Measuring concentration and diffusivity within biomolecular condensates using calibration-free scanning fluorescence...

Area of Science:

• Cell Biology
• Biophysics
• Biochemistry

Background:

• Cells utilize membrane-less organelles, or biomolecular condensates, to regulate biochemical activity.
• The precise mechanisms governing the interplay between phase separation and biochemical reactions within these condensates remain poorly understood.
• Accurate characterization of molecular concentrations and diffusivities in the dense phase is crucial for understanding biochemical regulation in condensates.

Purpose of the Study:

• To develop a calibration-free method for quantifying molecular concentrations and diffusivities in both dilute and dense phases of biomolecular condensates.
• To elucidate the relationship between molecular concentration, diffusivity, and phase behavior within condensates.
• To investigate the stability of aggregation-prone proteins within dense phases.

Main Methods:

• Utilized temporal line scan fluorescence correlation spectroscopy (tLS-FCS) and sinusoidal scan fluorescence correlation spectroscopy (ss-FCS).
• Developed a novel calibration-free approach to overcome limitations of traditional fluorescence correlation spectroscopies.
• Applied the method to measure the phase diagram of the DEAD-box protein Ddx4 and diffusivities of recruited client molecules.

Main Results:

• Successfully quantified concentrations and diffusivities of molecules in dilute and dense phases without calibration standards.
• Determined the complete phase diagram for the intrinsically disordered region of the DEAD-box protein Ddx4.
• Observed a significant decrease in client molecule diffusivity with increasing concentration in the dense phase.

Conclusions:

• The developed calibration-free FCS methods provide essential tools for studying biochemical regulation in phase-separated condensates.
• The observed decrease in diffusivity with concentration may explain the stability of aggregation-prone proteins in dense phases.
• This work advances the understanding of molecular dynamics within biomolecular condensates and their role in cellular regulation.