Steffen Kutter, Timo Eichner, Alexandra M. Deaconescu, Dorothee Kern

The molecular mechanism by which the microtubule associated protein tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. 17 of those phosphorylation sites precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, implicated in Alzheimer's disease, has been reported to be crucial for restoring tau's ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimer's associated phosphorylated tau in tubulin assembly [1,2]. Using turbidity assays, time-resolved small angle X-ray scattering (SAXS) and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin binding repeats, and a weaker interaction with the proline rich sequence and the termini of tau.