Conformational Switching in Ezrin Regulates

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 cross-linking ezrin-radixin-moesin 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 de-phosphorylation was pharmacologically inhibited displayed impaired microvillar dynamics, morphological polarization, and chemotaxis. Our data suggest a 2-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. B cell homing and recirculation are essential features of B cell immunity. B cells display varied responsiveness to chemokines such as B lymphocyte chemokine (BLC/ CXCL13), stromal cell-derived factor 1a (SDF-1a/CXCL12), and secondary lymphoid organ chemokine (SLC/CCL21) during their lifetime (1). Aberrant expression or signaling mediated by chemokines and their cognate receptors have been implicated in the pathogenesis of B cell disorders such as lupus (2), rheumatoid arthritis (3), leukemias (4, 5), and viral infections (6). Chemotaxis occurs when cells respond to gradients of chemokines displayed on endothelial cells lining the blood vessels or on stromal cells in secondary lymphoid organs (7). In situ imaging of lymph nodes from immunized mice using two-photon microscopy revealed spontaneously motile B cells that pause temporarily upon Ag binding, followed by preferential migration toward the T cell zone (8). During both forms of motility, B cells display a polarized morphology. Cell motility must require evolution of membrane– actin contacts at the leading edge that provide the protrusive force for migration (9). In addition, intra-and extravasation of B cells through blood vessels during homing to and from lymphoid organs must involve dynamic cell shape changes involving plasma membrane and cytoskeleton reorganization (10). The importance of cytoskeletal rearrangement in B cell polarization and migration was demonstrated using mice lacking proteins involved in chemokine receptor signaling. DOCK2-deficient mice exhibit poor B lymphocyte migration and disrupted lymphoid architecture due …