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.
The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.
Antigen presentation to T lymphocytes has been characterized extensively in terms of T lymphocyte activation and eventual cell death. In contrast, little is known about the consequences of antigen presentation for the antigen-presenting cell (APC). We have determined the outcome of major histocompatibility complex class II-restricted peptide presentation to a specific T cell. We demonstrate that specific T lymphocyte interaction with peptide-presenting APCs led to apoptosis in the APC population. In contrast, T lymphocyte interaction with nonpeptide-loaded APCs or APCs loaded with monosubstituted peptide failed to induce T lymphocyte secretion of interleukin-2 and APC apoptosis. Phosphatidylserine externalization and mitochondrial depolarization were used to evaluate APC apoptosis. Fas/Fas ligand interactions were not required, but cytoskeletal integrity and caspase activation were essential for APC apoptosis. Antigen presentation leading to T lymphocyte activation is therefore coordinated with apoptosis in the APC population and could provide a mechanism of immune response regulation by eliminating APCs, which have fulfilled their role as specific ligands for T lymphocyte activation. This pathway may have particular importance for APCs, which are not sensitive to death receptor-induced apoptosis.
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