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

Biomolecular condensates formed by liquid–liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.