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.
Shape, dynamics and viscoelastic properties of eukaryotic cells are largely governed by a thin reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of weakly adhered mesenchymal cells (fibroblasts) and epithelial cells (MDCK II) from parallel-plate compression and force relaxation experiments.We introduce an analytical expression for the compression and force relaxation based on the elastic-viscoelastic correspondence principle by treating the cell as a closed liquid-filled shell and assuming a power law to describe the change of surface area during deformation. This approach gives access to pre-stress, area compressibility modulus and the power law (fluidity) coefficient, which we modulate by interfering with myosin activity. We find that the fluidity of cells decreases with increasing intrinsic pre-stress as shown for isolated actin networks subject to external stress.
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