Dynamics and Statics in Phase Separating, Adhering Lipid Membranes

Abstract

When living cells adhere to one another or their environment they organize their cellular membrane components into physical structures responsible for biological functions. One striking example is the immunological synapse formed at the adhesion site of immune cells which exhibits dual heterogeneities—characterized by differences in proteins and lipids—that are at first dynamic for tens of seconds to minutes and then stable for tens of minutes to an hour (Grakoui et al., Science 285, 221-227 (1999)).We employ a minimalist biophysical approach to investigate the dynamic formation and static persistence of lipid and protein heterogeneities at the adhesion site of lipid membranes. In our experiments we adhere mixed-lipid model membrane vesicles to a supported bilayer substrate via biotin-avidin binding. In one set of experiments, our vesicle membranes are made of a lipid mixture near an Lo-Ld phase boundary but are kept above the miscibility transition temperature as measured in free floating vesicles (S. L. Veatch, S. L. Keller, Biophysical journal 85, 3074-3083 (2003)). When the membranes adhere, they form two coexisting heterogeneities at the adhesion site that are compositionally distinct from the non-adhered portion of the membrane: (1) a central region that excludes a membrane dye—which marks the Ld phase—and is devoid of binders and (2) a peripheral region that is enriched in the membrane dye and is dense with binders. In a second set of experiments, our vesicle membranes are made of binary lipid mixtures (DOPC and Cholesterol) that are reported to be miscible at all accessible temperatures. Remarkably, when these membranes adhere, they form domains that exclude the membrane dye at the adhesion site and the domains grow on seconds-to-minutes-long time scales. We suggest the domain growth in this system is a nonequilibrium phase transition.