Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching.

Area of Science:

• Cell Biology
• Biophysics

Background:

• Membraneless organelles form via liquid-liquid phase separation, crucial for cellular functions.
• Biomolecular mobility within these condensates influences cellular function and disease.
• Fluorescence recovery after photobleaching (FRAP) is a common method to study condensate dynamics.

Purpose of the Study:

• To systematically evaluate different models for FRAP analysis of protein condensates.
• To assess the impact of model choice and boundary conditions on measured values.
• To provide guidelines for appropriate modeling in FRAP experiments.

Main Methods:

• Performed FRAP experiments on both in vitro reconstituted droplets and intracellular condensates.
• Analyzed data using a systematic examination of various fitting models.
• Investigated the influence of different experimental boundary conditions (e.g., subregion vs. whole condensate bleaching).

Main Results:

• Model choice and boundary conditions significantly affect measured values of condensate fluidity and diffusion coefficients.
• Divergent results can arise depending on whether subregions or the entire condensate are bleached.
• The choice of model critically influences the interpretation of biomolecular mobility.

Conclusions:

• Careful consideration of modeling frameworks is essential for accurate FRAP analysis of protein condensates.
• Guidelines are proposed for selecting appropriate models to ensure reliable measurements.
• Further research is needed on molecular dynamics at condensate interfaces for quantitative insights.