Ezrin activation in vitro: Investigation of ezrin's conformation and the interaction between ezrin and F-actin

Abstract

The function of ezrin, a member of the ezrin-radixin-moesin (ERM) protein family, is to regulate the cell membrane architecture within the context of fundamental biological processes by linking the membrane and the actin cytoskeleton. In the inactive state, ezrin is conformationally masked by self-association of N- and C-terminal domains. Ezrin activation is thought to rely on a conformational change induced by binding to L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) and followed by phosphorylation of a conserved threonine (T) residue, thus rendering the binding site for filamentous actin (F-actin) accessible. However, the contribution of the individual activation factor is still controversially discussed. Moreover, several lines of evidence indicate that PIP2 binding might be sufficient to establish a stable interaction between ezrin and F-actin. In the first part of this work, different strategies for the preparation of PIP2-containing solid-supported lipid bilayers (SLBs) are presented overcoming often encountered spreading issues owing to the multiple negative charges of PIP2. Reflectometric interference spectroscopy (RIfS) and fluorescence microscopy were employed to verify the SLB formation. With regard to reliable formation of SLBs containing high PIP2 fractions and subsequent ezrin binding to control the accessibility of PIP2, only spreading at pH 4.8 gave satisfactory results, whereas calcium ions or increased ionic strength revealed to be not suitable. Solid-supported hybrid membranes, prepared by spreading of PIP2-containing vesicles on hydrophobically functionalized substrates, were characterized by comparably high lateral PIP2 mobility in fluorescence recovery after photobleaching (FRAP) experiments using a fluorescent PIP2 analogue as tracer. Binding of ezrin to PIP2 effectively reduced its diffusion, implicating that ezrin is able to maintain PIP2 clusters within a cellular context. The main part of this thesis is dedicated to investigating to what extent PIP2 and phosphorylation contribute to ezrin activation regarding on the one hand a possible height change of ezrin aggregates on SLBs due to the conformational change and on the other hand the strength of its interaction with F-actin. The influence of phosphorylation was assessed by using three ezrin variants, namely ezrin wildtype, ezrin T567A (nonphosphorylatable), and ezrin T567D (pseudophosphorylated). The influence of PIP2 on the activation was addressed by using two different ezrin immobilization strategies, either nickel nitrilotriacetic acid (NTA-Ni)- or PIP2-mediated. Only the combination of PIP2 binding and phosphorylation led to a significantly lower protein height level on the SLB as determined by RIfS and atomic force microscopy imaging. By colloidal probe microscopy (CPM), it was found that PIP2 is sufficient to significantly enhance the interaction between ezrin and F-actin in terms of increased surface adhesion energies, whereas phosphorylation contributes to a minor extent. Notably, the adhesion forces remained almost unchanged. From these findings, we inferred that ezrin activation generates rather many weak than few strong bonds between ezrin and F-actin, forming a stable multivalent interaction.