Probing Inhomogeneous Diffusion in the Microenvironments of Phase-Separated Polymers under Confinement

Area of Science:

• Cell Biology
• Biophysics
• Biochemistry

Background:

• Biomolecular condensates, formed via liquid-liquid phase separation, are increasingly recognized as vital cellular components.
• Their structural organization and biophysical properties, essential for function, remain poorly understood, especially at in vivo scales.
• Existing in vitro studies often use condensates much larger than those found within cells.

Purpose of the Study:

• To investigate the structural organization and biophysical properties of biomolecular condensates at physiologically relevant, in vivo-like sizes.
• To explore the dynamics of probe particles within confined condensates and relate them to condensate size and composition.

Main Methods:

• Utilized confinement microscopy to visualize and control the size of biomolecular condensates.
• Created specific confinement length scales mimicking cellular environments.
• Employed probe particle diffusion measurements to analyze internal dynamics.

Main Results:

• Observed anomalous diffusion of probe particles within confined condensates.
• Detected heterogeneous dynamics in both PEG/dextran and ribonucleoprotein (RNP) condensates.
• Proposed a hopping diffusion mechanism to explain the non-Gaussian dynamics.
• Found that probe particle diffusion in dextran-rich condensates, but not RNP condensates, is dependent on condensate size.

Conclusions:

• Confinement microscopy enables the study of biomolecular condensates at biologically relevant scales.
• Anomalous diffusion and heterogeneous dynamics suggest complex internal organization and transport mechanisms within condensates.
• Condensate size can influence internal dynamics, particularly in certain compositions like dextran-rich systems.