Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin‐binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell‐cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam‐1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP‐binding proteins induced a polarized phenotype in constitutively round‐shaped T cells with redistribution of ICAM‐3 and moesin to the uropod in an integrin‐dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF‐1α‐induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine‐induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.