The assembly of microtubules and drug-induced tubulin polymers : an X-ray diffraction and cryo-electron microscopy study.
Synchrotron Radiation (SR) X-ray solution scattering/diffraction and cryo-electron
microscopy have been used to characterise the structure of tubulin polymers assembled in a
variety of biochemical conditions. Cryo-microscopy has been used to image individual
structures and to interpret the X-ray diffraction profiles from the protein structures in solution.
The time-resolved X-ray data provided by SR has been analysed using correlation techniques
in order to elucidate the structural pathway of reactions triggered by temperature changes. The
results can be summarised as follows:
• The structure and temperature-induced assembly of microtubules polymerised from
microtubule protein (MTP), and purified tubulin (TB) preparations are very similar.
Differences in the assembly process concern the existence of prenucleation events, linked to
the presence of cold structured aggregates, only present in the MTP solutions. The only
structural difference is the presence of MAPs attached to the wall of MTP microtubules. When
TB microtubules were assembled in the presence of taxol the surface lattice was preserved but
the diameter was smaller, corresponding to an average loss of one proto filament.
• The vinblastine-induced self-assembly of TB can give rise to both, double-coiled spirals
that aggregate in a non-ordered fashion, and paracrystalline structures where the repeating
motif is a single-coiled spiral. The type of polymer is mainly detennined by the amount of
magnesium ion present in the preparation. Concentrations higher than 7.5 mM result in double
coils similar to those found in MAPs-containing preparations, pointing to a similarity of effect
possibly due to the positive charge of both MAPs and magnesium ions. At any magnesium
concentration, a temperature change induced a reversible change in the pitch of the spirals,
reflecting a temperature-induced confonnational change in the tubulin subunit.