Protein cargos anchored on the lipid membrane can be segregated by fluidic domain phase separation. Lipid membranes at certain compositions may separate into lipid domains to segregate cargos, and protein cargos themselves may be involved in protein condensate domain formation with multivalent binding proteins to segregate cargos. Recent studies suggest that these two driving forces of phase separation closely interact on the lipid membranes to promote codomain formation. In this report, we studied the effect of cargo density on the outcome of the cargo phase separation on giant unilamellar vesicles. Proteins and lipids are connected only by the anchored cargos, so it was originally hypothesized that higher cargo density would 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 a tug of war opposing each other. Cooperative domain formation is dominant under lower anchor density conditions, and above a threshold density, steric pressure was dominant opposing the domain formation. The result suggests that the cargo density is a key parameter affecting the outcome of cargo organization on the lipid membranes by phase separation.
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