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Taxol crystals can masquerade as stabilized microtubules.

Foss M, Wilcox BW, Alsop GB, Zhang D - PLoS ONE (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, Corvallis, Oregon, USA.

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.

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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.
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pone-0001476-g002: 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.

Mentions: To test our hypothesis, we prepared Taxol crystals, and exposed them to fluorescently labeled tubulin as follows. Stock Taxol in solvent was diluted in buffer to 20 µM, creating crystalline asters and bundles. The crystals were pipetted under oil onto a chamber slide. Ice-cold, fluorescently labeled tubulin subunits were micropipetted into the chamber, to one side of the crystals, allowing us to visualize their trajectory in real time using fluorescence microscopy. The crystals began to fluoresce almost instantly (Fig. 2 A), consistent with rapid binding of tubulin to the crystal rather than the slower assembly of microtubules on the crystal surface. In a separate experiment, tubulin was allowed to assemble into microtubules and the equilibrium solution of microtubules and tubulin subunits was pipetted near the crystal. Once again, the crystals were labeled almost instantly by the subunits, whereas the slower-moving microtubules were observed to diffuse past the crystal (Fig. 2 B). Both experiments suggested that crystals were decorated by free tubulin rather than by microtubules. Furthermore, since free Taxol binds the inner surface of microtubules, it would be topologically impossible for intact microtubules to bind the crystal via the previously characterized Taxol-binding site [5].


Taxol crystals can masquerade as stabilized microtubules.

Foss M, Wilcox BW, Alsop GB, Zhang D - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194920&req=5

pone-0001476-g002: 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.
Mentions: To test our hypothesis, we prepared Taxol crystals, and exposed them to fluorescently labeled tubulin as follows. Stock Taxol in solvent was diluted in buffer to 20 µM, creating crystalline asters and bundles. The crystals were pipetted under oil onto a chamber slide. Ice-cold, fluorescently labeled tubulin subunits were micropipetted into the chamber, to one side of the crystals, allowing us to visualize their trajectory in real time using fluorescence microscopy. The crystals began to fluoresce almost instantly (Fig. 2 A), consistent with rapid binding of tubulin to the crystal rather than the slower assembly of microtubules on the crystal surface. In a separate experiment, tubulin was allowed to assemble into microtubules and the equilibrium solution of microtubules and tubulin subunits was pipetted near the crystal. Once again, the crystals were labeled almost instantly by the subunits, whereas the slower-moving microtubules were observed to diffuse past the crystal (Fig. 2 B). Both experiments suggested that crystals were decorated by free tubulin rather than by microtubules. Furthermore, since free Taxol binds the inner surface of microtubules, it would be topologically impossible for intact microtubules to bind the crystal via the previously characterized Taxol-binding site [5].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Oregon State University, Corvallis, Oregon, USA.

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.

Show MeSH