ICAM-1 Dynamics in Endothelium Figure 8. Src-kinase has no effect on ICAM-1 dynamics. (A) Analysis of Src, Yes and Fyn-kinase (SYF)-deficient (SYF-/-) or SYF-/- with reconstituted Src (SYF-/-+Src) or control (CTRL) mouse embryonic fibroblast (MEF) cells by western blotting using antibodies against Src, Yes or Fyn. Tubulin was used for protein loading control. (B) Immunofluorescent images show localization of ICAM-1-GFP in filopodia in SYF-/- or SYF-/-+Src MEF cells. (C) Graph shows that the expression of Src had only minor effect on the ICAM-1 FRAP in the SYF-/- cells. (D) aICAM-1 antibody coated beads (size 3 mm) were allowed to adhere for 30 minutes on ICAM-1-GFP transfected SYF-/- or SYF-/-+Src cells. Number of beads per ICAM-1-GFP-positive cell was counted. No difference in adhesion of these beads the MEF cells is measured. Experiment was carried out three times. doi:10.1371/journal.pone.0011336.g008 Figure 7. Rac1 activity is required for optimal ICAM-1 dynamics. (A) Cells were pretreated with 100 mM NSC-23766 (NSC) for 30 minutes. Quantification of ICAM-1 recruitment using aICAM-1 antibody coated beads shows that ICAM-1-GFP recruitment is reduced upon inhibition of Rac1 activity by NSC. Experiment is repeated 3 times and data are mean 6 SEM. *p,0.05. (B) ICAM-1-GFP FRAP was analyzed as described in Materials and Methods and showed that NSC reduced FRAP of non-clustered and clustered ICAM-1-GFP. Experiment is done three times. Data are mean 6 SEM. *p,0.05. doi:10.1371/journal.pone.0011336.g007 23766 significantly reduced the dynamics of non-clustered ICAM- 1-GFP (Fig. 7B). This indicates that basal Rac1 activity is involved in ICAM-1 mobility in the membrane. In addition, the recovery of clustered ICAM-1-GFP was also significantly reduced upon Rac1 inhibition (Fig. 7B). These data underscore the importance of Rac1 signaling controlling the dynamics of non-clustered as well as clustered ICAM-1. A key molecule that transmits ICAM-1-induced intracellular signals is Src-kinase [30,31]. To assess the role of Src in ICAM-1 dynamics, we used Src-, Yes- and Fyn-kinase-deficient (SYF-/-) mouse embryonic fibroblast (MEF) cells or SYF-/- MEFs stably re-expressing Src (SYF-/-+Src) (Fig. 8A). ICAM-1-GFP was transfected into these cells and fluorescent analysis showed that ICAM-1-GFP was localized in filopodia-like structures in both cell types, albeit more clearly in the Src-reconstituted MEF cells (Fig. 8B). FRAP-analysis showed that the dynamics of non- clustered ICAM-1-GFP depended only minimally on the presence of Src (Fig. 8C). Moreover, depletion of Src did not affect the adhesion of anti-ICAM-1-antibody coated beads to ICAM-1-GFP-positive MEFs (Fig. 8D). In addition, the use of the classical inhibitors of Src-kinase PP1 and PP2 did not affect the adhesion of anti-ICAM-1-antibody coated beads to endo- thelial cells (data not shown). These data indicate that Src-kinase plays only a minor role in the regulation of the dynamics of ICAM-1. Finally, we tested if the lateral mobility of ICAM-1 affects its adhesive function under flow. The inhibitors used needed to be PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11336