Taxol crystals can masquerade as stabilized microtubules

Abstract

Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications.

Figure 1. Taxol crystals resemble asters and bundles formed in the presence of Taxol-stabilized microtubules.

(A) Two asters (top and middle) and one bundle (bottom) formed in the presence of Taxol and fluorescently labeled tubulin, as in text. Paired images with DIC (left) and fluorescent (right) optics. (B) DIC images of a Taxol crystal aster (top) and bundle (bottom), formed in the absence of tubulin. Bars, 10 µm.

Figure 2. Taxol crystals bind fluorescently labeled tubulin subunits.

(A) Fluorescently labeled tubulin (released from a micropipette to the left of the field) immediately accumulated on the preformed Taxol crystal aster, as shown in these sequential images. (B) An equilibrium solution of fluorescently labeled tubulin (green haze) and microtubules was released as in (A), and decorated a Taxol ‘bow-tie’ (i.e., nonspherical) crystal aster (half of aster is visible, in upper right). Again, the crystal was instantly labeled by subunits, which diffuse more rapidly than microtubules. Microtubules did not appear to contribute to the brightness of the tubulin-decorated crystal, as fluorescence intensity did not increase over time. Thus, microtubules did not appear to play a major role in decorating the Taxol crystal. The arrow tracks the path of a single microtubule that briefly made contact with the aster, but diffused away. The shallow aqueous puddle containing the crystal aster is outlined in the first panel. Time in seconds. Bars, 10 µm.

Figure 3. Concentration- and time-dependence of aster formation.

(A) Fluorescently labeled Taxol-induced asters (DIC and fluorescent optics) formed in polymerization solutions stabilized with a concentration gradient of Taxol (left to right): 0.92 µM, 1.0 µM, 10 µM, 20 µM. Asters were not visible at or below 0.88 µM Taxol. These results are consistent with the low solubility of Taxol in aqueous solution . (B) Taxol crystals, formed in 20 µM Taxol in aqueous buffer. Asters were visualized by DIC microscopy 24 hours after preparation. Compare to the smaller, less-dense aster in Fig. 1 B, which was visualized within 10–60 minutes. Crystals were not a solvent-induced artifact, as no structures were visible in a control solution lacking Taxol. Bars, 10 µm.