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.

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.

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Maintaining the integrity of the mitotic spindle in metaphase is essential to ensure normal cell division. We show here that depletion of microtubule-associated protein ATIP3 reduces metaphase spindle length. Mass spectrometry analyses identiied the microtubule minus-end depolymerizing kinesin Kif2A as an ATIP3 binding protein. We show that ATIP3 controls metaphase spindle length by interacting with Kif2A and its partner Dda3 in an Aurora kinase A-dependent manner. In the absence of ATIP3, Kif2A and Dda3 accumulate at spindle poles, which is consistent with reduced poleward microtubule lux and shortening of the spindle. ATIP3 silencing also limits Aurora A localization to the poles. Transfection of GFP-Aurora A, but not kinase-dead mutant, rescues the phenotype, indicating that ATIP3 maintains Aurora A activity on the poles to control Kif2A targeting and spindle size. Collectively, these data emphasize the pivotal role of Aurora kinase A and its mutual regulation with ATIP3 in controlling spindle length.

Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores (KTs) that are built on the centromeric region of sister chromatids and serve to capture mitotic spindle microtubules (MTs). In early mitosis, unattached KTs expand a crescent-shaped structure called fibrous corona whose function is to facilitate initial KT-MT attachments and chromosome transport by MTs. Subsequently, the fibrous corona must be timely disassembled to prevent segregation errors. Although recent studies provided new insights on the molecular content and mechanism of fibrous corona assembly, it remains unknown what triggers the disassembly of the outermost and dynamic layer of the KT. Here, we show that Aurora A and B kinases (AurA and AurB) phosphorylate CENP-E to release it from an autoinhibited state. At KTs, AurB phosphorylates CENP-E to prevent its premature removal together with other corona proteins by dynein. At the spindle poles, AurA phosphorylates CENP-E to promote chromosome congression and prevent accumulation of corona proteins at the centrosomes, allowing for their intracellular redistribution. Thus, the AurA/B-CENP-E axis establishes the long-sought mechanism of fibrous corona disassembly that is essential for accurate chromosome segregation.