ICAM-1 Dynamics in Endothelium Figure 3. Requirement of the intracellular domain of ICAM-1 for clustering. (A) ICAM-1-GFP full length or a construct lacking the intracellular tail (ICAM-1-DC) were transfected in HeLa cells and cultured on glass covers. Full-length ICAM-1 localizes to filopodia whereas the DC- mutant shows a more dispersed distribution. Images show ICAM-1 staining in green, F-actin in red and merge in yellow. Image on the right is a magnification of the merge. Bars, 20 mm. (B) ICAM-1-GFP full length or DC was transfected in Cos7 cells that lack basal filopodia and images were taken using a scanning electron microscope. Zoom shows filopodia in full-length or ICAM-1 DC expressing Cos7 cells, Bar, 1 mm. (C) Quantification of filopodia in Cos7 cells shows that ICAM-1 full length induces filopodia with an average length of 750nm. The DC-mutant shows smaller and fewer filopodia. Bar graph shows number of filopodia per Cos7 cell. Experiment is done three times. Data are mean 6 SEM. *p,0.01. **p,0.001. (D) Full length and DC ICAM-1-GFP are expressed in HeLa cells. Left panel shows FRAP in time and right panel shows FRAP quantification after 5 minutes of bleaching. ICAM-1-DC recovers significantly faster than full length ICAM-1. Experiment is done four times. Data are mean 6 SEM. *p,0.05. (E) Full length and DC ICAM-1-GFP are expressed in HeLa cells and ICAM-1 clustering was induced through aICAM-1 antibody-coated beads. Left panel shows FRAP in time and right panel shows FRAP quantification after 5 minutes of bleaching. Clustered ICAM-1-DC recovers significantly faster than full length. Experiment is done four times. Data are mean 6 SEM. *p,0.05. doi:10.1371/journal.pone.0011336.g003 intracellular domain of ICAM-1 plays an essential role in its function. Deleting the intracellular domain of ICAM-1 decreases transendothelial migration (TEM) of leukocytes [14,15]. This was further underscored by a study by Sans et al., in which they expressed ICAM-1 in CHO cells that lack endogenous ICAM-1 [16], which was sufficient to reconstitute TEM. However, expression of a C-terminally deleted mutant lf ICAM-1 severely impaired efficient TEM. Recently, using expression in Cos cells, Oh and colleagues defined the RKIKK motif within the intracellular domain of ICAM-1 as critical for ICAM-1 function [17]. These data point to the importance of ICAM-1 in TEM. ICAM-1 exists as a dimer and can freely diffuse in the plasma membrane [18]. Upon clustering by antibody-mediated cross- linking, ICAM-1 reduces its lateral mobility, most likely because of increased association of the intracellular tail to actin cytoskeleton- binding proteins [19]. Although all these efforts, it is still not known which signals and molecules regulate ICAM-1 function and mobility. In this study, we analysed ICAM-1 dynamics by using fluorescence recovery after photobleaching (FRAP) prior to and following ICAM-1 clustering. In addition, the adhesive function of ICAM-1 was studied under flow. We show that ICAM-1 function is regulated by the connection of its C-terminal intracellular domain to downstream, cytoskeleton-regulating proteins myosin-II and the small GTPase Rac1, but not Src-like kinases. Our data provide evidence for control of ICAM-1 function in leukocyte adhesion in an inside-out fashion through the modulation of ICAM-1 lateral mobility and linkage to the actin cytoskeleton. Materials and Methods Reagents and Abs Monoclonal antibody (mAb) against ICAM-1 was purchased from R&D Systems (Minneapolis, MN); Polyclonal Abs against ICAM-1 (for WB, no.7891) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ICAM-1-ALEXA 647 mAb (Clone 15.2) was purchased from Serotec (Oxford, UK). The GFP mAb was purchased from Invitrogen (Carlsbad, CA). Actin antibody and Cytochalasin B were purchased form Sigma-Aldrich (Zwijndrecht, the Netherlands). Recombinant Tumor-Necrosis- Factor (TNF)-a was purchased from PeproTech (Rocky Hill, NJ); Texas-Red Phalloidin, ALEXA 488-labelled GaM-Ig, ALEXA 568-labelled GaM-Ig, ALEXA 568-labelled GaR-Ig, ALEXA 488-labelled GaR-Ig and ALEXA 488-labelled streptavidin secondary Abs were from Molecular Probes (Leiden, The Netherlands) and all used in a 1:500 dilution. Jasplakinolide was purchased from Merck KGaA (Darmstadt, Germany). Expression Vectors ICAM-1-wt-GFP was a kind gift from Dr. Sanchez-Madrid (Madrid, Spain). The C-terminal deletion mutant was generated by deleting the 27 aa at the C-terminus. Cell cultures, treatments and transfections Primary HUVECs (Human Umbilican Vein Endothelial cells) were obtained from Cambrex (East Rutherford, NJ) and cultured as described previously [10]. Endothelial cells were activated with 10 ng/ml TNF-a overnight to mimic inflammation. All cell lines were cultured or incubated at 37uC at 5% CO2. The HL60 pro- myelocytic cell line was routinely cultured in IMDM, supplement- ed with 10% fetal bovine serum (Sigma). Where indicated, DMSO-differentiated HL60 cells were used. Differentiation to a neutrophil-like phenotype was achieved by including 1.3% DMSO in the medium for 3–5 days which resulted in increased expression of the LFA-1 and Mac-1 integrins [20]. Cos7 and Hela cells were maintained in IMDM with 10% fetal bovine serum. Cells were transiently transfected with the expression vectors indicated in each experiment according to the manufacturer’s protocol using LipofectAMINE PLUS (Invitrogen) or Fugene6 (Roche, Basel, Swiss). Immunofluorescence (IF) Cells were cultured on FN-coated-glass cover slips, fixed and immunostained with indicated primary Abs as described previ- ously [7]. Subsequent visualization was performed with Alexa- conjugated secondary Abs (Invitrogen). F-actin was visualized with fluorescently-labeled Phalloidin (Invitrogen). Images were record- ed with a ZEISS LSM510 confocal microscope with appropriate filter settings. Crosstalk between the different channels was avoided by use of sequential scanning. Scanning electron microscopy Transfected cells were grown on glass coverslips, fixed in 2.5% glutaraldehyde/PBS for 30 min at room temperature, and processed for scanning EM as described previously [10]. Cells were examined on a scanning electron microscope (model 820; JEOL) at 15 kV. Antibody-coated beads 3- or 10-mm polystyrene beads (Polysciences, Inc.) were pretreated with 8% glutaraldehyde overnight, washed five times with PBS, and were incubated with 300 mg/ml ICAM-1 mAb (R&D systems) or polyclonal rabbit Ab (Santa Cruz) according to the manufacturer’s protocol. Magnetic goat-anti-mouse IgG Ab- coated Dynabeads (Invitrogen) were coated with ICAM-1 or IgG isotype control Abs according to the manufacturers’ protocol. Adhesion under flow HUVECs were cultured on FN-coated glass cover slips, incubated with inhibitors as indicated, together with 1 mg/mL aICAM-1 antibody-coated beads for 30 minutes. Next, shear flow is introduced at 0.25dyn/cm2 (200 mL/minute) for 5 minutes, and then increased to 1.25dyn/cm2 for 5 minutes. Next, flow was increased every 5 minutes with 1.25dyn/cm2 up to 6.25dyn/cm2 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11336