ICAM-1 Dynamics in Endothelium Figure 1. ICAM-1 clusters to leukocyte adhesion sites. (A) Left panel shows localization of endogenous ICAM-1 in green on endothelial filopodia (Arrowheads). Middle panel shows localization of ICAM-1-GFP, expressed in HeLa cells on filopodia. Right panel shows scanning electron microscopy (SEM) recording of TNF-a-pretreated HUVECs visualising small filopodia protruding out of the apical plane of the membrane (arrowheads). Bars, 10 mm (B) Cos7 cells expressing GFP (left panel) or ICAM-1-GFP (right panel) were cultured on glass cover slips and analyzed by SEM. ICAM-1-GFP but not GFP only-expressing Cos7 cells showed multiple filopodia (arrowheads). Bar, 10 mm. (C) TNF-a-pretreated HUVECs were cultured on glass cover slips and calcein-red labelled differentiated HL60 cells were allowed to adhere for 30 minutes. Arrowheads show recruitment of ICAM-1 to adherent HL60 cells. Images show ICAM-1 in green and HL60 cells in red. Right panel shows ICAM-1-GFP-transfected HeLa cells, cultured on glass cover slips and calcein-red labelled differentiated HL60 cells adhered for 30 minutes. Bar, 10 mm. (D) ICAM-1-GFP-transfected Cos7 cells were cultured on glass covers and aICAM-1-antibody coated beads (size 3 mm) adhered for 30 minutes. Arrowheads show recruitment of ICAM-1-GFP to adhered beads. Images show ICAM-1-GFP in green, F-actin in red, merge in yellow and the beads in white. Note that the beads specifically adhered to ICAM-1-GFP expressing cells. The asterisk shows non-transfected cell with no bead adhesion. Bar, 20 mm. (E) ICAM-1-GFP (green) is expressed in HeLa cells and recruited (arrowheads) to aICAM-1-antibody coated beads (size 10 mm; asterisks). Quantification of ICAM-1 recruitment on the right shows that ICAM-1-GFP is recruited to 25% of the beads after 36 minutes. Data are mean 6 SEM from four experiments. doi:10.1371/journal.pone.0011336.g001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11336