Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces

Abstract

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

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

“…Our observation that the effect of KIF18A depletion on spindle elongation velocity could be modulated further by EG5 inhibition indicates that EG5 and KIF18A could form a functionally redundant partnership during anaphase B, although not to the same extent as KIF4A and EG5, as spindle elongation was not blocked after combined KIF18A-EG5 perturbations. KIF18A was recently reported to localize to metaphase bridging microtubules ( Jagrić et al., 2021 ), and we also observed clear localization of KIF18A to the spindle midzone from anaphase onset, similar to a previous study ( Stumpff et al., 2012 ). Kinesin-8 Kip3 has been reported to slide antiparallel microtubules during mitosis in the budding yeast ( Su et al., 2013 ), indicating a possible similar role of the human homolog KIF18A that could be an interesting topic of future studies.…”

“…Similarly, efficient depletion of KIF18A/kinesin-8 by 24 h siRNA treatment ( Figures S2 A and S2B), a microtubule dynamics regulator, which localizes to the antiparallel bridging microtubules that link sister k-fibers during metaphase ( Jagrić et al., 2021 ; Kajtez et al., 2016 ), early ( Figure S2 A) and late anaphase ( Stumpff et al., 2008 ), and slides microtubules in yeast ( Su et al., 2013 ), did not impact spindle elongation during early anaphase ( Figures 1 D and S1 A–S1C). Also, efficient depletion of the minus-end-directed HSET/KIFC1/kinesin-14 ( Figures S2 A and S2B), previously implicated in the maintenance of stable midzone structure during cytokinesis ( Cai et al., 2010 ), did not affect spindle elongation ( Figures 1 D and S1 A–S1C).…”

Coordinated poleward flux of sister kinetochore fibers drives chromosome alignment

“…Because our experiments provide the first measurement of poleward flux of bridging microtubules in human spindles, we decided to validate our method of identification of speckles in the bridging fiber. We used PRC1 siRNA, which is known to specifically reduce the number of bridging microtubule to ∼50% of the original number (Jagrić et al, 2021; Polak et al, 2017). In cells treated with PRC1 siRNA, we observed 10 speckles on bridging microtubules in 11 cells, which is roughly a 2-fold reduction in comparison with untreated cells, where 43 speckles were observed in 27 cells, providing support for our method of identifications of speckles on bridging fibers ( Fig.…”

“…How poleward flux of interpolar microtubules transmitted to k-fibers regulates forces acting on kinetochores has been explored in a theoretical model, which suggests that flux promotes tension uniformity on kinetochores, in agreement with experiments showing large variability in kinetochore tension in cells with abolished flux (Matos et al, 2009). Interestingly, physical coupling between k-fibers and the associated interpolar bundles (bridging fibers) is important not only for tension but also for chromosome alignment, given that optogenetic perturbation of bridging fibers led to chromosome misalignment (Jagrić et al, 2021). In these experiments, chromosome misalignment was accompanied by elongation of bridging microtubule overlaps.…”

Hyaluronan‐Mediated Motility Receptor Governs Chromosome Segregation by Regulating Microtubules Sliding Within the Bridging Fiber

“…Loss of bridging fiber induced a thicker metaphase plate width, which indicated a reduced chromosome alignment. [ 95 ] These studies suggest that bridging fiber plays different roles in different mitotic stages. Here in our study, apart from the reduced MTs sliding and suppressed segregation in anaphase, we also found the misaligned kinetochore in the HMMR‐depleted cells during metaphase, suggesting that HMMR may be a regulator of the bridging fiber in chromosome alignment.…”