Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

Jagrić, Mihaela; Risteski, Patrik; Martinčić, Jelena; Milas, Ana; Tolić, Iva Marija

doi:10.1101/865394

Cited by 6 publications

(19 citation statements)

References 81 publications

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“…Treatment of cells with a microtubule-destabilizing agent that results in thinner k-fibers causes a reduction of the interkinetochore tension (Dudka et al, 2018), supporting the former possibility. However, a similar effect on interkinetochore tension was observed upon perturbation of the bridging fiber by removing the microtubule crosslinker PRC1 (Jagrić et al, 2020;Kajtez et al, 2016;Polak et al, 2017), in agreement with the latter possibility. Our observation that the interkinetochore tension was most severely affected in the inner part of the spindle, where the bridging fibers were most impaired, suggests a link between interkinetochore tension and augmin-generated bridging microtubules.…”

Section: Discussionsupporting

confidence: 79%

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: Discussionsupporting

confidence: 79%

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 found that inactivation of Eg5 or depletion of Kif18A abolished the left-handed twist, with presence of right-handed twist in a subset of spindles after Kif18A depletion. Both of these motors are known to exert torque on the microtubules in vitro (Bormuth et al, 2012;Mitra et al, 2018;Yajima et al, 2008), and are found within the antiparallel overlaps of bridging microtubules in the spindle (Jagrić et al, 2020;Kajtez et al, 2016;Mann and Wadsworth, 2018). Thus, we suggest that they generate the twisted shape of the bundle by rotating the antiparallel microtubules within the bundle around each other (Fig.…”

Section: Mechanisms That Generate Spindle Twistmentioning

confidence: 73%

“…As it was previously shown, removal of the PRC1 from the spindle partially disassembles bridging fibers and affects spindle shape (Jagrić et al, 2020). Without PRC1, spindles have less curved and more diamond-like shape (Jagrić et al, 2020;Kajtez et al, 2016), which led us to hypothesize that the twist might also be affected. Surprisingly, when we depleted PRC1, the spindles had right-handed twist of 0.26 ± 0.15 °/µm (n = 14) ( Fig.…”

Section: Depletion Of Prc1 Makes the Spindles Twist In A Right-handedmentioning

confidence: 79%

“…PRC1 protein is a key regulator of cytokinesis, but also a main crosslinking protein of antiparallel microtubules (Kajtez et al, 2016;Polak et al, 2017). As it was previously shown, removal of the PRC1 from the spindle partially disassembles bridging fibers and affects spindle shape (Jagrić et al, 2020). Without PRC1, spindles have less curved and more diamond-like shape (Jagrić et al, 2020;Kajtez et al, 2016), which led us to hypothesize that the twist might also be affected.…”

Section: Depletion Of Prc1 Makes the Spindles Twist In A Right-handedmentioning

confidence: 88%

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The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Trupinić

Kokanović

Ponjavić

et al. 2020

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Mechanical forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for the movement of chromosomes and their segregation into the emerging daughter cells. In addition to linear forces, rotational forces are present in the spindle, reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the molecular origins of spindle chirality are unknown. Here we show that spindles are most twisted at the beginning of anaphase, and reveal multiple molecular players involved in spindle chirality. Inhibition of Eg5/kinesin-5 in a non-cancer cell line abolished spindle twist and depletion of Kif18A/kinesin-8 resulted in a right-handed twist, implying that these motors regulate twist likely by rotating the microtubules around one another within the antiparallel overlaps of bridging fibers. Depletion of the crosslinker PRC1 resulted in a right-handed twist, indicating that PRC1 may contribute to the twist by constraining free rotation of microtubules. Overexpression of PRC1 abolished twist, possibly due to increased torsional rigidity of the bundles. Depletion of augmin led to a right-handed twist, suggesting that twist depends on the geometry of microtubule nucleation. Round spindles were more twisted than elongated ones, a notion that we directly tested by compressing the spindle along its axis, which resulted in stronger left-handed twist, indicating a correlation between bending moments and twist. We conclude that spindle twist is controlled by multiple molecular mechanisms acting at different locations within the spindle as well as forces, and propose a potential physiological role of twist in promoting passive mechanical response of the spindle to forces during metaphase.

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“…To perturb specifically bridging fibers, we depleted PRC1 ( Fig. S1), which results in ~50% decrease of the number of microtubules in the bridging fiber without affecting the length of their antiparallel overlaps (Jagrić et al, 2020). We found that depletion of PRC1 did not change the flux velocity of bridging or kinetochore microtubules (Fig.…”

Section: Sliding Forces From the Bridging Fiber Are Transmitted Onto mentioning

confidence: 91%

Coordinated poleward flux of sister kinetochore fibers drives chromosome alignment

Risteski

Božan

Jagrić

et al. 2021

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Chromosome alignment at the spindle equator during metaphase is the most remarkable feature of mitosis, which promotes proper chromosome segregation and depends on the forces exerted at the plus end of kinetochore microtubules and polar ejection forces. However, forces arising from lateral mechanical coupling of kinetochore fibers with non-kinetochore microtubules play a role in chromosome alignment, but the mechanism is unclear. Here we develop a speckle microscopy assay to measure the poleward flux of individual microtubules in spindles of human cells and show that bridging microtubules slide apart and undergo poleward flux at a higher rate than kinetochore microtubules. Depletion of the microtubule coupler NuMa increased the difference in the flux velocity of kinetochore and bridging microtubules, suggesting that sliding forces from the bridging fiber are transmitted largely through NuMa onto the associated kinetochore fibers. Depletions of Kif18A/kinesin-8, Kif4A/kinesin-4, as well as double depletions of Kif18A together with Kif4A or Kif18A together with the crosslinker of antiparallel microtubules PRC1 increased the flux velocity of kinetochore fibers up to the velocity of bridging fibers, due to the increased coupling resulting from the extended antiparallel overlap regions. We found severe kinetochore misalignment after double Kif18A and Kif4A as well as Kif18A and PRC1 depletions compared to a single Kif18A depletion, suggesting that forces within the bridging fiber have a centering effect on the kinetochores. We propose that lateral length-dependent sliding forces that the bridging fiber exerts onto kinetochore fibers drive the movement of kinetochores towards the spindle center, thereby promoting chromosome alignment.

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Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

Cited by 6 publications

References 81 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

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Coordinated poleward flux of sister kinetochore fibers drives chromosome alignment

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