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Mobility of Taxol in Tight Microtubule Bundles

Taxol is a small molecule that binds on the inner surface of microtubules and stabilizes them against depolymerization. Using a fluorescent derivative of taxol, BODIPY-taxol (from Molecular Probes), we were able to visualize fluorescently labeled microtubules using epi-fluorescence. We bundled the microtubules around Sephadex beads that were melted to the slide surface by flowing microtubules in a flow cell. The beads pegged and bundled the microtubules (as seen the the figure below).
FIGURE: Microtubules bundled against a Sephadex bead by flowing. The bundle is labeled with rhodamine labeled taxol (red), but the intensified CDD only sees in black and white (as in the next figure).

 MT bundle

In this study, we examined how taxol diffuses through microtubule bundles using fluorescence recovery after photobleaching (FRAP). This method uses a bright light to "bleach" the fluorophore, which breaks a chemical bond in the fluorescent part of the molecule. The bleached molecules are first localized to a small region which expands and dissapates as the dark molecules move out of the region and are replaced by surrounding bright ones. We recorded the "recovery of fluorescence" using an intensified CCD and a computer.
FIGURE: Fluorescent recovery after photobleaching a microtubule bundle. The first picture is immediately after the bleach. The second picture is about 5 minutes later, and recovery can be seen. The third picture is 20 minutes after the initial bleach, and the bundle has recovered to its maximum final intensity.

 Taxol moves

The recovery of fluorescence was a single exponential decay with one characteristic time for recovery. As the figure above shows, the dark spot does not get wider over time, implying that we are not seeing very much diffusive spreading. The fact that we see a single exponential decay in fluorescence amplitude combined with little spreading indicates that we are observing the binding reaction and not a diffusive one. In addition, we found the fluorescence of BODIPY-taxol to increase 40-fold upon binding to microtubules, implying that we are only seeing bound taxol in the figure above.

We compared the characteristic recovery times for "GDP microtubules," microtubules with GDP at the E-site, and "GMPCPP microtubules," microtubules with GMPCPP, a non-hydrolyzable analog of GTP, at the E-site. We also varied the concentration of taxol for each of these types of microtubules in order to vary the proportion of filled binding sites. In this way we could test how the number of binding sites affects the mobility of a diffusing ligand (molecule that can bind to a specific site).

We found that the recovery times depend on bundle thickness and packing density, taxol concetration, and E-site nucleotide.

For the abstract and a PDF version of this paper, please click: Publications

For more details about the results or methods of this research, please click

Taxol Mobility

MT bundle Background Information

MT bundle Mobility of Taxol in Tight Microtubule Bundles

MT bundle Mobility in Tau-Coated Microtubule Bundles

MT bundle How Tau Affects Taxol Mobility