Eg5 Inhibitors have Contrasting Effects on Microtubule Stability and Spindle Integrity Depending on their Modes of Action

Abstract

The goal of this work was to investigate the mechanisms underlying different Eg5 (kinesin-5) inhibitors and to assess their effects on Eg5-mediated stabilization of dynamic microtubules. Antimitotic Eg5 inhibitors were grouped into those that bind Loop-5 and produce an ADP-like state (monastrol, STLC, ispinesib and filanesib) and those that produce a rigor-like state (BRD9876). To understand their contrasting effects on Eg5 mechanics, inhibitors were analyzed in mixed-motor gliding assays consisting of different ratios of kinesin-1 and Eg5 motors, in which Eg5 “braking” dominates motility. Loop-5 inhibitors increased gliding speeds, consistent with the compounds inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with Eg5 being locked in a rigor state. Quantitative critical detachment force analysis (Arpag et al. Biophys J. 2014) indicates that L5 inhibitors enhance Eg5 detachment whereas the rigor inhibitor BRD9876 generates an Eg5 species with decreased susceptibility of detachment. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with a KI of 4 nM. Consistent with its recently reported microtubule polymerase activity (Chen and Hancock, Nature Comm. 2015:8160) and its dominant two-head-bound strong-binding population (Chen et al., JBC 2016:M116), Eg5 was shown to stabilize microtubules against depolymerization in vitro following taxol washout. This stabilization was eliminated in monastrol but was enhanced by BRD9876, consistent with strong binding of Eg5 to microtubules enhancing microtubule stability. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors inhibit spindle formation through contrasting mechanisms and rigor inhibitors are predicted to paradoxically stabilize microtubule arrays in cells.