Elucidating Local Confinement in Crowded Polymer Solutions Within Giant Unilamellar Vesicles (GUVs) Through Single Particle Tracking Toward Deeper Understanding of Cells

In a crowded environment, macromolecules occupy a significant proportion volume of cells to repulse other molecules in H 2 O-rich phase domains. These H 2 O-rich phase domains have been found to significantly influence material transportation and biochemical reactions. However, the accurate quantification of the size of these domains remains a challenge. Here, formulas are set up to calculate the anomalous diffusion exponent ( α ), the concentration threshold ( c p ), and the radius of the H 2 O-rich phase domain ( r 0 ) to characterize the crowded solutions. Fitting coefficient ( R 2 ) of the r 0 fitted curves are 0.9989 for PEG-8k Da and 0.9901 for PEG-20k Da, respectively, which confirms the formulas to be suitable for quantifying the crowding degree. The values of α , r 0 , and c p of three different cell lysates is are calculated using these formulas. The r 0 values of the cytosol from eukaryotic cells are 1.22 µm for HEK-293T and 1.46 µm for S. Cerevisiae , respectively, which are smaller than that (2.13 µm) from prokaryotic cells ( E. coli ). This may be due to the more complex components, with higher molecular weight but lower concentration in the eukaryotic cells. This method for quantifying the H 2 O-rich phase in a crowded solution helps to have a deeper understanding of the biochemical mechanism inside cells.