Quantitative analysis of ezrin turnover dynamics in the actin cortex

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.