Correspondence to Francisco S á nchez-Madrid: fsanchez.hlpr@salud.madrid.org Abbreviations used in this paper: ACF, autocorrelation function; EAP, endothelial adhesive platform; FCS, fl uorescence correlation spectroscopy; FLIM, fl uorescence lifetime imaging microscopy; FN, fi bronectin; FRET, F ö rster resonance energy transfer; FRETeff, FRET effi ciency; 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.The online version of this article contains supplemental material.

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation. The interaction between T lymphocytes and antigen-presenting cells (APCs) is essential for the initiation of the immune response. The dynamic structure formed at cell-to-cell contacts between T cells and APCs, called the immune synapse (IS), is characterized by controlled recruitment of membrane receptors to specific subcellular sites (1). Upon activation by an APC, T cell molecules involved in the IS redistribute in highly organized structures at the T cell-APC contact (2). The T cell receptor (TCR) and associated molecules concatenate into the central area (central supramolecular activation cluster [cSMAC]), whereas adhesion receptors rearrange in a surrounding external ring called the peripheral supramolecular activation cluster (pSMAC) (3). During IS formation, preclustered TCR "protein islands" converge into larger aggregates that translocate toward the cSMAC (4, 5), from where they are internalized and degraded (6). The balance between the generation and degradation of TCR microclusters is critical for sustained T cell activation (5, 7) and is modulated by ligand mobility (8). However, the mechanisms regulating protein receptor movement and the basis for IS molecular segregation are still poorly understood.A plethora of molecules are translocated to the IS during T cell activation (9). These include the tetraspanins CD81 (10) and CD82 (11), which are known to associate with several IS components such as major histocompatibility complex class II (MHCII) molecules, CD4, and LF...

Data presented here provide evidence for a new direct interaction of the GTPase Rac with the C-terminal cytoplasmic domain of tetraspanin CD81. Tetraspanin-enriched, microdomain-dependent compartmentalization is a novel regulatory mechanism of Rac activity turnover, which provides a novel mechanism for regulation of cell motility by tetraspanins.