Small molecule allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin-5 during mitotic spindle assembly

Abstract

Human Kinesin-5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small-molecule inhibitors of Eg5 are candidate anti-cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S-trityl-L-cysteine inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity. Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5-targeting inhibitors of Kinesin-5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three-dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.

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

“…We argue that direct pushing by plus-end polymerization is excluded as a major force driving spindle elongation because KIF4A and EG5 promote microtubule depolymerization ( Kim et al., 2019 ; Bringmann et al., 2004 ); thus, in their absence microtubules are expected to polymerize more, although this was not found in our experiments, and promote spindle elongation as reported during late anaphase ( Hu et al., 2011 ), which is the opposite from the block of spindle elongation we observed. Similarly, cortical forces exerted by dynein on astral microtubules most likely do not drive spindle elongation in early anaphase, based on our results that astral microtubules were not perturbed when spindle elongation was blocked, and on the fact that the role of dynein in chromosome segregation is restricted to late anaphase events, as shown by perturbation of specific anaphase cortical adaptors of dynein ( Kiyomitsu and Cheeseman, 2013 ).…”

“…Moreover, chromosome segregation and spindle elongation velocities strongly correlated with sliding rates across conditions ( Figures 7 C, 7D, S7 D, and S7E), which suggests that the origin of blocked spindle elongation seen after perturbations of KIF4A and EG5 is a result of a defective microtubule sliding. Given that human KIF4A and EG5 promote microtubule depolymerization ( Kim et al., 2019 ; Bringmann et al., 2004 ), it is unlikely that these proteins drive spindle elongation via microtubule polymerization forces ( Figure 1 A, right) because their depletion is expected to promote spindle elongation rather than spindle elongation block as observed here. We conclude that KIF4A and EG5 drive anaphase spindle elongation together by moving along the antiparallel microtubules in the midzone to push them apart ( Figures 1 A left and 7 E).…”

“…Moreover, double perturbation often resulted in catastrophic failure of chromosome segregation by either complete absence of cytokinesis ( Figure S3 F) or transfer of the whole spindle to one daughter cell during the ongoing furrowing process ( Figure 3 C), similar to what we observed after PRC1 KO and EG5 inhibition ( Figures 2 C and S2 N). In KIF4A-depleted cells, the blocked spindle elongation phenotype was also observed when EG5 was inhibited by using 100-μM monastrol ( Figures 3 A, 3B, S1 A, and S3 D) ( Mayer et al., 1999 ), which unlike STLC does not inhibit microtubule-depolymerizing activity of EG5 ( Kim et al., 2019 ), indicating that the EG5 role during anaphase B is mediated by its motility rather than microtubule-depolymerizing function. Interestingly, spindle elongation could be reactivated when STLC was washed out during early anaphase, suggesting that the block in spindle elongation is reversible ( Figures S3 G and S3H).…”

Integrated Machine Learning and Chemoinformatics-Based Screening of Mycotic Compounds against Kinesin Spindle ProteinEg5 for Lung Cancer Therapy

“…Kinesin superfamily proteins or KIF11/Eg5 are microtubule-based motor proteins that generate directional movement along microtubules. KIFs are core proteins, and these are not only essential for intracellular transport but are also important for various cellular and morphology functions [ 7 ]. KIF11/Eg5 is also identified as a prognostic factor.…”

Antitumor Potential of the Isoflavonoids (+)- and (−)-2,3,9-Trimethoxypterocarpan: Mechanism-of-Action Studies

“…Such properties resemble those reported for monastrol (MON), a synthetically derived hetereocycle that targets the kinesin-5 or kinesin spindle protein Eg5. This protein plays an important role, inter alia, in the establishment of spindle bipolarity, a key facet of effective cell division. , As such, inhibitors of Eg5 are of considerable interest as potential therapeutic agents. , …”