Membrane Cargo Density-Dependent Interaction between Proteinand Lipid Domains on the Giant Unilamellar Vesicles

Protein cargos anchored on the lipid membrane canbe segregated byfluidic domain phase separation. Lipidmembranes at certain compositions may separate into lipiddomains to segregate cargos, and protein cargos themselves maybe involved in protein condensate domain formation withmultivalent binding proteins to segregate cargos. Recent studiessuggest that these two driving forces of phase separation closelyinteract on the lipid membranes to promote codomain formation.In this report, we studied the effect of cargo density on theoutcome of the cargo phase separation on giant unilamellarvesicles. Proteins and lipids are connected only by the anchoredcargos, so it was originally hypothesized that higher cargo densitywould increase the degree of interaction between the lipid and protein domains, promoting more phase separation. However,fluorescence image analysis on different cargo densities showed that the cooperative domain formation and steric pressure are at atug of war opposing each other. Cooperative domain formation is dominant under lower anchor density conditions, and above athreshold density, steric pressure was dominant opposing the domain formation. The result suggests that the cargo density is a keyparameter affecting the outcome of cargo organization on the lipid membranes by phase separation.