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 µg/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 µm. (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 ± 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 ± SEM. *p<0.05. (D) ICAM-1-GFP is expressed in HeLa cells and recruited to αICAM-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 ± SEM. *p<0.05. (E) TNF-α-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 µM). 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.