ICAM-1 Dynamics in Endothelium *p,0.05. (C) Cells were transfected as described in B. ICAM-1-Ab coated beads were allowed to adhere for 30 minutes to cluster ICAM-1. FRAP was measured at sites of bead adhesion in A7 (left panel) and M2 cells (right panel). Lower panel shows FRAP quantification. The data show that ICAM-1 requires filamin and its intracellular tail to shift to the immobile fraction upon clustering. Experiment is done four times. Data are mean 6 SEM. *p,0.05. doi:10.1371/journal.pone.0011336.g005 We previously showed that the actin-binding protein filamin binds to clustered rather than to non-clustered ICAM-1. To study if, in parallel, actin is recruited to clustered ICAM-1, we used magnetic beads, coated with the ICAM-1 antibody, to pull-out associating proteins. To do this, the beads were allowed to adhere and to cluster ICAM-1 on intact, TNF-a-treated HUVEC for 30 minutes. Next, the beads were isolated by extraction from cell lysates as described in Materials and Methods. As a control, the magnetic beads were added post-lysis, preventing ICAM-1 clustering. Western blot analysis showed that the clustering of ICAM-1 induced binding to actin (Fig. 2C). As a positive control, we showed that filamin A and B bind selectively to clustered ICAM-1, in line with our previous findings [22]. Filamin and actin were also recruited to exogenous ICAM-1-GFP (Fig. S1C). Moreover, by limiting the ICAM-1 clustering time, we could show that actin and filamin were recruited in a time-dependent manner to ICAM-1. After five minutes of clustering, no actin and filamin was associated to ICAM-1. However, after 20 minutes of clustering, actin and filamin were both precipitated with ICAM-1 (Fig. S1D). These data showed that clustering of ICAM-1 promotes binding to the actin cytoskeleton, possibly through filamin A or B. To determine whether the intracellular tail of ICAM-1 is required for the shift of clustered ICAM-1 to the immobile fraction, we used an ICAM-1-GFP mutant that lacks the intracellular domain (ICAM-1-DC-GFP). ICAM-1-DC-GFP lo- calized only to a limited extent to filopodia-like structures, as compared to full-length ICAM-1 (Fig. 3A). Using scanning electron microscopy, we found that Cos7 cells transfected with full length ICAM-1 expressed large numbers of filopodia with an approximate length of 750 nm (Fig. 3B and 3C). The number of dorsal filopodia in the Cos7 cells that were transfected with ICAM- 1-DC was significantly reduced (Fig. 3B and 3C). In addition to that, the average length of these filopodia was reduced to approximately 200 nm (Fig. 3C). Interestingly, FRAP-analysis showed that the mobility of non-clustered ICAM-1-DC was significantly increased compared to full length ICAM-1 (Fig. 3D). Moreover, a similar difference was observed when ICAM-1 was clustered with aICAM-1-antibody coated beads, i.e. increased recovery after bleaching of ICAM-1-DC compared to ICAM-1 full length (Fig. 3E). In line with this, we found that 3767% of ICAM- 1-DC resides in the immobile fraction as compared to 6566% for full length ICAM-1. These results underscore the importance of the intracellular tail in the shift of ICAM-1 to the immobile fraction. Clustering of ICAM-1 resulted in increased accumulation of F- actin at sites of binding of anti-ICAM-1-antibody coated beads or adhesion of leukocytes [23]. Carman and co-workers already showed by using cytochalasin B (CytoB) that F-actin is required for the induction of apical cups, surrounding adherent lymphocytes [24]. Our data showed that interfering with actin polymerization using CytoB drastically reduced the number of ICAM-1-positive dorsal filopodia (Fig. 4A). Next, we studied the role of actin polymerization in the dynamics of ICAM-1-GFP. FRAP analysis showed that basal dynamics of ICAM-1-GFP were not altered following CytoB treatment (Fig. 4B). Interestingly, FRAP of ICAM-1-GFP at sites of adhesion was significantly increased upon inhibition of actin polymerization (Fig. 4B). Recalculation of the immobile fraction showed that CytoB significantly reduced the immobile fraction from 6367% to 3466%. Figure 4C showed the quantification of the FRAP. In addition, when aICAM-1-antibody coated beads were used to cluster ICAM-1-GFP, the recruitment of ICAM-1 to sites of adhesion was delayed in CytoB-treated cells as compared to untreated cells (Fig. 4D). Nevertheless, after approximately 36 minutes of beads adhesion, the number of beads that recruited ICAM-1 was similar in CytoB-treated and in control cells. To study if blocking of actin polymerization affected the linkage of ICAM-1 with actin, induced by clustering, a pull-out assay using anti-ICAM-1-Ab coated magnetic beads was per- formed, which showed that CytoB treatment prevented the interaction of ICAM-1 with actin upon ICAM-1 clustering (Fig. 4E). Interestingly, stabilizing actin filaments by jasplakinolide promoted the interaction of ICAM-1 with actin upon clustering (Fig. 4E). Thus, actin polymerization is required for ICAM-1 mobility and for efficient recruitment of ICAM-1 to sites of adhesion. We have previously shown that filamin links ICAM-1 to the actin cytoskeleton [22]. In addition, reduction of filamin by siRNA prevented the interaction of ICAM-1 with actin upon clustering (Fig. 5A). These data indicate that filamin plays an important role in the clustering-induced ICAM-1-actin association. To study if filamin is also required in ICAM-1 motility, ICAM-1-GFP is expressed in M2 melanoma cells. These cells lack the expression of filamin and have been widely used in studies on actin regulation [25,26]. As a control, filamin has been reconstituted in A7 melanoma cells. FRAP analysis of non-clustered ICAM-1-GFP showed increased FRAP in M2 cells, indicating that filamin is required for ICAM-1 motility (Fig. 5B). To study the importance of the intracellular tail of ICAM-1 for its motility, we expressed ICAM-1-DC-GFP into M2 and A7 cells and analyzed ICAM-1 motility by FRAP. Our data showed that no difference in the motility of ICAM-1-DC-GFP was measured in the presence or absence of filamin (Fig. 5B). To study the role of filamin after ICAM-1 clustering, beads coated with ICAM-1 Ab were used. FRAP analysis showed reduced motility of full length ICAM-1- GFP in A7 cells, compared to the motility of ICAM-1-DC-GFP (Fig. 5C). Interestingly, no difference in motility was measured when ICAM-1-full length or DC were expressed in M2 cells (Fig. 5C). In addition, these experiments have been repeated in endothelial cells, in which filamin A expression was reduced using siRNA. Similar results were obtained in these cells (data not shown). Together, these data indicate that ICAM-1 needs its intracellular tail and filamin for the mobility upon clustering. Myosins are actin-based motors that regulate actin dynamics [27]. Myosin-II co-localized with ICAM-1 at sites of aICAM-1 antibody-coated beads (Fig. 6A). Inhibiting myosin-II using blebbistatin reduced ICAM-1-GFP dynamics, irrespective of whether ICAM-1 was clustered or not (Fig. 6B). Tilghman and Hoover showed that ICAM-1 shifts to lipid raft-rich membrane domains upon clustering [12]. Preventing the formation of lipid rafts by chelating cholesterol with methyl-b-cyclodextrin (b-CD) reduced the dynamics of clustered ICAM-1-GFP significantly (Fig. 6C). Interestingly, FRAP of non-clustered ICAM-1 was unaltered (Fig. 6C). These data, together with results from the ICAM-1-GFP FRAP analysis on CytoB-treated cells indicate that the clustering of ICAM-1 depends on membrane composition and PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11336