Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

Abstract

Summary Proper chromosome segregation into two future daughter cells requires the mitotic spindle to elongate in anaphase. However, although some candidate proteins are implicated in this process, the molecular mechanism that drives spindle elongation in human cells is unknown. Using combined depletion and inactivation assays together with CRISPR technology to explore redundancy between multiple targets, we discovered that the force-generating mechanism of spindle elongation consists of EG5/kinesin-5 together with the PRC1-dependent motor KIF4A/kinesin-4, with contribution from kinesin-6 and kinesin-8. Disruption of EG5 and KIF4A leads to total failure of chromosome segregation due to blocked spindle elongation, despite poleward chromosome motion. Tubulin photoactivation, stimulated emission depletion (STED), and expansion microscopy show that perturbation of both proteins impairs midzone microtubule sliding without affecting microtubule stability. Thus, two mechanistically distinct sliding modules, one based on a self-sustained and the other on a crosslinker-assisted motor, power the mechanism that drives spindle elongation in human cells.

An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors

“…These findings are at odds with the live-cell data presented here and are difficult to reconcile with the fact that Mklp2 is required for Kif4A accumulation at the spindle midzone ( Nunes Bastos et al., 2013 ; see also Figures S3 E–S3G), and Kif4A mediates Aurora B recruitment to this location ( Kurasawa et al., 2004 ). Moreover, our close inspection of spindle midzone MTs in the absence of Mklp2 by super-resolution CH-STED microscopy ( Pereira et al., 2019 ) failed to detect significant differences in MT density in the vicinity of anaphase lagging chromosomes (as well as any significant effect on non-KT MT half-life and spindle elongation capacity, as inferred from spinning disk confocal recordings), in agreement with recent expansion microscopy analysis of human cells depleted of PRC1, which is required for Kif4A recruitment to midzone MTs ( Kurasawa et al., 2004 ; Vukušić et al., 2021 ). One possibility is that Kif4A spatially controls the completion of NER on lagging chromosomes by regulating condensin-I-mediated chromosome condensation ( Afonso et al., 2014 ; Mazumdar et al., 2004 ; Poonperm et al., 2017 ; Samejima et al., 2012 ; Takahashi et al., 2016 ).…”

Coordinated poleward flux of sister kinetochore fibers drives chromosome alignment

“…3.6 ± 0.3 µm/min, given that the spindle length is constant during metaphase. This rate is comparable to the sliding rate of bridging microtubules in early anaphase measured by tubulin photoactivation, which is roughly 4.5 µm/min (Vukušić et al, 2021), suggesting that the bridging microtubule sliding may be driven by a similar mechanism in metaphase and early anaphase.…”

Astral microtubule crosslinking safeguards uniform nuclear distribution in the Drosophila syncytium

“…In a sliding assay of taxol-stabilized microtubules in which microtubules in solution and glass-immobilized microtubules form pairs cross-linked by Prc1, the antiparallel pairs of microtubules are slid apart by Kif4 (Wijeratne and Subramanian, 2018). This is reminiscent of plus-end directed sliding of Kinesin-5 (Eg5) (Kapitein et al, 2005) and explains the requirement of Prc1 orthologs for spindle elongation in several species (Khmelinskii et al, 2009;Schuyler et al, 2003;Vukušić et al, 2021;Wang et al, 2015;Zhu et al, 2006). Indeed, plus-end overlapping microtubules have an apparent mechanical stiffness that is governed by molecular friction and motor activity (Forth et al, 2014;Wijeratne and Subramanian, 2018).…”

“…Still, Klp61F and Feo can functionally cooperate in crosslinking astral microtubules because both proteins recognize and bind to microtubule pairs, though with different preference for microtubule orientation (Bieling et al, 2010;Kapitein et al, 2005; E. H. Kellogg et al, 2016;Subramanian et al, 2010). In human cells, Prc1-dependent Kif4A motor activity and the microtubule sliding by Eg5 are redundant for spindle elongation during anaphase (Vukušić et al, 2021). Interestingly, in Drosophila, while Feo modulates binding and localization of Klp61F at the spindle midzone in anaphase, Klp61F cannot functionally rescue the absence of Feo (Wang et al, 2015).…”