LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

Vav2 lacks Ca2+ entry-promoting scaffolding functions unique to Vav1 and inhibits T cell activation via Cdc42.

Vav family guanine nucleotide exchange factors (GEFs) are essential regulators of immune function. Despite their structural similarity, Vav1 promotes and Vav2 opposes T cell receptor (TCR)-induced Ca2+ entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca2+ entry via non-catalytic scaffolding functions that are encoded by the catalytic core of Vav1 and flanking linker regions. We implicate, in this scaffolding function, a previously undescribed polybasic motif that is strictly conserved in Vav1 and absent from Vav2 in tetrapods. Conversely, the catalytic activity of Vav2 contributes to the suppression of TCR-mediated Ca2+ entry. By performing an in vivo 'GEF trapping' assay in intact cells, we demonstrate that Cdc42 interacts with the catalytic surface of Vav2 but not Vav1, and that Vav1 discriminates Cdc42 from Rac1 via F56 (W56 in Rac1). Finally, the Cdc42-specific inhibitor ZCL278 and the shRNA-mediated suppression of Cdc42 each prevent the inhibition of TCR-induced Ca2+ entry by Vav2. These findings define stark differences in the functions of Vav1 and Vav2, and provide an explanation for the differential usage of these Vav isoforms by immune subpopulations.

Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses.

The guanine nucleotide exchange factor (GEF) Vav1 synergizes with the adaptor protein SLP-76 (Src homology 2 domain--containing leukocyte phosphoprotein of 76 kD) to support T cell development and activation. In response to ligation of the T cell receptor (TCR), SLP-76 is assembled into microclusters that provide an essential platform for the signaling events that drive T cell activation. We found that Vav1 selectively entered SLP-76 microclusters, rather than TCR microclusters, influencing their stability and function. The carboxyl terminus of Vav1, which consists of Src homology domains, was both necessary and sufficient for the entry of Vav1 into SLP-76 microclusters; however, this fragment of Vav1 was insufficient to stabilize the microclusters, and it potently suppressed T cell activation. This indicated that the amino terminus of Vav1, which has the GEF domain, also contributed to the integrity of SLP-76 microclusters and thereby to T cell activation. These microcluster-stabilizing functions were independent of the GEF activity in the amino terminus of Vav1 and were unaffected if the GEF function of Vav1 was either inactivated or constitutively activated by mutation. In contrast, Vav1 deletion mutants lacking either the calponin homology domain or the catalytic core of the GEF exhibited mild scaffolding defects, but they differentially affected TCR-dependent calcium ion (Ca²+) responses. We conclude that multiple GEF-independent scaffolding functions distributed throughout the amino terminus of Vav1 contribute to the activation of T cells by acting synergistically to increase the stability and function of SLP-76 microclusters.

T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76.

Antigen-dependent T cell activation drives the formation of signaling microclusters containing the adaptor SLP-76. Costimulatory integrins regulate SLP-76 phosphorylation and could influence SLP-76 microclusters in the integrin-rich periphery of the immune synapse. We report that costimulation by the integrin VLA-4 (alpha4beta1) required SLP-76 domains implicated in microcluster assembly. Pro-adhesive ligands enlarged the contact and increased the number of SLP-76 microclusters regardless of their costimulatory potential. Costimulatory VLA-4 ligands also prevented the centralization of SLP-76, promoted microcluster persistence, prolonged lateral interactions between SLP-76 and its upstream kinase, ZAP-70, and retained SLP-76 in tyrosine-phosphorylated peripheral structures. SLP-76 centralization was driven by dynamic actin polymerization and was correlated with inward actin flows. VLA-4 ligation retarded these flows, even in the absence of SLP-76. These data suggest a widely applicable model of costimulation, in which integrins promote sustained signaling by attenuating cytoskeletal movements that drive the centralization and inactivation of SLP-76 microclusters.