ICAM-2 Limits Metastasis Figure 2. ICAM-2 expression affected the subcellular distribution of actin fibers and cell motility in two types of in vitro assays. A) Confocal fluorescence microscopic images of actin fibers visualized by FITC-phalloidan (green) staining demonstrate that ICAM-2 expression redistributed F-actin from a transverse to a juxtamembrane localization. Cell nuclei were visualized using DAPI (blue). B) Competitive peptides to inhibit ICAM-2/a-actinin binding or a-actinin/actin binding reversed the ICAM-2-mediated juxtamembrane distribution of actin fibers in neuroblastoma cells. C) In scratch assays, ICAM-2 transfectants migrated more slowly than control transfectants. The width of scratches at 0 hours for pIRESneo and pIRES.ICAM2 transfectants were 1127 and 1044 pixels, respectively. At 72 hours, no gap remained in wells containing SK-N- ASpIRESneo cells, while a scratch distance of ,450 pixel width remained in wells containing SK-N-ASpIRES.ICAM2 cells. D) In modified Boyden chamber assays, ICAM-2 expression inhibited invasion of SK-N-AS cells through Matrigel to the distal side of porous membranes, as detected by Diff- Quick (IMEB, Inc., San Marcos, CA), and quantitated using an Alpha Imager software, as detailed in Materials and Methods. doi:10.1371/journal.pone.0003629.g002 ing the antibody that recognized the N-terminus of ICAM-2 (Fig. 3B) demonstrated that neither a-actinin nor actin associated with ICAM-2 in control transfectants or in transfectants expressing the truncated form of ICAM-2, and FITC-phalloidan staining (Fig. 3C) confirmed that expression of the truncated form of ICAM-2 lacking the a-actinin binding domain did not affect actin fiber distribution. Consistent with these observations, SK-N- pIRESneo and pIRES.ICAM2DCD transfectants migrated at similar rates in scratch assays, closing initial scratch widths of ,525 pixels within 45 hours (Fig. 3D). The data show that ICAM-2-mediated redistribution of actin fibers and inhibition of tumor cell motility depended on an intact intracellular domain. PLoS ONE | www.plosone.org 4 November 2008 | Volume 3 | Issue 11 | e3629