Tau, a developmentally regulated protein localized to the axon of mature neurons, stabilizes axonal microtubules but has been implicated in many neurodegenerative diseases ("tauopathies") including Alzheimer's, Pick's, and, more recently, chronic traumatic encephalopathy. Despite its importance in both development and disease, difficulty in understanding Tau is due, in part, to its intrinsically disordered nature; Tau does not assume a secondary structure in solution. However, lack of structure does not imply lack of function, as Tau binds to microtubules, thereby regulating microtubule assembly/stability and affecting inter-microtubule interactions, although the latter remains controversial. Herein, through microscopy and synchrotron small-angle X-ray scattering of cell free Tau/microtubule reconstitutions under various conditions, we report on the nature of the Tau structure and Tau-mediated interactions between microtubules.

By examining the force-response of Tau-coated paclitaxel-stabilized microtubules by osmotic depletants, we observed that longer isoforms of Tau at high (and physiologically relevant) coverage on microtubules more effectively sterically stabilize microtubules against microtubule bundling. This steric stabilization occurs by the amino-terminal tail of Tau assuming the conformational (and repulsive) properties of a polyelectrolyte brush. Furthermore, the coverage at which this transition into a polyelectrolyte brush occurs gives the first direct measurement of the size of the longer isoforms of Tau (20-23 nm) on microtubule surfaces.

To understand the molecular mechanism of Tau-mediated inter-microtubule interactions in dissipative, out-of-equilibrium conditions, we co-polymerized Tau with microtubules in the absence of stabilizing agents (i.e. paclitaxel) and found that Tau mediates microtubule bundles with resultant architectures mimicking fascicles of microtubules found in the axonal initial segment. These bundles confirmed an attractive component to the Tau-mediated microtubule interaction through an aggregate of sub-kBT interactions along the microtubule length heretofore unreported in intrinsically disordered systems. The interaction dependence on microtubule length reconciles previous (unsuccessful) attempts at reconstituting Tau-mediated bundles, as stabilizing agents often promoted more, but shorter, microtubules.

These novel biophysical characterizations of Tau on the microtubule surface give insight to the physiological function of Tau inside the neuronal axon and represent possible properties to investigate the role of mutations and post-translational modifications of Tau that lead to neurodegenerative disease.