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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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 MT dynamics 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. We show that both of these activities work in coordination with MTcrosslinking motors kinesin-5/EG5 and kinesin-12/KIF15. Our data further indicate that MT-flux driving force is transmitted from non-KT MTs to KT-MTs via MT-coupling by HSET and NuMA.Moreover, we found that MT-flux rate correlates with spindle size and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we revealed that flux is required to counteract the kinesin 13/MCAK-dependent MT-depolymerization to regulate spindle length.Thus, our study demonstrates that MT-flux in human cells is driven by the coordinated action of four kinesins, and is required to regulate mitotic spindle size in response to MCAK-mediated MTdepolymerizing activity at KTs. Keywords Kinesins / Kinetochore / Microtubules / Mitosis / Mitotic spindleWe thank Duane Compton and Rene Medema for providing the U2OS PA-GFP-tubulin and U2OS PA-GFP-tubulin/mCherry-tubulin cell lines, respectively. We thank Claire Walczak for providing the GFP-HSET and GFP-HSET N593K plasmids. We thank Martina Barisic for technical assistance. Author contributions YS, GR, AJP, HM and MB designed experiments; YS generated tools and performed and analyzed most of the experiments; YS and MB performed photoactivation experiments, image quantification and analysis. GR performed and analyzed the spindle size-related experiments. MO performed and analyzed initial photoactivation experiments; YS, AJP and HM performed CH-STED experiments and analysis; SG provided reagents; SE contributed to designing and analyzing the experiments; MB, YS and GR wrote the manuscript, with contributions from all authors; MB conceived and coordinated the project.

Incorrect kinetochore–microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination—a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore–microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore–microtubule attachments, thereby promoting mitotic fidelity.

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Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

Microtubule-targeting agents (MTAs) have been used for decades to treat different hematologic and solid cancers. The mode of action of these drugs mainly relies on their ability to bind tubulin subunits and/or microtubules and interfere with microtubule dynamics. In addition to its MTH1-inhibiting activity, TH588 has been recently identified as an MTA, whose anticancer properties were shown to largely depend on its microtubule-targeting ability. Although TH588 inhibited tubulin polymerization in vitro and reduced microtubule plus-end mobility in interphase cells, its effect on microtubule dynamics within the mitotic spindle of dividing cells remained unknown. Here, we performed an in-depth analysis of the impact of TH588 on spindle-associated microtubules and compared it to the effect of low-dose nocodazole. We show that both treatments reduce microtubule turnover within the mitotic spindle. This microtubule-stabilizing effect leads to premature formation of kinetochore-microtubule end-on attachments on uncongressed chromosomes, which consequently cannot be transported to the cell equator, thereby delaying cell division and leading to cell death or division with uncongressed chromosomes.

Continuous poleward motion of microtubules in metazoan mitotic spindles has been fascinating generations of cell biologists over the last several decades. In human cells, this so-called poleward flux was recently shown to be driven by the coordinated action of four mitotic kinesins. The sliding activities of kinesin-5/EG5 and kinesin-12/KIF15 are sequentially supported by kinesin-7/CENP-E at kinetochores and kinesin-4/KIF4A on chromosome arms, with the individual contributions peaking during prometaphase and metaphase, respectively. Although recent data elucidate the molecular mechanism underlying this cellular phenomenon, the functional roles of microtubule poleward flux during cell division remain largely elusive. Here, we discuss potential contribution of microtubule flux engine to various essential processes at different stages of mitosis.