LAUSR

Our study examined the steric pressure-induced mixing and reversibility of phase-separated phospholipid regions. It has been previously demonstrated that steric pressure among crowded proteins can induce mixing of lipid domains. However, prior to our work, the dynamics of this process had yet to be investigated. The influence of protein steric pressure on dynamics is important for understanding time-dependent processes in cell membrane-derived biomaterials and highly transient lipid rafts in cells. Therefore we designed experiments that allowed us to examine the process by which protein steric pressure dissolved lipid membranes. Specifically, fluorescence microscopy was used to examine the targeted binding of proteinaceous particles to phase-separated, supported lipid bilayers. Highly localized binding of these particles within the liquid ordered (Lo) domains resulted in a highly crowded environment, leading to a build-up of steric pressure. The alleviation of this steric pressure induced mixing of the Lo domains with the surrounding liquid disordered (Ld) phase. With the development of a first-principles mass transfer model, the dynamics of this mixing transition were observed as a function of the steric pressure among proteins. At sufficiently high steric pressure, rapid dissolution of Lo domains occurred. As steric pressure was decreased, mixing became more gradual and occurred in a step-wise process where small lipid clusters were initially ejected from Lo domains. Time-dependent dissolution data for Lo domains was in quantitative agreement with the mass-transfer model. Diffusion coefficients derived from the model indicate that lipids undergo diffusion as clusters, rather than single lipids. We also examined systems in which proteins bound to the Ld phase. Our results strongly suggest that the degree of protein crowding and the phase to which proteins are targeted control the rate and mechanism of steric pressure-induced mixing. This phenomena is applicable to the development of many biologically derived materials, including high-density arrays, microfluidic networks, and biosensors. Controlled mixing also provides fundamental insights to the impact of proteins on the stability and dynamics of lipid rafts in cells.