ICAM-1 Dynamics in Endothelium Figure 4. Actin polymerization is required for the shift of ICAM-1 to the immobile fraction. (A) HUVECs were treated with Cytochalasin B (CytoB; 5 mg/mL) for 10 minutes to block actin polymerization, fixed and analyzed by fluorescent microscopy. CytoB prevents ICAM-1 localization to filopodia. Images show ICAM-1 staining in green and F-actin in red. Merge shows co-localization of ICAM-1 and F-actin. Bar, 20 mm. (B) ICAM-1-GFP is expressed in HeLa cells and treated or not with CytoB. ICAM-1 is clustered as described in B. Left panel shows no difference in FRAP of non-clustered ICAM-1 upon CytoB treatment. Panel at the right shows increased ICAM-1 FRAP when actin polymerization was blocked. Experiment is done three times. Data are mean 6 SEM. *p,0.05. (C) Quantification of clustered and non-clustered ICAM-1 shows that ICAM-1-GFP recruitment upon clustering is delayed upon CytoB treatment but not in non-clustered conditions. Experiment is repeated 3 times and data are mean 6 SEM. *p,0.05. (D) ICAM- 1-GFP is expressed in HeLa cells and recruited to aICAM-1-antibody coated beads as described under 1E. Quantification graph shows that ICAM-1- GFP recruitment is delayed when actin polymerization is blocked. Experiment is repeated 3 times and data are mean 6 SEM. *p,0.05. (E) TNF-a- pretreated HUVECs were cultured on FN-coated surfaces and prior to anti-ICAM-1-coated magnetic beads-induced clustering, cells were pretreated with CytoB or jasplakinolide (10 mM). Next, beads were allowed to adhere to the endothelium for 30 minutes, and samples were processed as described in the legend of figure 2C. Panels on the left show magnetic beads pull down. ICAM-1 was precipitated equally under all conditions. Clustering of ICAM-1 induced the link to actin, which was prevented by CytoB pre-treatment. Stabilizing actin filaments by jasplakinolide promoted the link of ICAM-1 with actin upon clustering. Panels on the right show the expression of indicated proteins in the cell lysates. doi:10.1371/journal.pone.0011336.g004 (5 mL/minute) finally. Images were collected and the number of adherent leukocytes was counted per field of view. Note that the inhibitors were included in the medium and thus were present throughout the experiment. Immunoprecipitation and Western blot analysis Cells were grown to confluency on FN-coated dishes (50 cm2), washed twice gently with ice-cold Ca2+-and Mg2+-containing PBS and lysed in 300 mL of lysis buffer (25 mM Tris, 150 mM NaCl, 10 mM MgCl2, 2 mM EDTA, 0.02% SDS, 0.2% deoxycholate, 1% NP-40, 0.5 mM orthovanadate with the addition of fresh protease-inhibitor-cocktail tablets (Boehringer) pH 7.4). After 10 minutes on ice, cell lysates were collected and pre-cleared for 30 minutes at 4uC with Streptavidin beads (Sigma-Aldrich, 15 ml for each sample). The supernatant, cleared by centrifugation (14.000*g, 5 minutes at 4uC) was incubated with 15 ml protein Streptavidin beads that were coated with the biotin-tagged S27D or the control peptide, for 1 h at 4uC under continuous mixing. The beads were washed 3 times in lysis buffer and proteins were eluted by boiling in SDS-sample buffer containing 4% 2- mercaptoethanol (Bio-Rad). The samples were analyzed by 10% SDS-PAGE. Proteins were transferred to 0.45 mm nitro-cellulose (Schleicher and Schnell Inc., NH) and the blots were first blocked with 5% (w/v) low-fat milk in TBST for 1 h, subsequently incubated at room temperature with the appropriate Abs for 1 h, followed by 30 minutes incubation with HRP-conjugated secondary Abs at RT. Between the various incubation steps, the blots were washed 5 times with TBST and finally developed with an enhanced chemiluminescence (ECL) detection system (Amersham). Statistics Student-T test was used to calculate statistical significance. Results In the course of our studies on ICAM-1 dynamics in endothelial cells, we noticed that ICAM-1 on the surface of endothelial cells as well as ICAM-1-GFP, transfected in HeLa cells, localizes to small, fibrillar structures (Fig. 1A). Subsequent scanning electron microscopy analysis showed that the surface of the apical membrane of the endothelium forms small filopodia-like protru- sions (Fig. 1A), reminiscent of the ICAM-1-positive fibrils, identified by fluorescent microscopy. Interestingly, transfection of ICAM-1-GFP into Cos7 cells, which normally do not expose filopodia [21], resulted in the induction of multiple filopodia (Fig. 1B). These data showed that ICAM-1 induces the formation of dorsal filopodia. Live-cell imaging of transfected HeLa cells showed that ICAM-1-GFP-positive dorsal filopodia are highly motile (Movie S1). ICAM-1 is well recognized for its adhesive function in binding leukocytes, and we and others have shown ICAM-1 to be concentrated in ring-like structures surrounding adherent leukocytes. In contrast, there is relatively little knowledge about the dynamics of ICAM-1 recruitment upon binding to an adhered leukocyte. To study this, leukocytes were allowed to adhere to TNF-a-treated endothelial cells and ICAM-1 distribu- tion was analyzed. Using HL60 cells, we found ICAM-1 recruitment to sites of leukocyte adhesion (Fig. 1C). The same effect was observed when leukocytes adhered to HeLa cells expressing ICAM-1-GFP (Fig. 1C). To mimic leukocyte-induced ICAM-1 clustering, we used polystyrene beads that were coated with anti-ICAM-1 antibodies [10]. These antibodies efficiently block leukocyte adhesion and thus target an essential epitope in the extracellular domain of the ICAM-1 molecule [7]. The anti- ICAM-1-antibody coated beads only adhered to ICAM-1-GFP expressing cells and recruited ICAM-1-GFP to sites of bead adhesion in a similar fashion as the leukocytes (Fig. 1D and 1E). Additional experiments showed that ICAM-1-GFP co-localized with endogenous ICAM-1 in endothelial cells and clustered around adhered anti-ICAM-1-antibody coated beads (Fig. S1A and S1B). To study the kinetics of ICAM-1 recruitment, the recruitment of ICAM-1-GFP to sites of bead adhesion was recorded in real-time using confocal laser scanning microscopy. ICAM-1-GFP was transfected into HeLa-cells to avoid dimeriza- tion with endogenous ICAM-1 in endothelial cells [18]. These experiments showed that after 30 minutes, approximately 25% of the anti-ICAM-1-antibody coated beads that adhered to ICAM-1- GFP-positive cells recruited ICAM-1-GFP (Fig. 1E). To focus in more detail on the dynamics of ICAM-1 distribution in the plasma membrane, we used a FRAP (Fluorescence Recovery After Photobleaching)-based approach. ICAM-1-GFP, expressed in HeLa cells, was bleached within a small area of the cell and the recovery of ICAM-1-GFP was monitored in real time. The data showed that levels of ICAM-1-GFP were restored to approximately 60% within 5 minutes after bleaching (Fig. 2A and 2B). This data shows that 30–35% of ICAM-1-GFP resides in the so-called ‘immobile fraction’. Similar results were obtained when ICAM-1-GFP was transiently transfected into endothelial cells (data not shown). Interestingly, recovery of clustered ICAM-1- GFP, induced by the anti-ICAM-1-antibody coated beads, was significantly reduced (3764%), compared to non-clustered ICAM- 1-GFP (6162%; Fig. 2B). To calculate the immobile fraction of ICAM-1-GFP, fluorescence recovery was measured when a plateau phase at 5 minutes was reached. These data showed that the immobile fraction of ICAM-1-GFP increased over 2-fold upon clustering (2364% for not clustered ICAM-1-GFP compared to 6265% for clustered ICAM-1-GFP). This indicates that clustering drives ICAM-1 into an immobile fraction. PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11336