Conformational Switching in Ezrin Regulates Morphological and Cytoskeletal Changes Required for B Cell Chemotaxis

Abstract

B cell chemotaxis occurs in response to specific chemokine gradients and is critical for homeostasis and immune response. The molecular regulation of B cell membrane-actin interactions during migration is poorly understood. In this study we report a role for ezrin, a member of the membrane-cytoskeleton crosslinking Ezrin-Radixin-Moesin (ERM) proteins, in the regulation of the earliest steps of B cell polarization and chemotaxis. We visualized chemokine-induced changes in murine B cell morphology using scanning electron microscopy, and spatiotemporal dynamics of ezrin in B cells using epifluorescence and total internal reflection microscopy. Upon chemokine stimulation ezrin is transiently dephosphorylated to assume an inactive conformation, and localizes to the lamellipodia. B cells expressing a phosphomimetic conformationally active mutant of ezrin, or those in which ezrin dephosphorylation was pharmacologically inhibited displayed impaired microvillar dynamics, morphological polarization and chemotaxis. Our data suggest a two-fold involvement of ezrin in B cell migration whereby it first undergoes chemokine-induced dephosphorylation to facilitate membrane flexibility, followed by relocalization to the actin-rich lamellipodia for dynamic forward protrusion of the cells.

Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice

“…Several in vitro studies support the view that the presence of phosphomimetic ERM protein (ie, ERM protein unable to dephosphorylate) impairs lymphocyte migration. 9,17,18 However, apparently conflicting findings have been reported in 2 other studies. 15,16 Discrepancies between studies, which may reflect heterogeneity in cell types studied and in amounts of phosphomimetic ERM protein expressed, left ambiguity regarding the role of ERM protein phosphorylation in cell migration.…”

“…12 They have been used in many biochemical and cell biologic studies of ERM protein activation/ function to probe the roles of ERM phosphorylation/dephosphorylation. 9,[13][14][15][16][17][18][19][20][21] For example, regulation of ERM protein phosphorylation has been implicated in functions as diverse as compaction in the mouse early embryo, 14 cell rounding in mitosis, 21 and promotion of uropod formation in lymphocytes. 15 Importance of regulated ERM protein phosphorylation in 1 or more of the events involved in lymphocyte recruitment from blood into tissue was suggested by findings that a key trigger of this process (chemokines) induced rapid dephosphorylation of ERM protein.…”

ERM proteins at a glance

“…Such 'phosphocycling' of ezrin has recently been shown to be necessary for the formation of apical microvilli (Viswanatha et al, 2012). In fact, expression of the phosphomimetic ezrinT567D can lead to both loss and exaggerated stabilization of apical microvilli (Brown et al, 2003;Göbel et al, 2004;Parameswaran et al, 2011;Saotome et al, 2004). Many kinases have been reported to phosphorylate this site, and particularly strong evidence implicates SLK family kinases in both flies and mammalian cells.…”

Actin Cytoskeleton and Related Proteins Role During Immune Cells Migration, Polarity and Activation: An Overview

“…Concerning B cells migration, authors have shown that SWAP-70, a Rac-interacting protein involved in actin rearrangement (Pearce et al, 2006) and ezrin, a member of the membrane-cytoskeleton cross-linking ezrin-radixin-moesin proteins (Parameswaran et al, 2011) play important role during B cells chemotaxis. In addition, Rap-1 and -2 GTPases play an important role in mediating adhesion and cytoskeletal reorganization during SDF-1a-dependent B cell migration (McLeod et al, 2002;Durand et al, 2006;Chen et al, 2008).…”