The creation of biologically inspired artificial lipid bilayers on planar supports provides a unique platform to study membrane-confined processes in a well-controlled setting. At the plasma membrane of mammalian cells, the linkage of the filamentous (F)-actin network is of pivotal importance leading to cell-specific and dynamic F-actin architectures, which are essential for the cell's shape, mechanical resilience, and biological function. These networks are established through the coordinated action of diverse actin-binding proteins and the presence of the plasma membrane. Here, we established phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P 2 )-doped supported planar lipid bilayers to which contractile actomyosin networks were bound via the membrane−actin linker ezrin. This membrane system, amenable to high-resolution fluorescence microscopy, enabled us to analyze the connectivity and contractility of the actomyosin network. We found that the network architecture and dynamics are not only a function of the PtdIns[4,5]P 2 concentration but also depend on the presence of negatively charged phosphatidylserine (PS). PS drives the attached network into a regime, where low but physiologically relevant connectivity to the membrane results in strong contractility of the actomyosin network, emphasizing the importance of the lipid composition of the membrane interface.

The connection between the actomyosin cortex and the plasma membrane of eukaryotic cells is investigated by creating a versatile, near-native model system that allows studying the architecture and contractility of the cortex as a function of lipid composition. We found that the concentration of phosphatidylserine, a characteristic lipid of the inner leaflet of mammalian plasma membranes, plays a pivotal role in the binding of the membrane-cytoskeleton linker protein ezrin and the resulting contractile behavior of an adjacent actin network. In addition to the specific receptor lipid for ezrin, i.e., PtdIns[4,5]P2 cross-linking the network to the inner leaflet, the presence of phosphatidylserine in the membrane is critical to enhancing the binding of ezrin to PtdIns[4,5]P2 and allows rapid local actin contraction at physiologically relevant concentrations in the regime of 1-3 mol% PtdIns[4,5]P2. In the presence of phosphatidylserine, the additional negative charges in the membrane may induce enhanced sliding of the filaments on the membrane surface due to repulsive interactions between F-actin and the bilayer readily leading to the emergence of contraction foci. Conversely, if phosphatidylserine is replaced by an increased PtdIns[4,5]P2 concentration of 5 or 8 mol%, a highly connected but non-contracting actin network is observed.