Mobility of taxol in microtubule bundles

Abstract

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With >63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k(off). It was found that 10k(off)(GMPCPP) approximately equal k(off)(GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With <63% sites occupied, recovery times decreased as approximately [Tax](-1/5) for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within approximately 7 nm of each other.

FIGURE 1

Schematic of the flow cell. Two holes (2-mm) drilled in the slide served as inlet and outlet ports. Parafilm wax cut to have a rectangular flow path seals the coverslip to the slide when heated as described. Sephadex beads are dusted on the flow path before sealing, providing obstacles to trap microtubules.

FIGURE 2

Schematic of the FRAP apparatus used. (Bleaching path) The upper Hg arc lamp is used for DIC imaging and bleaching of the sample. The illumination path goes through a green filter (550 nm) and a neutral density filter (ND 0.25), off of a mirror (M), and through an aperture to reduce the size of the spot. A shutter times the bleach. A 60× water-coupled objective (NA 0.9) is used as a condenser to focus the spot onto the sample. A shutter shields the intensified charge-coupled device camera during the bleach. (Observation path) The lower Hg arc follows an epi-fluorescence illumination path though two neutral density filters (ND 0.1 and 0.5) and a rhodamine excitation filter (XF, 535 nm). The light is reflected off a dichroic mirror (DM) and focused onto the sample with a 60× oil-coupled objective (NA 0.9) to excite the BODIPY fluorophore (565/571 nm). The same objective collects the emitted light that passes through the dichroic mirror and through an emission filter (MF, 630 nm) to the intensified charge-coupled device (CCD). The images are captured directly to RAM by the computer.

FIGURE 3

(A) Time series of fluorescence recovery after photobleaching on a bundle of GTP microtubules in 5 μM taxol; ROI example is shown as a dashed line. Initially, the bleach spot is very dark. After ∼5 min, the spot is brighter and wider, and after 20 min, the region has recovered to its maximum intensity. (B) Intensity profiles of the bleach spot at two different times. At t = 0, intensity profile is best fit by a Gaussian with negative amplitude and a width of 29 ± 0.1 μM. At t = 1214 s, the profile has decreased amplitude because the spot is brighter, but the width has changed little, 34 ± 0.4 μM. (C) Amplitude of the bleached spot decays exponentially with characteristic time τ = 329 ± 1.9 s.

FIGURE 4

Geometry of the bundle affects fluorescence recovery after photobleaching. (A) A GTP microtubule bundle viewed in fluorescence. The microtubule bundle begins as a single thick bundle in the upper right, and fans out into two parts: one with more microtubules (medium) than the other (thin). The thick region was bleached four times. The average recovery time was 24 ± 2 s. (B) Fluorescence recovery curves at the three specified locations in Fig. 4 A. Characteristic times are 8, 15, and 21 s, for the thin, medium, and thick bundles, respectively.

FIGURE 5

Average recovery times, τ, for GMPCPP microtubules (solid circles) and GDP microtubules (open circles) as a function of taxol concentration. Recovery times for GMPCPP microtubules are always slower than for GTP microtubules. GMPCPP microtubules in taxol concentrations from 25 pM–25 nM exhibit a similar power law behavior as GTP microtubules in 93 nM–55 μM taxol. For GMPCPP microtubules >25 nM, the trend stops and the recovery times are all ∼1000 s. The error in recovery times is the standard error due to averaging.