Liquid-Liquid Phase Separation of Alzheimer's Protein Tau

Abstract

Liquid-liquid phase separation (LLPS) is the reversible demixing of a homogenous solution into a dilute phase and a dense phase composed of droplets. These droplets have become increasingly important in understanding cellular biochemistry as membrane-less organelles. Tau, an intrinsically disordered, microtubule-associated protein expressed in neurons, undergoes LLPS in vitro. In Alzheimer’s disease, hyperphosphorylated tau accumulates in ageing neurons as neurofibrillary tangles. Droplets of disease-associated mutants of tau and phosphorylated tau exhibit reduced dynamics with time, transitioning from a normal liquid state to a viscous gel. Interestingly, these droplets also increase the aggregation kinetics of tau. Dynamically arrested droplets are now considered relevant in many neurodegenerative diseases as crucibles for protein aggregation. They may offer an alternative pathway for the assembly of fibrils, non-exclusive from the established nucleation dependent polymerisation. Altered dynamics in droplets could be driven by tau adopting different conformations inside them, and these remain to be elucidated. We intend to use pulsed hydrogen/deuterium exchange mass spectrometry (HXMS) to characterise the structural conformations of full-length tau in vitro droplets. How these conformations change with droplet maturation will shed light on the role of LLPS in tau aggregation. In this study, we isolated full-length tau to ~95% purity and standardised the initiation of LLPS in conditions described previously. We show that a certain concentration of salt abolishes LLPS and requires the presence of a molecular crowder to induce droplet formation. Preliminary FRAP data reveals that droplets are dynamic in nature. Standardised experiments will help estimate the timescale of the exchange of tau molecules between the two phases for the design of HXMS studies.