Phosphorylation-Induced Self-Coacervation versus RNA-Assisted Complex Coacervation of Tau Proteins

In this study, the
role of phosphorylation in the liquid–liquid
phase separation (LLPS) of tau, the underlying driving forces, and
the potential implications of this separation on protein conformation
and subsequent protein aggregation were investigated. We compared
in vivo-produced phosphorylated tau (p-tau) and nonphosphorylated
tau under different coacervation conditions without adding crowding
agents. Our findings revealed that spontaneous phase separation occurs
exclusively in p-tau, triggered by a temperature shift from 4 °C
to room temperature, and is driven by electrostatic and hydrophobic
interactions. The p-tau self-acervation is reversible with temperature
changes. Native mass spectrometry detects only two to nine phosphate
groups per p-tau molecule, highlighting the impact of phosphorylation
on tau’s structural flexibility. Cross-linking mass spectrometry
showed fewer long-range contacts in p-tau, suggesting a looser conformation
induced by phosphorylation. Phosphorylation-induced LLPS and RNA-induced
LLPS occurred at different timeframes. However, neither tau nor p-tau
formed fibrils without the addition of dextran sulfate or RNA as inducers.
Using human kidney epithelial cells expressing the tau R domain fused
with fluorescent proteins as reporter cells, we observed aggregates
in the nuclear envelope (NE) only in the cells treated with LLPS-state
p-tau, which correlates with NE occurrences reported in Alzheimer’s
disease brain sections. These findings provide deeper insights into
the impact of phosphorylation on tau aggregation through an intermediate
condensation phase, offering novel perspectives on neurodegenerative
disease mechanisms.