Sequential binding of Ezrin and Moesin to L-selectin regulates monocyte protrusive behaviour during transmigration

Abstract

Leukocyte transendothelial migration (TEM) is absolutely fundamental to the inflammatory response, involving initial pseudopod protrusion and subsequent polarised migration across inflamed endothelium. Ezrin/radixin/moesin (ERM) proteins are expressed in leukocytes and mediate cell shape change and polarity. The spatio-temporal organisation of ERM with their targets, and their individual contribution to protrusion during TEM, has never been explored. Here we show that blocking PIP2 binding reduces C-terminal phosphorylation of moesin during monocyte TEM, and that on/off cycling of ERM activity is essential for pseudopod protrusion into the subendothelial space. Reactivation of ERM within transmigrated pseudopods establishes rebinding to targets, such as L-selectin. Knockdown of ezrin, but not moesin, severely impaired recruitment to activated endothelial monolayers under flow, suggesting a unique role in early recruitment. Ezrin binds preferentially to L-selectin at rest and in early TEM. Moesin/L-selectin interaction enriches within transmigrated pseudopods as TEM proceeds, facilitating localised ectodomain shedding. Non-cleavable L-selectin mutant binds selectively with ezrin, driving multi-pseudopodial extensions. Taken together, ezrin and moesin play mutually exclusive roles in modulating L-selectin signalling and shedding to control protrusion dynamics and polarity during monocyte TEM.