The cortical cytoskeleton attached to the plasma membrane is an essential mechanical component within the cell. It regulates fundamental processes including cell division, motility and shape. In order to investigate how the individual components of the cortical cytoskeleton contribute to the mechanical properties of the plasma membrane, a model system was established. Pore spanning lipid bilayers (PSLBs) containing a specific receptor lipid were prepared on porous silicon nitride substrates. Subsequently, F-actin was bound to the PSLBs either directly via electrostatic interaction or via the linker protein ezrin. Fluorescence microscopy was utilized to visualize the prepared PSLBs and the F-actin binding. Indentation experiments were performed by atomic force microscopy (AFM) to elucidate the mechanical properties of the PSLBs. The apparent spring constants of the PSLBs were extracted from the measured force-indentation curves. In our system, the determined apparent spring constants directly correlate with the lateral tension of the PSLBs. We analyzed the lateral tension as a function of F-actin coverage.

Phosphoinositides and in particular L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) are key lipids controlling many cellular events and serve as receptors for a large number of intracellular proteins. To quantitatively analyze protein-PIP2 interactions in vitro in a time-resolved manner, planar membranes on solid substrates are highly desirable. Here, we describe an optimized protocol to form PIP2 containing planar solid supported membranes on silicon surfaces by vesicle spreading. Supported lipid bilayers (SLBs) were obtained by spreading POPC/PIP2 (92:8) small unilamellar vesicles onto hydrophilic silicon substrates at a low pH of 4.8. These membranes were capable of binding ezrin, resulting in large protein coverage as concluded from reflectometric interference spectroscopy and fluorescence microscopy. As deduced from fluorescence microscopy, only under low pH conditions, a homogeneously appearing distribution of fluorescently labeled PIP2 molecules in the membrane was achieved. Fluorescence recovery after photobleaching experiments revealed that PIP2 is not mobile in the bottom layer of the SLBs, while PIP2 is fully mobile in the top layer with diffusion coefficients of about 3 μm(2)/s. This diffusion coefficient was considerably reduced by a factor of about 3 if ezrin has been bound to PIP2 in the membrane.