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.

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

“…1 C-E). Consistent with previous results without Tau (40,41), MTs at high concentration undergo a transition from an orientationally ordered nematic state (N MT ) (Fig. 1C) to two distinct higher-ordered bundled phases ( Fig.…”

“…2D, bottom profile) (0-0.46 wt% of 20k PEO), where DIC shows the N MT phase, fits of background-subtracted scattering corresponded to a hollow cylinder form factor, with inner radii (∼8 nm) and cylinder thickness (4.9 nm) consistent with EM data of MTs (42). This behavior is similar to previous reports of bare MTs, where SAXS profiles of the N MT phase shows no evidence of a correlation peak (5,32,40,41,43,44). At intermediate osmotic pressures (∼1-3 wt% of 20k PEO), where DIC shows bundled MTs (Fig.…”

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

“…results in k long ϭ 4 N͞m (43), which is 40 times larger than the elastic constant measured in a transverse compression by an AFM tip, k trans ϭ 0.1 N͞m (7). Thus, a large amount of experimental data, including the assembly͞disassembly dynamics of MTs, electron crystallography maps (41,42), cryoelectron microscopy (19,38,44), and small-angle x-ray diffraction (21,45), suggest a pronounced mechanical anisotropy of MTs.…”

“…For forces larger than the thermal force, the compliance of the lateral connection between PFs might be eventually exceeded, leading to deformation of tubulin subunits. Higher forces can be obtained, for instance, by further increasing the osmotic pressure in the medium (45). Tubulin subunits have a high stiffness, typical of proteins, of Ϸ2 GPa (46).…”

“…Deformation of the stiff tubulin dimers might be the explanation of the postbuckling elastic response measured in ref. 45. The shear modulus G 13 may be considered as the result of two possible contributions: a vertical shear modulus (G 13 v ) arising from stretching of intra-PF tubulin, and a horizontal shear modulus (G 13 h ), determined by lateral bonds between PFs (Fig.…”