Radial compression of microtubules and the mechanism of action of taxol and associated proteins

Abstract

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.

FIGURE 1

Schematic illustration of the MT phases observed. For <0.4% (wt/wt) 20 k PEO (corresponding to an osmotic pressure of 600 Pa), the MTs are undistorted and form a nematic. Above this concentration of 20 k PEO, the MTs buckle to a noncircular cross section and form bundles with rectangular symmetry. The MTs distort further as the osmotic pressure increases. At 5% 20 k PEO (25,000 Pa), approximately c*, the polymer overlap concentration, the PEO is forced inside the lumen of the MTs, and the MTs convert to undistorted MTs in hexagonal bundles. At 18% 20 k PEO (400,000 Pa), all the MTs are in the hexagonal bundle phase.

FIGURE 2

Optical micrographs of MTs and MT bundles with 20 k PEO. Polarized microscopy of (A) MTs with no added PEO and (B) MTs with 0.42% (wt/wt) 20 k PEO (scale bar = 500 μm). Video-enhanced DIC microscopy of (C) MTs with 0.2% 20 k PEO and of (D) MTs with 1% 20 k PEO (scale bar = 10 μm). With low concentration of added 20 k PEO (A and C), MTs are oriented on μm to mm length scales, denoted N_MT. MTs form bundles with higher concentrations of added PEO (D) which display weaker large-scale orientation (B). X-ray scattering experiments demonstrate that these bundles are in the rectangular phase,

FIGURE 3

Small angle synchrotron x-ray diffraction scans of MTs with 20 k PEO. (A) The scattering patterns continuously evolve as the 20 k PEO concentration is increased from 0% (wt/wt) to 20%. All peaks, oscillations, and minima of the scattering can be accounted for by three structures: a nematic of single MTs (N^MT), bundles with rectangular symmetry (), and bundles with hexagonal symmetry (). (B) Up to nine peaks of the rectangular lattice and four peaks of the hexagonal lattice are visible, all of which can be indexed. The (1 0) rectangular peak is often difficult to discern but is more prominent with 25 mM added KCl (). Coexistence between bundles with rectangular and hexagonal symmetry is evident in many scans ( arrow indicates hexagonal (1 0) peak. All other peaks are from rectangular bundles, indices not shown). (C) Model calculations of the scattering from isolated MTs (unbuckled), unbundled, distorted MTs (buckled), bundles of distorted MTs with rectangular symmetry (), and bundles of undeformed MTs with hexagonal symmetry (). See text for modeling details.

FIGURE 4

The transition to rectangular bundles is reversible. (A) Small angle synchrotron x-ray diffraction scans of MTs with 1% (wt/wt) 20 k PEO show bundles with rectangular symmetry. If these MTs are spun into a pellet, the supernatant is removed and the MTs are resuspended in solution with 0.2% 20 k PEO, the solution reconverts to a nematic of undistorted, undamaged MT (A), as seen by the SAXRD scan showing form factor scattering and (A, inset, scale bar = 50 nm) confirmed by whole mount electron microscopy. (B) Small angle rotating anode x-ray scans of MTs, with lower resolution and greater noise, demonstrate that MTs that were buckled 1% 20 k PEG and 150 mM NaCl, unbuckle with the addition of 2% GA. Plastic embedded electron microscopy cross sections confirm that previously buckled MTs reconvert to undistorted, unbundled MTs (B, inset, scale bar = 50 nm).

FIGURE 5

Osmotic stressing polymers do not enter the MT lumen in the rectangular but do enter the MT lumen in the hexagonal phase. DIC microscopy, upper, and corresponding fluorescence microscopy, lower, of MTs with 1% 20 k PEO, 20% 20 k PEO, and 8% 500 kd dextran (scale bar = 10 μm). All samples contain 100 mM monovalent salt. Both PEO samples have 0.1% fluorescently labeled 20 k PEO. All of the dextran is fluorescently labeled. The bundles with 1% 20 k PEO and 8% 500 kd dextran, which x-ray indicates are rectangular, are dark in fluorescence showing that the stressing polymers are excluded from these bundles. The fluorescence images are uniform with 20% 20 k PEO, which are hexagonal, and this is consistent with PEO entering these bundles.

FIGURE 6

Small angle synchrotron x-ray diffraction scans of 20 k PEO with MTs, polymerized in the presence of partially purified MTP, which is ∼30% MT-associated protein and ∼70% tubulin. (A) MTs polymerized with 5% MTP form rectangular bundles but only with higher concentrations of 20 k PEO. (B) With 50% MTP hexagonal bundles are present for high 20 k PEO concentrations and rectangular bundles are not observed. (C) 100% MTP with no taxol and 150 mM KCl also display no rectangular bundles.

FIGURE 7

Osmotic pressure-MTP phase diagram measured with 20 k PEO. Tubulin (phosphocellulose chromatography purified) phase boundaries are indicated as 0.1% MTP. Dotted lines are guides to the eye. Regions where rectangular bundles are observed are shaded with a grid. Increasing the percentage of MTP results in a drastic increase in the pressure required to observe rectangular bundles. No rectangular bundles are observed for >10% MTP.

FIGURE 8

Small angle synchrotron x-ray diffraction scans of MTs with 20 k PEO in the presence of added (A) 25 mM KCl and (B) 250 mM KCl. Increasing KCl concentration drives the nematic to rectangular bundle transition to a lower concentration of 20 k PEO.

FIGURE 9

(A) Pressure-KCl and (B) pressure-taxol phase diagrams, near the nematic-rectangular bundle phase boundary, for MTs with 20 k PEO. Dotted lines are guides to eye. The pressure required to form rectangular bundles decreases with increasing KCl concentration but is unaffected by taxol concentration.

FIGURE 10

Continued deformation of rectangular bundles with increasing osmotic pressure, a_R and b_R, measured by small angle x-ray scattering are defined as in the cartoon. The slope of a_R versus the logarithm of pressure is the same for (A) all amounts of MTP, (B) all concentrations of KCl, and (C) all concentrations of taxol. (D) b_R and (E) the calculated MT perimeter versus osmotic pressure (see text).

FIGURE 11

In the hexagonal bundle phase, the MT spacing decreases with increasing osmotic pressure. The measured lattice spacing a_R (left), the MT center-to-center distance, can be used to obtain the MT wall-to-wall distance (right). The applied osmotic pressure (bottom) can be used to calculate the force per unit length between MTs (top). See text for details. The dashed line is a best fit to the data.