Sustained LFA‐1 cluster formation in the immune synapse requires the combined activities of L‐plastin and calmodulin

Abstract

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution -however, not TCR/CD3 relocalization -into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS. Key words: Actin cytoskeleton . Immunological synapse . T cells Supporting Information available online IntroductionDuring the activation of T cells the immune synapse (IS) is formed at the area of interaction between T cells and APC [1,2]. The IS is involved in enhancing, directing and terminating the T-cell immune response (for review, see [3][4][5][6][7]). Within the IS, surface receptors as well as intracellular signaling and scaffolding proteins are organized in distinct structures, which are called supramolecular activation clusters (SMAC). The inner cluster (central SMAC or cSMAC) contains PKCY and the TCR/CD3 complex. The outer cluster (peripheral SMAC or pSMAC) is composed of the integrin LFA-1 (CD11a/CD18) and Talin [8].It is clear that for the development of an IS the actin cytoskeleton is of special importance [2,[9][10][11]. For construction of an actin meshwork, as it is found in the IS, crosslinking and bundling of F-actin is indispensable to support F-actin rigidity. Here, we Its function for the formation of the IS, however, was so far unexplored. Plastins belongs to the fimbrin family. Plastins contain two tandemly organized actin-binding sites at the C-terminus, which enable them to form very tight actin bundles [12,13]. But despite having two actin-binding domains, it was reported that plastins bind only weakly to pre-existing actin filaments. An optimal binding occurs, if plastin binds during the process of actin polymerization [14,15]. Bundling of F-actin reduces the speed of the actin turnover, thereby making F-actin structures more stable -but not inflexible and stiff. In this regard, it was also reported that plastin binding can protect actin filaments from depolymerization by cofilin in vitro [16]. Thus, plastins control the length of actin fibers and the speed of G/F-actin turnover. Only very little is known about the regulation of plastins ...

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