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.

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IntroductionPlasma membrane contains small organized microdomains (lipid rafts) in which restricted repertoires of proteins are arranged together. 1,2 In resting cells, lipid rafts are estimated to be around 100 nm in diameter, including a few dozen proteins, and are distributed randomly on the cell surface, covering up to 50% of the plasma membrane. Upon cell activation, raft domains coalesce, recruiting and excluding different receptors, and allowing the proper organization of signaling complexes for efficient signal transduction. 1,2 Tetraspanins comprise a large number of small palmitoylated polypeptides that span the plasma membrane 4 times, [3][4][5][6] and form microdomains that contain a restricted repertoire of proteins. Biochemically, they share some properties with lipid rafts, but tetraspanin microdomains are based on protein-protein interactions. [7][8][9][10] Tetraspanins have a highly conserved structure with a short and a large extracellular loop (LEL) where 2 or 3 disulfide bonds can be formed. 11 This large loop interacts noncovalently with other tetraspanins and transmembrane proteins, including integrins and adhesion receptors of the immunoglobulin (Ig) superfamily. Although all mammalian cells express different tetraspanins, genetic approaches have been elusive and their function has not yet been fully elucidated. However, their role in antigen presentation and sperm-egg binding has been recently underscored. [12][13][14][15][16][17][18][19][20] The association of certain plasma membrane proteins to the cortical actin cytoskeleton is critical for their proper localization and function. Thus, the concentration of selectins and their ligands on the tip of microvilli 21,22 both at the leukocyte and the apical surface of endothelial cells favors their interaction during the rolling phase of leukocyte extravasation. Likewise, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which are relevant in the subsequent leukocyte firm adhesion step, are also displayed anchored to actin through ezrin-radixin-moesin proteins (ERMs) 23,24 at the apical surface on endothelial cells. Upon leukocyte firm adhesion, the engagement of VCAM-1 and ICAM-1 triggers the reorganization of the endothelial cortical actin cytoskeleton, building up a 3-dimensional docking structure that prevents the detachment of leukocytes by shear stress. 22,23 Here, we show that ICAM-1 and VCAM-1 are included in tetraspanin microdomains that regulate their membrane expression and the efficient adhesive function necessary for proper leukocyte transendothelial migration under flow conditions. Materials and methods Cells and cell culturesHuman umbilical vein endothelial cells (HUVECs) were obtained and cultured as previously described. 25 Cells were used up to the third passage in all assays. To activate HUVECs, tumor necrosis factor-␣ (TNF-␣; 20 ng/mL)(R&D Systems, Minneapolis, MN) was added to the culture media 20 hours before the assays were performed. T lymphoblasts were derived The online vers...