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Updated: Sep 28, 2025
In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening
Published on: November 20, 2018
In vitro characterization and molecular dynamics simulation reveal mechanism of 14-3-3ζ regulated phase separation of
Yue Han1, Haiqiong Ye1, Ping Li1
1Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei University of Technology, Wuhan 430068, China; Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China; Department of Biological Engineering, Hubei University of Technology, Wuhan 430068, China.
Abstract:
As a major microtubule-associated protein, tau is involved in the assembly of microtubules in the central nervous system. However, under pathological conditions tau assembles into amyloid filaments. Liquid droplets formed by liquid-liquid phase separation (LLPS) are a recently identified assembly state of tau and may have a major effect on the physiological function of tau and the formation of tau aggregates. 14-3-3 proteins are ubiquitously expressed in various tissues and regulate a wide variety of biological processes. In this work, we demonstrate that 14-3-3ζ is recruited into tau droplets and regulates tau LLPS by in vitro assays. While the mobility of tau molecules inside the droplets is not affected in the presence of 14-3-3ζ, the amount and size of droplets can vary significantly. Mechanistic studies reveal that 14-3-3ζ regulates tau LLPS by electrostatic interactions and hydrophobic interactions with the proline-rich domain and the microtubule-binding domain of tau. Surprisingly, the disordered C-terminal tail rather than the amphipathic binding groove of 14-3-3ζ plays a key role. Our findings not only provide a novel dimension to understand the interactions between 14-3-3 proteins and tau, but also suggest that 14-3-3 proteins may play an important role in regulating the LLPS of their binding partners.
Insights
14-3-3ζ protein regulates tau liquid-liquid phase separation (LLPS) by interacting with tau droplets. This interaction affects droplet size and amount, offering new insights into tau pathology.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Tau is a key microtubule-associated protein in the central nervous system.
- Pathological tau can form amyloid filaments, and liquid-liquid phase separation (LLPS) forms liquid tau droplets.
- 14-3-3 proteins are vital regulators of diverse biological processes.
Purpose of the Study:
- To investigate the role of 14-3-3ζ in tau liquid-liquid phase separation (LLPS).
- To elucidate the mechanism by which 14-3-3ζ influences tau droplet formation and properties.
Main Methods:
- In vitro assays to study tau LLPS.
- Biochemical analysis of protein-protein interactions.
- Characterization of droplet dynamics and morphology.
Main Results:
- 14-3-3ζ is recruited into tau droplets, modulating tau LLPS.
- While tau mobility within droplets remains unaffected, 14-3-3ζ significantly alters droplet amount and size.
- 14-3-3ζ interacts with tau's proline-rich and microtubule-binding domains via electrostatic and hydrophobic forces.
- The disordered C-terminal tail of 14-3-3ζ is crucial for this regulation.
Conclusions:
- 14-3-3ζ plays a significant role in regulating tau LLPS, impacting droplet characteristics.
- The findings reveal a novel interaction mechanism involving the C-terminal tail of 14-3-3ζ.
- 14-3-3 proteins may broadly regulate the LLPS of their binding partners, with implications for cellular function and disease.
