The small GTPase Rac1 and myosin-II activity are not only involved in ICAM-1 clustering, but appear to play a more general role in ICAM-1 dynamics. Interfering with the activity of one of these two molecules affects the lateral mobility of ICAM-1. Rac1 has not been implicated in ICAM-1 dynamics before, although many studies have shown involvement of Rac1 activity in membrane dynamics [28]. Interestingly, Heiska and colleagues showed that PIP2 is required for proper binding of the actin- binding protein ezrin to ICAM-1 [35]. Our lab and others have previously shown that Rac1 associates to PIP-5-Kinase, which may link Rac1 to PIP2-synthesis [36,37]. This suggests that Rac1 is involved, through the regulation of inositol lipid turnover and the regulation of ezrin, in linking ICAM-1 to the actin cytoskeleton upon clustering. Myosin-II has not previously been implicated in ICAM-1 dynamics. However, the fact that ICAM-1 localizes at dorsal filopodia and moves within the lateral plane of the cells’ plasma-membrane makes myosin-II a possible candidate to be involved in ICAM-1 dynamics. Recently, Khuon and colleagues showed that myosin-II in endothelial cells forms a ring-like structure that encapsulates an invading tumor cell [38]. Together these findings suggest that Rac1 and myosin-II activity are required for ICAM-1 dynamics. The adhesive capacity of ICAM-1 can be tested using flow. By applying increasing flow rates on an anti-ICAM-1 antibody-coated bead bound to endothelium, the minimal flow rate that is required for proper adhesion by ICAM-1 can be determined. We have used beads instead of leukocytes for two reasons: First, the inhibitors used need to be present throughout the experiment. This may affect the leukocytes in their ability to bind to the endothelium. Second, leukocytes adhere not only to ICAM-1 but also to ICAM- 2 and VCAM-1 and possibly other yet unidentified adhesion molecules on activated endothelial cells. Thus, to avoid confusion, we have used beads that specifically adhere to ICAM-1. Our results indicate that actin polymerization, Rac1 signaling and in particular myosin-II play a crucial role for optimal ICAM-1 adhesion (Fig. 10). The fact that actin polymerization is required for the shift of ICAM-1 to the immobile fraction may explain why ICAM-1 adhesion under flow is reduced following treatment of cells with cytochalasin B. Under these conditions, ICAM-1 is not efficiently anchored to the actin cytoskeleton which in turn results in lower adhesive capacity. It has been reported that blocking myosin light chain kinase inhibits leukocyte transendothelial migration [39]. The authors suggested that MLC-kinase acts on the cell-cell junctions and by blocking its activity the junctions remain intact, resulting in a reduced passage of leukocytes. We show that myosin-II is recruited to sites of ICAM-1 adhesion and that blocking myosin-II activity results in an impaired ICAM-1-mediated adhesion under flow. In line with our data, Khuon and colleagues showed that myosin-II activity is found at sites of adhesion [38]. These data underscore the notion that ICAM-1 dynamics within the apical endothelial membrane are essential for optimal ICAM-1 adhesive function. Yang and co-workers showed that Src and cortactin play an important role in the linking of ICAM-1 to the cytoskeleton [19]. These authors used pharmacological inhibitors to block Src activity. We choose to use MEF cells that lack three Src-like kinases Src, Yes and Fyn. Reconstitution of Src into these MEFs did not affect the ICAM-1 mobility in the plane of the membrane as determined by FRAP nor the adhesive function of ICAM-1. The latter finding is in line with the results reported by Yang and colleagues. Therefore, we concluded from these experiments that Src is not directly involved in the adhesive function of ICAM-1, but rather in transmitting intracellular signals downstream from ICAM-1 clustering, possibly to regulate cell-cell junction integrity [30]. ICAM-1 Dynamics in Endothelium In conclusion, we postulate that the dynamics of ICAM-1 within the plane of the membrane is vital for its function to induce firm adhesion under physiological flow conditions. Our data emphasize the contribution of ICAM-1 dynamics to its adhesive function and may therefore represent an interesting target for future therapies to reduce leukocyte accumulation to the vessel wall, as occurs in diseases such as atherosclerosis and rheumatoid arthritis. Supporting Information Figure S1 ICAM-1-GFP co-localizes with endogenous ICAM- 1 and associates to filamin and actin upon clustering. (A) TNF- a-stimulated endothelium was transiently transfected with ICAM-1-GFP, fixed and stained for ICAM-1-GFP in green, endogenous ICAM-1 in red. Merge shows co-localization in yellow and F-actin is in grayscale. Images were taken from the apical surface (upper panel) and at the baso-lateral plane (lower panel). Bars, 50 mm. (B) Beads, coated with ICAM-1 Ab, were allowed to adhere for 30 minutes to TNF-a-stimulated HUVECs. ICAM-1 was stained using an ICAM-1 Ab that is directly labelled with ALEXA 647. The results show that ICAM- 1-GFP (green) and endogenous ICAM-1 (red) co-localize (Arrowheads; Merge in yellow) at sites of bead adhesion. DIC image shows localization of the beads. Images were taken from the apical surface (upper panel) and at the baso-lateral plane (lower panel). Bars, 10 mm. (C) ICAM-1-GFP was transfected into HeLa cells and anti-ICAM-1-coated magnetic beads were allowed to adhere for 30 minutes, resulting in clustering of ICAM-1, after which the cells were lysed ands treated as described in the legend of figure 2C. The images on the left show that the magnetic beads efficiently precipitated ICAM-1- GFP and that ICAM-1 clustering resulted in the recruitment of actin and filamin A. Images on the right show the expression of indicated proteins in the cell lysates. (D) ICAM-1-GFP was transfected into HeLa cells and anti-ICAM-1-coated magnetic beads were allowed to adhere for 5 or 20 minutes, as indicated. ICAM-1 was precipitated at both time points, whereas actin and filamin A and B were associated to ICAM-1 only after 20 minutes of clustering. Images on the right show the expression of indicated proteins in the cell lysates. Found at: doi:10.1371/journal.pone.0011336.s001 (0.29 MB TIF) Figure S2 Rac1 activation downstream from ICAM-1 cluster- ing. (A) ICAM-1 was clustered using anti-ICAM-1-antibody coated beads for 30 minutes on TNF-a-stimulated HUVECs. Rac1 activity was measured based on a CRIB-pull down assay from cell lysates. The results show that Rac1 is activated upon clustering of ICAM-1 (Upper panel). Lower panel shows equal levels of Rac1 in total cell lysates. (B) NSC-27632 inhibits Rac1 activity. HEK293 cells were transfected with GFP-Trio-D1, the first DH/PH domain of the guanine nucleotide exchange factor Trio and known to activate Rac1. The inhibitor (100 mM) is incubated for 30 minutes and showed that Trio-D1-induced Rac1 activity was reduced. Rac1 activity was measured using a CRIB- based pull down assay. The upper panel shows Rac1 activity, the middle panel depicts the total Rac1 protein levels in the total cell lysates and the lower panel shows the expression of the transfected Trio-D1 constructs, stained with an anti-GFP antibody. Found at: doi:10.1371/journal.pone.0011336.s002 (0.18 MB TIF) Movie S1 Real-time recording of ICAM-1-GFP. HeLa cells were transfected with ICAM-1-GFP full length and real-time recording was taken from these cells for 45 minutes. Movie shows small filopodia at the apical surface of the cell. Found at: doi:10.1371/journal.pone.0011336.s003 (1.58 MB AVI) PLoS ONE | www.plosone.org 13 June 2010 | Volume 5 | Issue 6 | e11336