Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation

Abstract

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins thought to congregate in rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers may provide insights into the roles rafts play in cells. Multiple studies of PEG-conjugated lipids have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure. Furthermore, our recent work has shown that steric pressure on membrane surfaces can oppose the assembly of lipid domains. Based on these findings, this work investigates whether steric pressure among membrane-bound polymeric domains could oppose membrane phase separation. To address this question we created GUVs with a ternary composition (DPPC:DOPC:Chol), which separated into coexisting liquid ordered and disordered domains. DPPE-PEG lipids (1, 2, or 5 kDa PEG) were included at increasing molar concentrations. As the distance between PEG molecules on membrane surfaces decreased, the fraction of GUVs that phase separated decreased, suggesting that membrane-bound PEG chains opposed phase separation. Interestingly, for lipid mixtures with a Tm just above room temperature, domains were destabilized when the spacing among PEG chains was greater than their hydrodynamic diameter, suggesting that relatively little pressure was required. In contrast, when we examined GUVs with a Tm significantly above room temperature, domains were not destabilized until the PEG spacing was much less than their hydrodynamic diameter, suggesting that the polymer chains had to pass through the high pressure brush transition to drive lipid mixing. These results demonstrate that steric pressure among membrane-bound polymers can drive mixing of lipid domains and suggest that the ability of cellular lipid rafts to resist this pressure and concentrate polymeric domains likely depends heavily on their lipid composition.