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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
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Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of
Jian Liu1, Wenxin Yu1, Chaoqing Dong1
1School of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China.
The Analyst
|March 25, 2024
Summary
Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) offers improved reproducibility for studying protein phase separation dynamics, including self-fusion and recruitment behaviors, overcoming limitations of conventional methods.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Biology
Background:
- Protein phase separation is crucial for biological processes and disease development.
- Conventional fluorescence imaging and fluorescence correlation spectroscopy (FCS) have limitations in studying protein phase separation due to resolution and detection area constraints.
- These limitations hinder a full understanding of protein condensate behavior across various scales.
Purpose of the Study:
- To introduce and validate a novel objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) method for studying protein phase separation.
- To compare the efficacy of Scan-FCCS with conventional FCS for characterizing protein phase separation behaviors.
- To investigate the self-fusion and recruitment dynamics of CRDBP proteins and their response to environmental factors.
Main Methods:
- Development and application of objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS).
- Utilized CRDBP proteins fused with fluorescent proteins (EGFP and mCherry) as a model system.
- Compared Scan-FCCS with conventional FCS for analyzing protein phase separation.
Main Results:
- Scan-FCCS demonstrated significantly improved reproducibility compared to conventional FCS due to an expanded detection zone.
- Determined the phase change concentration of CRDBP to be 25 nM and observed complete fusion of mCherry-CRDBP and EGFP-CRDBP within 70 minutes at 500 nM.
- Identified that salt concentration inhibits CRDBP self-fusion, while molecular crowding agents enhance it.
- Characterized the recruitment behavior and dynamics of CRDBP to β-catenin proteins.
Conclusions:
- Scan-FCCS is a superior method for studying protein phase separation, offering enhanced reproducibility and a larger detection area.
- The method provides valuable insights into self-fusion and recruitment dynamics, crucial for understanding biological processes and disease mechanisms.
- Scan-FCCS overcomes the spatio-temporal resolution and detection area limitations of conventional techniques.
