Leukocyte transendothelial migration (TEM) is absolutely fundamental to the 
inflammatory response, and involves initial pseudopod protrusion and subsequent 
polarised migration across inflamed endothelium. Ezrin/radixin/moesin (ERM) 
proteins are expressed in leukocytes and mediate cell shape changes and 
polarity. The spatio-temporal organisation of ERM proteins with their targets, 
and their individual contribution to protrusion during TEM, has never been 
explored. Here, we show that blocking binding of moesin to phosphatidylinositol 
4,5-bisphosphate (PIP2) reduces its C-terminal phosphorylation during monocyte 
TEM, and that on-off cycling of ERM activity is essential for pseudopod 
protrusion into the subendothelial space. Reactivation of ERM proteins within 
transmigrated pseudopods re-establishes their binding to targets, such as 
L-selectin. Knockdown of ezrin, but not moesin, severely impaired the 
recruitment of monocytes to activated endothelial monolayers under flow, 
suggesting that this protein plays a unique role in the early recruitment 
process. Ezrin binds preferentially to L-selectin in resting cells and during 
early TEM. The moesin-L-selectin interaction increases within transmigrated 
pseudopods as TEM proceeds, facilitating localised L-selectin ectodomain 
shedding. In contrast, a non-cleavable L-selectin mutant binds selectively to 
ezrin, driving multi-pseudopodial extensions. Taken together, these results show 
that ezrin and moesin play mutually exclusive roles in modulating L-selectin 
signalling and shedding to control protrusion dynamics and polarity during 
monocyte TEM.