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Kinesin-13 motors regulate precise microtubule dynamics and limit microtubule length throughout metazoans by depolymerizing microtubule ends. Recently, the kinesin-13 motor family member MCAK (also known Kif2C) has been proposed to undergo large conformational changes during its catalytic cycle, as it switches from being in solution to being bound to microtubules. Here, we reveal that MCAK has a compact conformation in solution through crosslinking and electron microscopy experiments. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N-terminus and neck region of MCAK interacting with the microtubule. Interestingly, the region of MCAK that interacts with the microtubule is the region phosphorylated by Aurora B and contains an end binding (EB) protein-binding motif. The level of phosphorylation of the N-terminus results in a graded microtubule depolymerase activity. Here, we show that the N-terminus of MCAK forms a platform to integrate Aurora B kinase downstream signals and in response fine-tunes its depolymerase activity during mitosis. We propose that this allosteric control mechanism allows decoupling of the N-terminus from the motor domain of MCAK to allow MCAK depolymerase activity at kinetochores., Summary: The kinesin-13 MCAK has a compact conformation in solution but is extended when bound to microtubules. Aurora B phosphorylation of MCAK inhibits depolymerase activity by disrupting its extended conformation.

Kinesin-13 motors regulate precise microtubule dynamics and limit microtubulelength throughout metazoans by depolymerizing microtubule ends. Recently,MCAK has been proposed to undergo large conformational changes during itscatalytic cycle, as it switches from solution to bound state. Here, we reveal thatMCAK has a compact conformation in solution using crosslinking and electronmicroscopy. When MCAK is bound to the microtubule ends, it adopts anextended conformation with the N terminus and neck region of MCAK interactingwith the microtubule. Interestingly, the region of MCAK that interacts with themicrotubule is the region phosphorylated by Aurora B and contains an EBbindingmotif. The level of phosphorylation of the N terminus results in a gradedmicrotubule depolymerase activity. Here we show the N terminus of MCAK formsa platform to integrate Aurora B kinase downstream signals and in response finetunes its depolymerase activity during mitosis. We propose that this allosteric control mechanism allows decoupling of the N terminus from the motor domain of MCAK to allow MCAK depolymerase activity at kinetochores.

Kinesin-12 is a motor protein that has a role in the processes of mitotic spindle formation and maintenance. The human Kinesin-12, Kif15, has been shown to have some functional redundancy with Eg5, a Kinesin-5 that plays key roles in the formation of the bipolar spindle and is a potential target for anti-cancer drugs. Eg5 is thought to contribute to spindle formation by cross-linking and sliding microtubules, however little is known about the mechanism of Kif15.\ud \ud We have used laser tweezers to investigate the mechanical properties of Kif15 compared to those of kinesin-1. We have found that Kif15 is plus end directed and takes multiple steps along the microtubule without detaching. Full-length Kif15 walks faster and supports more load than full-length Eg5. Kif15 is less processive under load than kinesin-1, although it has a similar stall force. A second, diffusive, microtubule binding site in Kif15 supports processivity at zero load, and slows flyback following a detachment in the optical trap.\ud \ud The microtubule-associated protein, Tpx2, is necessary for the localisation of Kif15 to spindle microtubules. We find that Tpx2 binding arrests the motion of Kif15 and creates a stable binding state that resists both assisting and hindering loads. We also find evidence of a tail-mediated auto-inhibitory mechanism that creates a stable MT binding state and causes pausing during processive runs. C-terminal truncation of the Kif15 tail relieves this inhibition leading to faster overall stepping and abrogates the effects of Tpx2.\ud \ud We examined the detachment behaviour of Kif15 from microtubules, under assisting and hindering loads. We find that assisting loads cause single Kif15 and Kinesin-1 motors to detach from the microtubule more easily than hindering loads. Kif15 shows a much more asymmetric response to load in low levels of ATP than Kinesin-1, and both show more asymmetry than Eg5: previous work has shown that the behaviour of Eg5 does not change dramatically with differing loading directions. This has interesting implications for the roles of Kif15 and Eg5 motors in both parallel and anti-parallel microtubule bundles. Overall our data supports an in vivo mechanism for Kif15 that it distinct from that of Eg5.\ud \ud We investigated the load-dependent detachment of Kinesin-1 and Kif15 in millimolar concentrations of ADP, AMPPNP and micromolar concentrations of ATP. Kinesin-1 in ADP detached at low loads, and in AMPPNP at two different loads, both higher than in ADP. These two AMPPNP states of Kinesin1 likely corresponding to single and double headed microtubule binding, as proposed by Ishiwata and colleagues. Kif15 behaved broadly similarly. At micromolar ATP concentrations and hindering loads, both Kinesin-1 and Kif15 again showed two different high load detachment states. This is inconsistent with the model proposed by Ishiwata and possible modifications are discussed.

Centromere association of the chromosomal passenger complex (CPC; Borealin-Survivin-INCENP-Aurora B) and Sgo1 is crucial for chromosome biorientation, a process essential for error-free chromosome segregation. Phosphorylated histone H3 Thr3 (H3T3ph; directly recognized by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognized via Sgo1), together with CPC’s intrinsic nucleosome-binding ability, facilitate CPC centromere recruitment. However, the molecular basis for CPC–Sgo1 binding and how their physical interaction influences CPC centromere localization are lacking. Here, using an integrative structure-function approach, we show that the “histone H3-like” Sgo1 N-terminal tail-Survivin BIR domain interaction acts as a hotspot essential for CPC–Sgo1 assembly, while downstream Sgo1 residues and Borealin contribute for high-affinity binding. Disrupting Sgo1–Survivin interaction abolished CPC–Sgo1 assembly and perturbed CPC centromere localization and function. Our findings reveal that Sgo1 and H3T3ph use the same surface on Survivin to bind CPC. Hence, it is likely that these interactions take place in a spatiotemporally restricted manner, providing a rationale for the Sgo1-mediated “kinetochore-proximal” CPC centromere pool.