Fig 1.
Thermodynamic analysis of stathmin-tubulin interaction.
Vinflunine increases stathmin binding to tubulin whereas paclitaxel does not. Typical curves of: A: raw data obtained for 25–30 injections, each of 10 μL of stathmin (80 μM) into the sample cell containing tubulin (10 μM) in the presence or the absence of MTAs. The three titration curves (which were translated upward for clarity purposes) were obtained during one set of experiments; curve A corresponds to stathmin binding to tubulin in the absence of MTA, curve B corresponds to stathmin binding to tubulin in the presence of vinflunine (10μM), whereas curve C corresponds to stathmin binding to tubulin in the presence of paclitaxel (10μM). B: integrated heats of reaction (symbols) in the absence of MTAs (squares), in the presence of vinflunine (circles) and in the presence of paclitaxel (triangles) with the best fit to the data (lines). The measurements were carried out at 37°C in PM buffer (20 mM NaPi, GTP 0.1 mM MgCl2 4mM pH 6.5).
Fig 2.
Effect of stathmin on in vitro microtubules polymerization in PM buffer followed by turbidity.
Various concentrations of stathmin ranging from 0 to 15μM were added at the arrow to 10μM tubulin after (panel A) or before (panel B) addition of 10μM paclitaxel. Panel A and B are representative turbidimetric curves of samples treated with 0, 1, 2, 3, or 5μM stathmin. Panel C represents the average of at least three experiment for each stathmin concentration (0, 1, 2, 3, 5, 10 and 15μM). Solid line corresponds to addition of stathmin after paclitaxel (panel A conditions) and dotted line corresponds to addition of stathmin before paclitaxel (panel B conditions) showing that the effect of stathmin on polymerization is similar whether stathmin is added before or after paclitaxel.
Fig 3.
Effect of the buffer on stathmin depolymerizing activity.
Samples containing various concentrations of stahtmin (0–15μM) added to paclitaxel stabilized microtubules (10μM tubulin) were sedimented and the amount of tubulin in the supernatants (SN) and pellets (P) was analyzed by SDS PAGE. The two representative gels displayed above the graph correspond to samples in the presence of 0, 5, 10 or 15μM stathmin, in PEM buffer (upper gel) or PM buffer (lower gel). Quantification of sedimentable tubulin polymers assembled in PM buffer (black squares) vs. PEM buffer (black circle) corresponds to averages of 0, 1, 2, 3, 5, 7.5, 10 and 15 μM stathmin in PM buffer and 0, 2, 5, 7.5, 10 and 15 μM stathmin in PEM buffer.
Fig 4.
Model of the molecular mechanism of stathmin interplay with Vinca alkaloids and taxanes.
Left side of the cartoon depicts a situation in which the level of expression is normal, i.e. not higher in the tumor that in the surrounding cells: stabilizing effect of paclitaxel and destabilizing effect of Vinca alkaloids, while right side depicts a situation where stathmin is overexpressed: loss of paclitaxel stabilizing effect and increase of Vinca alkaloids destabilizing effect.