Termination of Protofilament Elongation by Eribulin Induces Lattice Defects that Promote Microtubule Catastrophes

Abstract

Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.

Kinesin-4 KIF21B is a potent microtubule pausing factor

“…Noteworthy in this respect is our observation that when a MT continued growing after KIF21B was stalled, its growth was often strongly perturbed: we observed switching between growth and shortening episodes, as well as strong MT bending after the point of KIF21B attachment (Figure 7J). These data are reminiscent of our work on the effect of binding of a protofilament-blocking drug, Eribulin (Doodhi et al, 2016). We recently showed that attachment of a single Eribulin molecule, which, based on structural data can inhibit growth of only one MT protofilament, was sufficient to either cause a catastrophe or induce MT growth perturbation, suggesting elongation of an incomplete MT (Doodhi et al, 2016).…”

“…These data are reminiscent of our work on the effect of binding of a protofilament-blocking drug, Eribulin (Doodhi et al, 2016). We recently showed that attachment of a single Eribulin molecule, which, based on structural data can inhibit growth of only one MT protofilament, was sufficient to either cause a catastrophe or induce MT growth perturbation, suggesting elongation of an incomplete MT (Doodhi et al, 2016). It is tempting to speculate that similar to Eribulin, a KIF21B molecule stalled at the MT tip would be sufficient to block a small number of protofilaments, and this would result in inefficient elongation of the remaining protofilaments.…”

Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs

“…The second group including drugs such as vinca alkaloids and eribulin (a halichondrin) bind primarily to microtubule ends and are often referred to as microtubule destabilizers because at high concentrations, they block the addition of new tubulin subunits to microtubule ends, thus leading to microtubule depolymerization. However, their most potent activity is stabilization and inhibition of microtubule dynamic instability, which occurs with binding of low numbers of drug molecules at microtubule ends (7)(8)(9). Despite sharing the ability to suppress microtubule dynamic instability, we found that these two classes of drugs exert very different effects on microtubulebased transport.…”

“…5,[7][8][9]. The first group is sometimes referred to as microtubule stabilizers because by binding along the microtubule lattice, they stabilize microtubule dynamic instability and inhibit microtubule depolymerization.…”

Design, synthesis, and structure–activity relationships of pyrimido[4,5- b ]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells

“…7 Eribulin (Figure 1), a simplified analog of halichondrin B, 8 binds within the vinca domain, and has unique effects on microtubule dynamics. 8,9 Structurally diverse natural products including rhizoxin and maytansine (Figure 1) bind to a different site on β-tubulin, referred to as the maytansine site. 10 The occupancy of this site by maytansine causes microtubule depolymerization by inhibiting longitudinal tubulin interactions.…”