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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}salud.madrid.org
Abstract:
VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruitedto cell–cell contact sites on the apical membrane of activatedendothelial cells. In this study, we show that this recruitmentis independent of ligand engagement, actin cytoskeleton anchorage,and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruitedby inclusion within specialized preformed tetraspanin-enrichedmicrodomains, which act as endothelial adhesive platforms (EAPs).Using advanced analytical fluorescence techniques, we have characterizedthe diffusion properties at the single-molecule level, nanoscaleorganization, and specific intradomain molecular interactionsof EAPs in living primary endothelial cells. This study providescompelling evidence for the existence of EAPs as physical entitiesat the plasma membrane, distinct from lipid rafts. Scanningelectron microscopy of immunogold-labeled samples treated witha specific tetraspanin-blocking peptide identify nanoclusteringof VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecularorganization that regulates the leukocyte integrin–bindingcapacity of both endothelial receptors during extravasation.
Abbreviations used in this paper: ACF, autocorrelation function;EAP, endothelial adhesive platform; FCS, fluorescence correlationspectroscopy; 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 nearestneighbor; LEL, large extracellular loop; mEGFP, monomeric EGFP;TEM, tetraspanin-enriched microdomain; VE-cadherin, vascularendothelial cadherin.
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