X‐ray fiber diffraction analysis shows dynamic changes in axial tubulin repeats in native microtubules depending on paclitaxel content, temperature and GTP‐hydrolysis

Abstract

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations.

Kinesin-binding–triggered conformation switching of microtubules contributes to polarized transport

“…The shorter pitch of GDP microtubules without KIF5C is in agreement with our previous results with GDP microtubules with a substoichiometric concentration of Taxol measured using the same method (Kamimura et al, 2016). The longer pitch with KIF5C is closer to the pitch of the GMPCPP microtubules measured at the same temperature (37°C).…”

Molecular modeling study on the differential microtubule‐stabilizing effect in singly‐ and doubly‐bonded complexes with peloruside A and paclitaxel

“…Axial repeats r (Å) for each tubulin dimer were calculated to evaluate the compaction of the protein in the MT models along the simulation run, and were computed as the distance between the centers of mass of each subunit in the α 1 β 1 and α 2 β 2 dimers (Figure ). Average axial repeats for tubulin dimers in all systems under study lie in the range between 40.5 and 41.4 Å, which are in close agreement with previous resports . Despite no significant differences were observed among average axial repeats, distribution data revealed that PLA association impairs the axial repeat distribution between α 1 β 1 and α 2 β 2 dimers, unlike LAU and TX, which exhibit similar distribution patterns in both dimers.…”

“…Average axial repeats for tubulin dimers in all systems under study lie in the range between 40.5 and 41.4 Å, which are in close agreement with previous resports. 33 ORCID Verónica A. Jiménez https://orcid.org/0000-0002-6783-5657…”

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes

“…Final concentrations of protein, nucleotide and compounds were 50 µM tubulin, 1 mM GTP and 100 µM of compound. Samples were centrifuged for 10 s at 2000 g to eliminate air bubbles and transferred to a share-flow device (29,60).…”