Ezrin is a member of the ERM (Ezrin, Radixin, Moesin) family of proteins. It is a membrane-cytoskeleton linker protein, which is heavily involved in membrane remodelling. Through this remodelling behaviour, ezrin is shown to form cellular protrusions (such as stereocillia and microvilli) which have crucial roles in many pathologies and cancer metastasis.
Ezrin is know to interact with cellular membranes, specifically, the phospholipid PIP2. This interaction is still not well understood. Cosedimentation and TIRF microscopy have been employed to understand the kinetics of this this interaction. In order to utilise fluorescence microscopy, site directed mutagenesis was used to generate mutants amenable to site specific cysteine labelling. Mulit-angle laser light scattering and circular dichroism spectroscopy were used to ensure the behaviour of cysteine mutants was comparable to that of the wild type.
In preliminary cosedimentation experiments, specific binding of ezrin to PIP2 containing vesicles was observed and mutants for use in TIRF microscopy were shown to bind in a similar manner. Having developed and confirmed our labelling construct was valid, TIRF microscopy experiments were performed to assess the kinetics of ezrin binding to lipid vesicles of various concentrations at a single molecule level. Understanding the mode of this interaction and the kinetics of ezrin membrane binding, we hope to elucidate the underlying biochemical pathways involved in membrane remodelling events and their pathological manifestations.