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 H₂O-rich phase domains. These H₂O-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 H₂O-rich phase domain (r₀) to characterize the crowded solutions. Fitting coefficient (R²) of the r₀ 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, r₀, and c_p of three different cell lysates is are calculated using these formulas. The r₀ 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 H₂O-rich phase in a crowded solution helps to have a deeper understanding of the biochemical mechanism inside cells.