Membrane-induced 2D phase separation of focal adhesion proteins

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol.