Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterolmediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains <120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (<150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation. cholera toxin | membrane heterogeneity | near-field scanning optical microscopy | raft ganglioside GM1 | single-molecule detection

Lipid rafts, defined as domains rich in cholesterol and sphingolipids, are involved in many important plasma membrane functions. Recent studies suggest that the way cells handle membrane cholesterol is fundamental in the formation of such lateral heterogeneities. We propose to model the plasma membrane as a nonequilibrium phase-separating system where cholesterol is dynamically incorporated and released. The model shows how cellular regulation of membrane cholesterol may determine the nanoscale lipid organization when the lipid mixture is close to a phase separation boundary, providing a plausible mechanism for raft formation in vivo.

We present a nonequilibrium approach for the study of a two-dimensional phase-separating ternary mixture. When the component that promotes phase separation is dynamically exchanged with the medium, the separation process is halted and actively maintained finite-size segregation domains appear in the system. In addition to this effect, already reported in our earlier work [J. Gómez, F. Sagués, and R. Reigada, Phys. Rev. E 77, 021907 (2008)], the use of a generic Ginzburg-Landau formalism and the inclusion of thermal fluctuations provide a more dynamic description of the resulting domain organization. Its size, shape, and stability properties are studied. Larger and more circular and stable domains are formed when decreasing the recycling rate, increasing the mobility of the exchanged component, and the mixture is quenched deeper. We expect this outcome to be of applicability in raft phenomenology in plasmatic cell membranes.

The existence of lipid rafts is a controversial issue. The affinity of cholesterol for saturated lipids is manifested in macroscopic phase separation in model membranes, and is believed to be the thermodynamic driving force for raft formation. However, there is no clear reason to explain the small (nanometric) size of raft domains in cell membranes. In a recent paper Yethiraj and Weisshaar [Biophys. J. 93, 3113 (2007)] proposed that the effect of neutral integral membrane proteins may prevent from the formation of large lipid domains. In this paper we extend this approach by studying the effect of the protein size, as well as the lipid-protein interaction. Depending on these factors, two different mechanisms for nanodomain stabilization are shown to be possible for static proteins. The application of these results to a biological context is discussed.

Preferential affinity of cholesterol for saturated rather than unsaturated lipids underlies the thermodynamic process of the formation of lipid nanostructures in cell membranes, that is, of rafts. In this context, phase segregation of two-dimensional ternary lipid mixtures is formally studied from two different perspectives. The simplest approach is based on Monte Carlo simulations of an Ising model corresponding to two interconnected lattices, from which the basic features of the phenomenon are investigated. Then, the coarse-graining mean field procedure of the discrete Hamiltonian is adapted and a Ginzburg-Landau-like free energy expression is obtained. From this latter description, we construct kinetic equations that enable us to perform numerical simulations and to establish analytical phase separation criteria. Application of our formalism in the biological context is also discussed.