Lipopolymer Crowding in Polymer-Tethered Lipid Bilayers Alters Lipid Mixing Behavior and Protein Sequestration in the Presence of Raft-Mimicking Lipid Mixtures

Abstract

It is now well established that the cytoplasm and plasma membrane of cells are characterized by high concentrations of proteins. Consequently, macromolecular crowding and confinement effects are believed to play important roles in the organization, dynamics, and function of proteins in cellular environments. However, the functional significance of molecular crowding remains unclear and conclusions are still controversial, due to the complexity of cellular systems. To address this experimental challenge, here we introduce a model membrane system on the basis of a polymer-tethered lipid bilayer, in which lipopolymers act as crowding agents. Our experiments show that changes in lipopolymer concentration have a profound effect on the lipid mixing behavior of raft-mimicking lipid mixtures forming micron-size liquid-ordered (lo) and liquid-disordered (ld) phase separations. Complementary fluorescence correlation spectrometry (FCS), confocal fluorescence intensity analysis, and photon counting histogram (PCH) analysis experiments demonstrate that lipopolymer-mediated molecular crowding also influences the lateral diffusion of lipids and membrane proteins, as well as membrane protein sequestration in raft-mimicking lipid mixtures. Interestingly, our experiments confirm that elevated lipopolymer concentrations cause the formation of buckling structures, which can be seen as a stress relaxation phenomenon. Notably, the extent of membrane buckling is substantially different in lo and ld lipid regions.