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First published online 28 September 2004
doi: 10.1242/jcs.01380
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Research Article |
1 INSERM U396, Hôpital Saint Louis, 1 avenue Claude Vellefaux 75010 Paris, France
2 Unité d'Immunologie Virale, Institut Pasteur, 25, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France
3 Département de Biologie Cellulaire, Institut Cochin, INSERM U567, CNRS UMR 8104, Université Paris V. 22, rue Méchain, 75014 Paris, France
4 INSERM U268, Institut Andre-Lwoff, Hopital Paul-Brousse, 7 rue Guy Moquet, BP 8, 94801 Villejuif CEDEX, France
* Author for correspondence (e-mail: conjeaud{at}cochin.inserm.fr)
Accepted 24 June 2004
T-cell activation is initiated by the concerted engagement of the T-cell receptor and different co-stimulatory molecules, and requires cytoskeleton-dependent membrane dynamics. Here, we have studied the relationships between tetraspanins, cytoskeleton and raft microdomains, and their relevance in T-cell signaling. Localization studies and density-gradient flotation experiments indicate that part of tetraspanins localizes in raft microdomains linked to the actin cytoskeleton. First, partial coalescence of lipid raft is triggered by tetraspanin cross-linking and results in large caps in which F-actin also concentrates. Second, the amount of tetraspanins, which are recovered in the cholesterol-dependent insoluble fractions of low and intermediate density, and which appears to be membrane vesicles by electron microscopy, is under cytoskeletal influence. Disruption of actin filaments enhances the amount of tetraspanins recovered in typical raft fractions, whereas F-actin-stabilizing agents induce the opposite effect. Our data also reveal that CD82 constitutes a link between raft domains and the actin cytoskeleton, which is functionally relevant. First, tetraspanin signaling induces a selective translocation of CD82 from detergent-resistant membrane fractions to the cytoskeleton-associated pellet. Second, all functional effects linked to CD82 engagement, such as adhesion to culture plates, formation of actin bundles and early events of tyrosine phosphorylation, are abolished, or strongly reduced, by cholesterol depletion. We also show that dynamic relocalization of CD82 and F-actin at the periphery of the immune synapse is induced upon contact of T cells with antigen-presenting cells. This suggests that the tetraspanin web might participate in the membrane dynamics required for proper T-cell signaling. More generally, the interaction of tetraspanins with raft domains and with the actin cytoskeleton might relate with their role in many cellular functions as membrane organizers.
Key words: Tetraspanin, Rafts, Cytoskeleton, CD82, T-cell signalling
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