Synchrotron X-ray Diffraction Study of Microtubules Buckling and Bundling under Osmotic Stress: A Probe of Interprotofilament Interactions

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Figure 1

A MT with a tubulin heterodimer and protofilament outlined. DIC of (a) a nematic of MTs with no PEO, (b) rectangular bundles of MTs with 1% (w/w) 20k PEO, and (c) MTs in hexagonal bundles with 20% 20k PEO. DIC (d) and the corresponding fluorescence (e) of the rectangular phase with fluorescently labeled 20k PEO $10\mu \mathrm{m}$ scale bars.

Figure 2

SAXRD of MTs with 20k PEO. MTs in the nematic phase with 0.2% (w/w) 20k PEO $(N_{\mathrm{M}\mathrm{T}})$. With 1% 20k PEO ( $R_{\mathrm{M}\mathrm{T}}^{\mathrm{B}}$, lower plot), SAXRD shows a rectangular lattice. With 25 mM KCl and 1% 20k PEO ( $R_{\mathrm{M}\mathrm{T}}^{\mathrm{B}}$, upper plot) the peaks become sharper and the (1 0) peak is clearly visible. With 7.5% 20k PEO $(R_{\mathrm{M}\mathrm{T}}^{\mathrm{B}}+H_{\mathrm{M}\mathrm{T}})$, there is coexistence between the hexagonal [(1 0) peak indicated by arrow] and rectangular bundles. With 20% 20k PEO $(H_{\mathrm{M}\mathrm{T}})$, only the hexagonal phase is present. Cartoons display MT cross sections from the bundle phases.

Figure 3

Phase diagram of MTs with 20k PEO as a function of osmotic pressure. The lattice constants of the bundle phases change with pressure. For rectangular bundles, $a_R$ defines the smaller center-to-center distance (circles, left axis) and $b_R$ is the larger center-to-center distance (squares, right axis). $a_H$ is the center-to-center distance in the hexagonal phase (triangles, left axis).

Figure 4

The effect of solution conditions on the critical buckling pressure of MTs, $P_{\mathrm{c}\mathrm{r}}$. Added KCl (upward triangles) decreases $P_{\mathrm{c}\mathrm{r}}$. Polymerizing highly purified tubulin with MT protein ($\mathrm{M}\mathrm{T}\mathrm{P}=20\%$ $\mathrm{M}\mathrm{A}\mathrm{P}/80\%\mathrm{\text{tubulin}}\mathrm{w}/\mathrm{w}$) leads to a large increase in $P_{\mathrm{c}\mathrm{r}}$ (downwards triangles, percent MTP by weight). $P_{\mathrm{c}\mathrm{r}}$ is unaffected by changes in the concentration of the chemotherapy drug taxol (circles). Cartoons display a single MT cross section.