores. Such a mechanism, among others, creates treadmilling,id esta net poleward movement of microtubules called poleward flux. This flux is involved in many roles, chromosome congression in prometaphase, chromosome misattachment detection and correction, spindle length maintenance in metaphase, and synchronous segregation of sister chromatids in anaphase. Interestingly, no poleward flux was measured in theCaenorhabditis eleganssingle-cell embryo, despite it is equipped with all homologous proteins involved in this mechanism in other organisms. To investigate this peculiarity, we labelled the microtubules and photobleached them in a rectangular region. Surprisingly, we observed that both edges of the bleached zone (fronts) move inwards, closing the dark area. However, the middle of the bleached zone does not move clearly, confirming the absence of a global poleward flow. The dynamics of the microtubules emanating from the centrosomes combined with the diffraction due to microscopy imaging account for the apparent movement of the front on the centrosome side. Therefore, we suggest no flux of the centrosome-anchored (spindle) microtubules. In contrast, on the chromosome side, we observed a front moving poleward, faster than the one on the other side, and dependent on proteins ensuring the attachment and growth of microtubules at kinetochores, NDC-80, CLS-2CLASP, and ZYG-9XMAP215. Besides, we found that the depletion of the depolymerase KLP-7MCAKdoes not impair this poleward recovery. Finally, the faster recovery is restricted to the spindle region close to the chromosomes. Therefore, we suggest that the kinetochore microtubules undergo a poleward flux, moving with respect to spindle microtubules. Because the kinetochore microtubules are shorter than the half-spindle, this flux is localised close to the chromosomes. Furthermore, it may not rely on treadmilling as KLP-7MCAKis dispensable. This spatially restricted flux found in the nematode may be related to the slow elongation of the spindle during metaphase and may buffer the strong pulling forces exerted by the cortical force generators at the spindle poles.

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Length-dependent poleward flux of sister kinetochore fibers promotes chromosome alignment

Cited by 15 publications

References 63 publications

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers

Kinetochore microtubules flux poleward along fixed centrosome-anchored microtubules during the metaphase ofC. elegansone-cell embryo

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