Crowder Chain Length Variability and Excluded Volume Effect on the Phase Separation Behavior of Mucin.

Phase separation within cellular membranes, a critical process underpinning diverse cellular functions, is significantly influenced by transmembrane proteins. Therefore, elucidating the behavior of a transmembrane protein in its phase-separated state is of utmost importance. Our study explores mucin behavior in the cellular milieu, aiming to determine the role of crowder chain length and excluded volume in phase separation. Confocal microscopy images demonstrate the strong partitioning of mucin into the condensed phase influenced by hydrophobic and electrostatic interactions. Fluorescence recovery after photobleaching analysis revealed increased mobility in the presence of shorter chain length crowders, indicating the dynamic behavior of protein within condensed phases. Excluded volume calculation using the theoretical model emphasizes its importance in mucin phase separation under crowded conditions. Our findings underscore the ability of mucin to phase-separate under crowded conditions, highlighting the crucial role of excluded volume and enhancing our understanding of its involvement in cancer progression.