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J. Cell Biol. 183 (3): 527-542

Copyright © 2008 by the Rockefeller University Press.


Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms

Olga Barreiro1,2, Moreno Zamai4,5, María Yáñez-Mó1,2, Emilio Tejera1,2, Pedro López-Romero3, Peter N. Monk6, Enrico Gratton7, Valeria R. Caiolfa2,4,5, , and Francisco Sánchez-Madrid1,2

1 Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain
2 Departamento de Biología Vascular e Inflamación and 3 Unidad de Genómica, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain
4 Department of Molecular Biology and Functional Genomics and 5 Italian Institute of Technology Network Research, Unit of Molecular Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy
6 Academic Neurology Unit, School of Medicine and Biomedical Science, University of Sheffield, Sheffield S10 2RX, England, UK
7 Laboratory for Fluorescence Dynamics, Biomedical Engineering Department, University of California, Irvine, Irvine, CA 92697

Correspondence to Francisco Sánchez-Madrid: fsanchez.hlpr{at}

Abstract: VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell–cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin–binding capacity of both endothelial receptors during extravasation.

Abbreviations used in this paper: ACF, autocorrelation function; EAP, endothelial adhesive platform; FCS, fluorescence correlation spectroscopy; FLIM, fluorescence lifetime imaging microscopy; FN, fibronectin; FRET, Förster resonance energy transfer; FRETeff, FRET efficiency; GPI, glycosylphosphatidylinositol; HUVEC, human umbilical vein endothelial cell; knn, k nearest neighbor; LEL, large extracellular loop; mEGFP, monomeric EGFP; TEM, tetraspanin-enriched microdomain; VE-cadherin, vascular endothelial cadherin.

© 2008 Barreiro et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at

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