Acknowledgments We thank Dr. Sanchez-Madrid and Dr. Barreiro (Madrid, Spain) for supplying us with the ICAM-1-GFP construct. References 1. Carman CV, Springer TA (2004) A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them. J Cell Biol 167: 377–88. 2. Kluger MS (2004) Vascular endothelial cell adhesion and signaling during leukocyte recruitment. Adv.Dermatol 20: 163–201. 3. van Buul JD, Kanters E, Hordijk PL (2007) Endothelial signaling by Ig-like cell adhesion molecules. Arterioscler Thromb Vasc Biol 27: 1870–1876. 4. Vestweber D (2007) Molecular mechanisms that control leukocyte extravasation through endothelial cell contacts. Ernst Schering Found Symp Proc 3: 151–167. 5. Wittchen ES (2009) Endothelial signaling in paracellular and transcellular leukocyte transmigration. Front Biosci 14: 2522–2545. 6. Thompson PW, Randi AM, Ridley AJ (2002) Intercellular adhesion molecule (ICAM)-1, but not ICAM-2, activates RhoA and stimulates c-fos and rhoA transcription in endothelial cells. J Immunol 169: 1007–1013. 7. van Buul JD, Voermans C, van den Berg V, Anthony EC, Mul FP, et al. (2002) Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin. J Immunol 168: 588–596. 8. Burridge K, Wennerberg K (2004) Rho and Rac take center stage. Cell 116: 167–179. 9. Etienne S, Adamson P, Greenwood J, Strosberg AD, Cazaubon S, Couraud PO (1998) ICAM-1 signaling pathways associated with Rho activation in microvascular brain endothelial cells. J Immunol 161: 5755–5761. 10. van Buul JD, Allingham MJ, Samson T, Meller J, Boulter E, et al. (2007) RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte trans-endothelial migration. J Cell Biol 178: 1279–1293. 11. Wojciak-Stothard B, Williams L, Ridley AJ (1999) Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol 145: 1293–1307. 12. Tilghman RW, Hoover RL (2002) E-selectin and ICAM-1 are incorporated into detergent-insoluble membrane domains following clustering in endothelial cells. FEBS Lett 525: 83–87. 13. Barreiro O, Yanez-Mo M, Serrador JM, Montoya MC, Vicente-Manzanares M, et al. (2002) Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes. J Cell Biol 157: 1233–1245. 14. Greenwood J, Amos CL, Walters CE, Couraud PO, Lyck R, et al. (2003) Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration. J Immunol 171: 2099–2108. 15. Lyck R, Reiss Y, Gerwin N, Greenwood J, Adamson P, Engelhardt B (2003) T- cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothe- lial migration of T cells. Blood 102: 3675–3683. 16. Sans E, Delachanal E, Duperray A (2001) Analysis of the roles of ICAM-1 in neutrophil transmigration using a reconstituted mammalian cell expression model: implication of ICAM-1 cytoplasmic domain and Rho-dependent signaling pathway. J Immunol 166: 544–551. 17. Oh HM, Lee S, Na BR, Wee H, Kim SH, et al. (2007) RKIKK motif in the intracellular domain is critical for spatial and dynamic organization of ICAM-1: functional implication for the leukocyte adhesion and transmigration. Mol Biol Cell 18: 2322–2335. 18. Chen X, Kim TD, Carman CV, Mi LZ, Song G, Springer TA (2007) Structural plasticity in Ig superfamily domain 4 of ICAM-1 mediates cell surface dimerization. Proc Natl Acad Sci USA 104: 15358–15363. 19. Yang L, Kowalski JR, Yacono P, Bajmoczi M, Shaw SK, et al. (2006) Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration. J Immunol 177: 6440–6449. ICAM-1 Dynamics in Endothelium Author Contributions Conceived and designed the experiments: JDVB JvM PH. Performed the experiments: JDVB JVR FPJvA MH ST KAH EM. Analyzed the data: JDVB JVR JvM EM. Wrote the paper: JDVB PH. 20. Back AL, Gollahon KA, Hickstein DD (1992) Regulation of expression of the leukocyte integrin CD11a (LFA-1) molecule during differentiation of HL-60 cells along the monocyte/macrophage pathway. J Immunol 148: 710–714. 21. Bohil AB, Robertson BW, Cheney RE (2006) Myosin-X is a molecular motor that functions in filopodia formation. Proc Natl Acad Sci USA 103: 12411–12416. 22. Kanters E, van Rijssel J, Hensbergen PJ, Hondius D, Mul FP, et al. (2008) Filamin B mediates ICAM-1-driven leukocyte transendothelial migration. J Biol Chem 283: 31830–31839. 23. van Buul JD, Hordijk PL (2009) Endothelial adapter proteins in leukocyte transmigration. Thromb Haemost 101: 649–655. 24. Carman CV, Jun CD, Salas A, Springer TA (2003) Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1. J Immunol 171: 6135–6144. 25. Nakamura F, Osborn TM, Hartemink CA, Hartwig JH, Stossel TP (2007) Structural basis of filamin A functions. J Cell Biol 179: 1011–1025. 26. Sverdlov M, Shinin V, Place AT, Castellon M, Minshall RD (2009) Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells. Mol Biol Cell 20: 4531–4540. 27. Berg JS, Powell BC, Cheney RE (2001) A millennial myosin census. Mol Biol Cell 12: 780–794. 28. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21: 247–269. 29. Bouquier N, Vignal E, Charrasse S, Weill M, Schmidt S, et al. (2009) A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway. Chem Biol 16: 657–666. 30. Allingham MJ, van Buul JD, Burridge K (2007) ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration. J Immunol 179: 4053–4064. 31. Durieu-Trautmann O, Chaverot N, Cazaubon S, Strosberg AD, Couraud PO (1994) Intercellular adhesion molecule 1 activation induces tyrosine phosphor- ylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelial cells. J Biol Chem 269: 12536–12540. 32. Alcaide P, Auerbach S, Luscinskas FW (2009) Neutrophil recruitment under shear flow: it’s all about endothelial cell rings and gaps. Microcirculation 16: 43–57. 33. Millan J, Hewlett L, Glyn M, Toomre D, Clark P, Ridley AJ (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat Cell Biol 8: 113–123. 34. Celli L, Ryckewaert JJ, Delachanal E, Duperray A (2006) Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis. J Immunol 177: 4113–4121. 35. Heiska L, Alfthan K, Gronholm M, Vilja P, Vaheri A, Carpen O (1998) Association of ezrin with intercellular adhesion molecule-1 and -2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4, 5-bisphosphate. J Biol.Chem 273: 21893–21900. 36. Tolias KF, Rameh LE, Ishihara H, Shibasaki Y, Chen J, et al. (1998) Type I phosphatidylinositol-4-phosphate 5-kinases synthesize the novel lipids phospha- tidylinositol 3,5-bisphosphate and phosphatidylinositol 5-phosphate. J Biol Chem 273: 18040–18046. 37. van Hennik PB, ten Klooster JP, Halstead JR, Voermans C, Anthony EC, et al. (2003) The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity. J Biol Chem 278: 39166–39175. 38. Khuon S, Liang L, Dettman RW, Sporn PH, Wysolmerski RB, Chew TL (2010) Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study. J Cell Sci 123: 431–440. 39. Saito H, Minamiya Y, Saito S, Ogawa J (2002) Endothelial Rho and Rho kinase regulate neutrophil migration via endothelial myosin light chain phosphoryla- tion. J Leukoc Biol 72: 829–836. PLoS ONE | www.plosone.org 14 June 2010 | Volume 5 | Issue 6 | e11336