FIGURE 3.

WTS seeds significantly impair Tau-promoted MT assembly. A, scheme of the in vitro MT assembly assay. MTs are formed from tubulin dimers in the presence of GTP and Tau at 37 °C accompanied by increasing turbidity of the solution. Turbidity assays were performed in the presence of α-Syn at α-Syn/Tau ratios of 4 or 3. In all cases, control was a turbidity assay performed in the absence of α-Syn. The MT assembly-promoting drug taxol was added to promote MT formation under inhibiting conditions. B–D, representative diagrams of three independent experiments with either α-Syn monomers (B) or α-Syn multimers (C) at an α-Syn/Tau ratio of 4 and with WTS seeds at an α-Syn/Tau ratio of 3 (D). WTS seeds, but not oligomers or fibrils and not WTS monomers, inhibited Tau-promoted MT assembly. Adding more Tau to the assay can rescue an inhibition of MT assembly caused by WTS seeds. Each curve represents one measurement. Taxol was added to each reaction with monomers to induce formation of MTs (40 min time point; B). Similarly, taxol increased turbidity when added to α-Syn multimeric assays (data not shown). E and F, normalized assembly levels calculated as turbidity values at the 30 min time point for WTS and each α-Syn variant in monomeric (E) or multimeric stage (F) at an α-Syn/Tau ratio of 4. The means of three independent measurements ± S.D. (error bars) are shown. Only WTS seeds significantly inhibited Tau-promoted MT formation. *, p < 0.05; ns, not significant. G and H, MTs were assembled from tubulin in the presence of Tau and different concentrations of WTS monomers (G) or WTS seeds (H). Each curve represents one measurement. Representative diagrams of three independent experiments are shown. I, WTS seeds but not monomers dose-dependently inhibited Tau-promoted formation of MTs. Normalized assembly levels calculated as turbidity values at the 30 min time point under different WTS concentrations as means of three independent measurements ± S.D. are shown. R², a linear regression coefficient.