Abstract
ABSTRACTLeukocyte 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.
Highlights
An essential feature of acute inflammation is the migration of circulating innate immune cells, such as monocytes, towards damaged/infected tissue (Nourshargh and Alon, 2014; Schimmel et al, 2017)
Regulation of ERM protein activity during transendothelial migration (TEM) To monitor the subcellular organisation of ERM proteins during TEM, the human monocyte-like cell line THP-1 was subjected to lentiviral transduction with short hairpin RNA to deplete endogenous levels of moesin (Fig. S1A–D)
To address the impact of PIP2 binding on moesin activation during TEM, a series of lysine (K) to asparagine (N) mutations at positions 253, 254, 262 and 263 (K253N, K254N, K262N and K263N), which are known to be important for PIP2 binding (Barret et al, 2000), were engineered into the moesin–GFP FERM domain and stably reconstituted into cells lacking endogenous moesin
Summary
An essential feature of acute inflammation is the migration of circulating innate immune cells, such as monocytes, towards damaged/infected tissue (Nourshargh and Alon, 2014; Schimmel et al, 2017). The ezrin/radixin/moesin (ERM) proteins link the plasma membrane and the underlying cortical actin-based cytoskeleton to mediate changes in cell shape (Fehon et al, 2010; Ivetic and Ridley, 2004a). By anchoring receptors to the underlying actin cortex, ERM proteins restrict lateral mobility and modulate clustering for signal transduction (Treanor et al, 2011). In this regard, ERM proteins play an essential role in modulating diverse signal transduction events. ERM proteins play an essential role in modulating diverse signal transduction events Despite their high level of similarity, different ERM protein members exhibit non-redundant roles in multiple cell types, including leukocytes (Ilani et al, 2007)
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