Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments. In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments. Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement.

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.

Small molecule sensing is of great importance in pharmaceutical research. While there exist well established screening methods such as EMSA (electrophoretic motility shift assay) or biointeraction chromatography to report on successful binding interactions, there are only a few techniques that allow studying and quantifying the interaction of low molecular weight analytes with a binding partner directly. We report on a binding assay for small molecules based on the reflectivity change of a porous transparent film upon immobilisation of an absorbing substance on the pore walls. The porous matrix acts as a thin optical transparent film to produce interference fringes and accumulates molecules at the inner wall to amplify the sensor response. The benefits and limits of the assay are demonstrated by investigating the binding of biotin labelled with an atto dye to avidin physisorbed within an anodic aluminium oxide membrane.

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.