Lateral compression of lipids drives transbilayer coupling of protein condensates.

Abstract

Protein phase separation that is restricted to a two-dimensional lipid membrane has recently gained much attention due to its involvement in various cellular processes. Two popular in vitro model membrane systems, giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs), have been used to study it. However, due to the accessibility to only one side of the membrane in these systems, protein phase separation on both sides of the membrane remains unexplored. Here, we introduce a freestanding planar lipid membrane array as an effective platform to study membrane-associated protein phase separation. Protein condensates on the membrane display liquid-like features, and environmental stimuli, such as salt concentration and temperature, effectively control the observed phase separation results in our system with the application of several intrinsically disordered proteins. Interestingly, we observe transbilayer coupling of protein condensates such that protein-rich domains on one side of the membrane are found to colocalize with those on the other side of the membrane. How do these protein phases communicate across the lipid bilayer? Based on data from lipid probe partitioning and photobleaching experiments, we hypothesize that protein condensation on the membrane exerts lateral compressive stress on the underlying lipids, driving the formation of lipid domains with reduced fluidity. Specifically, lipid domains with reduced fluidity and increased lipid packing, formed by protein condensation on each membrane leaflet, are attracted to one another, leading to transbilayer coupling of protein condensates. These findings suggest a previously unknown mechanism by which cellular signals from one membrane leaflet, triggered by membrane-associated protein phase separation, can be transferred to the opposing leaflet.