Abstract
BackgroundDuring transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.Principal FindingsDetailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.ConclusionsTogether, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.
Highlights
Leukocytes use integrins to firmly adhere to activated endothelium through adhesion molecules such as VCAM-1 and ICAM-1 prior to extravasation
Blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton
These data indicate that signaling events within the endothelium are required for efficient ICAM-1mediated leukocyte adhesion
Summary
Leukocytes use integrins to firmly adhere to activated endothelium through adhesion molecules such as VCAM-1 and ICAM-1 prior to extravasation. We and others have shown increased stress fiber formation after ICAM-1 antibody crosslinking [6,7] These changes in the actin cytoskeleton imply an important role for Rho-like small GTPases [8]. In agreement with this notion, various studies have shown that RhoA, which induces stress fibers and contractility, is activated downstream from ICAM-1 clustering [6,9,10]. Blocking RhoA activity by C3 toxin or overexpression of a dominant negative mutant (RhoAT19N) prevents antibody-mediated and monocyte-induced clustering of ICAM-1, monocyte adhesion and subsequent stress fiber formation [11]. We studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions
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