BPS2026 – Molecular dynamics simulations elucidate the biophysical mechanisms of transbilayer condensate coupling

Many cellular functions rely on the ability to reorder or reshape the lipid bilayer. For example, stages of cell signaling pathways involve the organization of protein domains on both sides of the membrane. Liquid-liquid phase separation of proteins occurs on biological membranes, where it is involved in some of these transmembrane processes. Recent work has demonstrated colocalization between protein condensates on opposing leaflets of the bilayer, without the need for membrane-spanning proteins. These results suggest that protein phase separation on one side of the membrane is able to spontaneously couple with phase separation on the opposite side. This coupling could arise from reorganization of lipids when proteins phase separate on their surfaces. However, the detailed molecular mechanism behind this coupling phenomenon remains poorly understood. Here, we seek to understand the protein-protein and protein-membrane interactions that give rise to this behavior. We began by performing coarse grained molecular dynamics simulations of a system with a pre-formed condensate on either leaflet. In this condition, we observed stably coupled diffusion of the condensates for the duration of the simulation. Next, we constructed a system with proteins in a dispersed phase on either side and allowed protein condensates to form and coarsen over time. Here, again we found that the resulting condensates were stably coupled. We next analyzed the impact on lipid organization, packing, and order to elucidate potential entropic effects. Finally, we examined the interactions between the proteins and lipids to probe possible energetic drivers of coupling. Our work provides insight on the role that protein condensates play in trans-bilayer processes, potentially identifying new biophysical mechanisms by which phase separation of proteins can transfer information across membrane boundaries.