Study on Phase Separation of Fused in Sarcoma by Fluorescence Correlation Spectroscopy.
Langmuir
; 40(2): 1266-1276, 2024 01 16.
Article
en En
| MEDLINE
| ID: mdl-38157426
ABSTRACT
Liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) has emerged as a fundamental principle underpinning cellular function and malfunction. However, we know little about the FUS phase transition process from individual molecules to nanoscale condensates, which plays important roles in neurodegenerative diseases. Here, we propose the fluorescence correlation spectroscopy (FCS) method to quantitatively study the phase separation process of FUS protein with the fluorescent tag-enhanced green fluorescent protein (EGFP), from individual molecules to nanoscale condensates. The characteristic diffusion time (τD) of the protein condensates can be obtained from the FCS curve, which increases with the growth of the protein hydration radius. The bigger the τD value of the protein condensates, the larger the condensates formed by the phase separation of FUS. By this method, we discovered that the critical concentration for FUS to phase separation was 20 nM. We then plotted FUS phase diagrams based on τD under different concentrations of NaCl and found that both low-salt and high-salt concentrations tended to promote FUS-EGFP phase separation. Our results showed that ATP has a good inhibitory effect on FUS phase separation, and its inhibition constant IC50 was 3.2 mM. Finally, we evaluated the inhibition efficiency of single-stranded DNA sequences (ssDNA) on FUS phase separation and demonstrated that ssDNA containing three copies of TCCCCGT had relatively strong inhibition efficiency. In summary, our work provides detailed insight into the FUS phase transition process from individual molecules to nanoscale condensates at nanomolar concentrations and can be exploited for drug screening of neurodegenerative diseases.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteína FUS de Unión a ARN
Límite:
Humans
Idioma:
En
Revista:
Langmuir
Asunto de la revista:
QUIMICA
Año:
2024
Tipo del documento:
Article
Pais de publicación:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
/
UNITED STATES OF AMERICA
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US
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USA