ICAM-1 Dynamics in Endothelium Figure 6. Myosin and lipid raft formation are required for the shift of ICAM-1 to the immobile fraction. (A) TNF-a-pretreated HUVECs were cultured on glass covers and images show ICAM-1 staining in green using ICAM-1-FITC to avoid cross-talk with the beads, myosin-II in red, merge of ICAM-1 and myosin-II in yellow, F-actin in white and DIC is included to depict the beads (arrowheads). Myosin-II together with ICAM-1 is clustered around the aICAM-1-antibody coated beads that were allowed to adhere for 30 minutes. Bar, 20 mm. Lower panel shows X-Z projection of ICAM-1 in green and myosin-II in red (arrowheads) around the adhered aICAM-1-antibody coated beads (asterisk). White depicts F-actin. Bar, 5 mm. (B) Cells were pretreated with 8 mM Blebbistatin to inhibit myosin-II for 30 minutes (Blebbi). ICAM-1-GFP FRAP was analyzed as described above and showed that Blebbistatin significantly decreased the FRAP of non-clustered and clustered ICAM-1-GFP. Experiment is done three times. Data are mean 6 SEM. *p,0.05. (C) Cells were pretreated with 5mM methyl-b-cyclodextrin for 30 minutes (b-CD). ICAM-1-GFP FRAP was analyzed as described above and showed that b-CD did not affect ICAM-1-GFP FRAP of non-clustered ICAM1-GFP but significantly reduced the FRAP of clustered ICAM-1- GFP. Experiment is done three times. Data are mean 6 SEM. *p,0.01. doi:10.1371/journal.pone.0011336.g006 is controlled by proteins that are involved in cytoskeletal remodeling. The small GTPase Rac1 regulates the remodelling of the actin cytoskeleton and induces membrane ruffles [28]. We recently showed that Rac1 is activated upon clustering of ICAM-1 ([10]; Fig. S2A). To elucidate the role of Rac1 activity in ICAM-1 dynamics, we tested the Rac1-GEF inhibitor NSC-23766 on the kinetics of ICAM-1 clustering. Using Trio-GEF-D1, a catalytic active mutant of the exchange factor Trio, we showed that the inhibitor indeed blocked Rac1 activity (Fig. S2B), as was also shown by others [29]. Rac1 inhibition reduced the clustering of ICAM-1-GFP to aICAM-1-antibody coated beads by approxi- mately 50% (Fig. 7A). Next, we assessed the effects of this inhibitor on ICAM-1-GFP dynamics by FRAP analysis. Interestingly, NSC- PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11336