Details

Autor(en) / Beteiligte

• Fukuyama, Mao
• Nishinami, Suguru
• Maruyama, Yoko
• Ozawa, Taiki
• Tomita, Shunsuke
• Ohhashi, Yumiko
• Kasuya, Motohiro
• Gen, Masao
• Chatani, Eri
• Shiraki, Kentaro
• Hibara, Akihide

Titel

Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid–Liquid Phase Separation

Ist Teil von

• Analytical chemistry (Washington), 2023-07, Vol.95 (26), p.9855-9862

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2023

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Elucidating the link between amyloid fibril formation and liquid–liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.