present throughout the experiment. Therefore, anti-ICAM-1 antibody-coated beads were used instead of leukocytes, since the inhibitors may affect the function of the leukocytes. The beads were allowed to adhere to the endothelium for 20 minutes, followed by incubation with the inhibitors for 10 minutes. Then flow was introduced, which gradually increased in time, as described in Materials and Methods. These studies showed that increasing the flow-rate resulted in a decreased adhesion of aICAM-1 antibody-coated beads to the endothelium, indicating that flow negatively regulates ICAM-1 function (Fig. 9A). Subsequent blocking of actin polymerization or Rac1 activation indicated that these are both needed for ICAM-1 function under flow (Fig. 9A). Using blebbistatin or cyclodextrin, a steep decrease in leukocyte adhesion was observed after an increase in the flow-rate from 0.25dyn/cm2 to 1.25dyn/cm2 (Fig. 9B). Since all inhibitors used in the flow assay reduced lateral mobility of clustered ICAM-1, we conclude that optimal ICAM-1 mobility is a prerequisite for ICAM-1 function to bind leukocytes to the endothelium under physiological flow conditions. The findings are summarized in figure 10. ICAM-1 Dynamics in Endothelium Figure 10. Schematic overview of ICAM-1 dynamics. ICAM-1 dynamics require Rac1 and myosin-II activity. Upon clustering, actin polymerization and lipid raft formation play a pivotal role. These signalling modules all affect the adhesive capacity of ICAM-1 under flow conditions. doi:10.1371/journal.pone.0011336.g010 Discussion Leukocytes bind through their integrins LFA-1/Mac-1 to endothelial ICAM-1 to establish firm adhesion. Upon leukocyte binding, ICAM-1 translocates to sites of adhesion, forming a typical ring structure around the adhered leukocyte [10,19,32,33]. Here, we show that upon clustering ICAM-1 shifts to an immobile fraction in an actin-dependent manner, which also requires its intracellular domain. In addition, actin-regulating proteins such as Rac1 and myosin-II are involved in ICAM-1 dynamics, whereas the polymerization of actin and the formation of lipid rafts are necessary for the clustering. Our recent work showed that filamin associates to ICAM-1 upon clustering [22]. In this report, we show that clustering of ICAM-1 not only recruits filamin, but also beta-actin, illustrated in figure 10. a-Actinin-1 and -4, other actin-binding proteins, are reported to bind to the intracellular domain of ICAM-1 [34]. In addition, these authors showed that a-actinin is required for proper leukocyte extravasation. Through this molecule, ICAM-1 may be linked to the actin cytoskeleton. The family of ERM-proteins has been implicated in the direct link of ICAM-1 to the actin cytoskeleton as well [17]. Deletion of ezrin results in reduced ICAM-1-positive filopodia. The authors furthermore show that deletion of the intracellular domain of ICAM-1 prevents the interaction with ezrin and also reduces filopodia. Our study indicates that deletion of the intracellular domain of ICAM-1 results in smaller and reduced numbers of filopodia. Adhesion of the leukocytes to tail-less ICAM-1 was not affected under static or low shear stress conditions. However, increasing the flow induces a loss of leukocyte adhesion to tail-less ICAM-1, but not full length ICAM-1 [17]. These data indicate that the intracellular domain plays an important role in ICAM-1 function, in line with previous reports [14,15]. Our current data show that the intracellular domain of ICAM-1 is required for the shift to the immobile fraction. From these data, it is clear that the intracellular tail of ICAM-1, as well as the actin cytoskeleton is required for proper ICAM-1 clustering, in order to function efficiently as an adhesion molecule. Figure 9. Quantification of ICAM-1 adhesion. (A) aICAM-1- antibody coated-beads were allowed to pre-adhere to TNF-a-stimulated endothelium for 5 minutes. Experiment was carried out as described in 2 Materials and Methods. Briefly, low shear was introduced (0.25dyn/cm ) and flow was increased stepwise with 1.25dyn/cm2 at the time points, 2 indicated with arrows. The final shear corresponded to 6.25dyn/cm . The aICAM-1-antibody coated beads detached from the endothelium when shear increased. Pre-treatment of the endothelium with cytochalasin B (CytoB) or NSC-23766 (NSC) showed a steeper decline of detached beads upon increased shear flow. Experiment is done three times. Data are mean 6 SEM. *p,0.05. (B) Experiment carried out as described under A. Pre-treatment of the endothelial cells with either blebbistatin (Blebbi) or cyclodextrin (b-CD) show a decrease in adhesion of the beads to the endothelium, already after the first increase in shear flow. Experiment is done three times in duplicate. Data are mean 6 SEM. *p,0.01. doi:10.1371/journal.pone.0011336.g009 PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e11336