Reduced microtubule-nucleation activity of tau after dephosphorylation.

Based on video-enhanced differential interference contrast (DIC) microscopy, we developed a small-scale method which is capable of measuring both the lengths and the number densities of microtubules (MTs) assembled in vitro. With this method, effect of dephosphorylation on the activity of bovine brain tau protein to promote the assembly of tubulin at physiological concentration (15 microM) was quantitatively analyzed. The MT number density was selectively reduced when tau isolated directly in the presence of phosphatase inhibitors (N-tau) was dephosphorylated in vitro (DP-tau), without significant changes in the mean MT length or the binding affinity toward preformed MTs. Tau obtained from brain MTs (MT-tau) also exhibited lower nucleation activity in spite of its high MT-binding affinity. The results indicate that nucleation, elongation and MT-binding are distinct aspects of tau function which are differentially affected by the phosphorylation state of tau.