Detecting and quantifying liquid-liquid phase separation in living cells by model-free calibrated half-bleaching

Area of Science:

• Cell biology
• Biophysics

Background:

• Cells utilize numerous substructures, with liquid-liquid phase separation (LLPS) being a proposed formation mechanism.
• Reliably distinguishing LLPS from other molecular organization principles remains a significant challenge in cell biology.

Purpose of the Study:

• To benchmark methods for distinguishing LLPS from alternative molecular organization mechanisms.
• To introduce a robust workflow for analyzing the interfacial properties of biomolecular condensates.

Main Methods:

• Benchmarking of 1,6-hexanediol treatment and fluorescence recovery after photobleaching (FRAP) using in vitro and in vivo model systems.
• Utilizing half-bleach experiments to assess preferential internal mixing as a distinguishing feature.
• Introducing model-free calibrated half-FRAP (MOCHA-FRAP) to quantify interfacial barriers.

Main Results:

• 1,6-hexanediol treatment and classical FRAP were found to be unreliable for distinguishing LLPS from clustered binding sites.
• Half-bleach experiments demonstrated robust differentiation between LLPS and clustered binding sites.
• MOCHA-FRAP revealed an increasing strength of the interfacial barrier in heterochromatin foci, nucleoli, stress granules, and nuage granules.

Conclusions:

• Preferential internal mixing, as assessed by half-bleach experiments, is a reliable indicator of LLPS.
• MOCHA-FRAP provides a powerful tool for characterizing the biophysical properties of biomolecular condensates.
• This work offers a new approach to understanding the formation and function of cellular substructures.