Tau induces cooperative Taxol binding to microtubules

Abstract

Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds beta tubulin in the MT interior. Tau is a MT-associated protein that binds both alpha and beta tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of approximately 2 microM, and determines the escape rate of Taxol through one pore to be 1.7 x 10(3) (M x s)(-1). Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.

Fig. 1.

MTs were fluorescently labeled with botax and flow-aligned into densely packed bundles around a Sephadex bead. Panels present images of the same bundle as viewed by differential interference contrast (A and C) and fluorescence (B and D) before (A and B) and after (C and D) unlabeled tau diffused into the area. (E) Image of tau infiltrating a bundle. To the left of the tau front (arrow), tau has bound and spaced the MTs.

Fig. 2.

Representative biexponential fluorescence recovery curve for acrylodan-labeled four-repeat tau (circles). Recovery curves for all types of samples overlap this data. The fit (dashed line) yields a short time constant, ≈80 sec, corresponding to diffusion of unbound tau around the MTs or fast dissociation of loosely bound tau. The long time constant, ≈2,500 sec, corresponds to the characteristic dissociation time for tau.

Fig. 3.

Representative fill ratio curves for botax binding to MTs in the presence (filled circles) and absence (open squares) of tau. The two curves overlap, indicating that tau does not affect the equilibrium binding of botax. The dashed line curve is the average fit(n = 3) curve with fit parameters given.

Fig. 4.

Recovery time, τ_R, as a function of free Taxol concentration for tau to tubulin molar ratios of 0 and 1:50 (A), 1:10 (B), and 1:1 (C) with fit parameters from the model with a Hill coefficient to estimate the cooperativity. (A) At low tau concentrations, the model fits well without cooperativity n_H = 1. (B) At intermediate tau concentration, τ_R becomes more sensitive to Taxol concentration with n_H = 4.9. (C) At saturating amounts of tau, Taxol appears very cooperative with n_H = 15. Fit parameters for each curve are listed inside the plot.