Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Long, Alexandra F.; Suresh, Pooja; Dumont, Sophie

doi:10.1101/846154

Cited by 9 publications

(10 citation statements)

References 78 publications

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“…Thus, MT-flux might regulate the fine balance between MCAK-dependent depolymerization and CLASPs-promoting polymerization of k-fibers to ensure the steady-state length of the mitotic spindle. Interestingly, a recent elegant study used microneedlebased mechanical manipulation to pull on photomarked k-fibers and showed that the applied pulling force on KTs suppressed depolymerization of k-fibers, resulting in their elongation via MT plus-end polymerization (Long et al, 2020). Likewise, the sliding activities of four MT-flux driving motors might exert similar forces on KTs to promote MT plus-end polymerization.…”

Section: Microtubule Fluxmentioning

confidence: 99%

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

et al. 2020

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Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MTdynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/ KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MTflux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MTdepolymerization and regulate spindle length.

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Section: Microtubule Fluxmentioning

confidence: 99%

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

et al. 2020

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“…Furthermore, evidence also suggests that lateral cross-links along the length of the k-fiber help to reinforce the k-fiber and alter its mechanical properties. K-fiber bundles appear to be much straighter and stiffer than individual microtubules (Goshima et al, 2005) and rupture collectively when subjected to sufficient force (Long et al, 2020).…”

Section: Introductionmentioning

confidence: 96%

“…K-fiber bundles appear to be much straighter and stiffer than individual microtubules ( Goshima et al. , 2005 ) and rupture collectively when subjected to sufficient force ( Long et al. , 2020 ).…”

Section: Introductionmentioning

confidence: 99%

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

Begley

Solon

Davis

et al. 2021

MBoC

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The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

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“…This drop, together with the action of the phosphatase PPP2CA, causes NuMA's phosphorylation state to drop. This precipitates the translocation of significant amounts of NuMA from spindle poles and the spindle to the cortex, where it, together with bound dynein, drives spindle elongation during anaphase [66][67][68][69][70]. Given all this, one would predict that in the absence of Myo10-dependent WEE1 sequestration (i.e.…”

Section: Discussionmentioning

confidence: 99%

Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering

Yim

Pedrosa

et al. 2022

Preprint

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Myosin 10 (Myo10) is a member of the MyTH4/FERM domain family of unconventional, actin-based motor proteins. Studies have implicated Myo10 in supporting cell adhesion via its integrin-binding FERM domain, and spindle positioning and spindle pole integrity via its microtubule-binding MyTH4 domain. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from a Myo10 knockout mouse. Most notably, both of these knockout cells exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is PCM fragmentation, which creates y-tubulin-positive, centriole-negative microtubule asters that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing centrosome clustering. These results argue, therefore, that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Finally, we present evidence that the defect in spindle pole integrity in Myo10 knockout cells is likely due to a defect in pole stability rather than pole maturation, and that Myo10 promotes supernumerary centrosome clustering at least in part by promoting cell adhesion during mitosis.

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Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Cited by 9 publications

References 78 publications

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering

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