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

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 ß-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.

Characterization of a human ßV-tubulin antibody and expression of this isotype in normal and malignant human tissue.

There are seven distinct ß-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. ßV-tubulin, like ßIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human ßV-tubulin. The antibody did not cross-react with mouse ßV-tubulin or other human ß-tubulin isotypes and specifically labeled ßV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that ßV-tubulin was expressed in endothelial cells, myocytes and cells with muscle differentiation, structures with transport and/or secretory function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretory function such as prostate. ßV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. Initial studies in breast, lung and ovarian cancers indicated aberrant expression of ßV-tubulin, suggesting that this isoform may be associated with tumorigenesis. Thus, ßV-tubulin expression is a potentially promising prognostic marker of malignancy.