Divalent Cations Induce Non-Equilibrium Phase Transitions in Tau-Mediated Microtubule Bundles Undergoing Dynamic Instability

Abstract

The microtubule-associated protein tau is known for its ability to alter microtubule (MT) dynamic instability in developing and mature neurons and for its role in the pathology of many neurodegenerative diseases. Bundling of MTs into linear arrays (i.e. MT fascicles) is thought to be a cardinal feature of the axon initial segment of neurons, and tau's role in mediating MT-MT interactions in these arrays is thought to be critical for neuron function; however, the exact mechanism by which tau links and dictates the wide spacing of bundled MTs remains unclear. To help elucidate the nature of tau's role in physiological bundling of MTs, we have expanded on a recent platform for studying paclitaxel-free in vitro reaction mixtures of tubulin, GTP, and wild-type tau under dissipative, out-of-equilibrium conditions at 37 C. Using synchrotron small-angle x-ray scattering and transmission electron microscopy, we show that Ca2+ or Mg2+ in the mM concentration range can induce changes in the assembly of tubulin structures, revealing a phase transition between the previously reported widely spaced bundles and a novel transient bundled state with intermediate spacing. The latter bundled state is followed by rapid MT depolymerization and the formation of tubulin oligomers and rings. The transitions between each of these phases and their dependence on both time and cation concentration will be discussed.