T Cell Costimulation via the Integrin VLA-4 Inhibits the Actin-Dependent Centralization of Signaling Microclusters Containing the Adaptor SLP-76

Abstract

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.

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

“…TEM studies of Wilson et al in mast cells also support the interaction of the actin cytoskeleton with receptor clusters (Wilson et al 2001). Furthermore, inhibitor studies showed that actin is essential for the formation of microclusters Douglass and Vale 2005;Varma et al 2006;Nguyen et al 2008). An intact cytoskeleton is also required for dynamic translocation of clusters once they are formed and it has been proposed that retrograde actin flow drives centripetal cluster movement Varma et al 2006;Kaizuka et al 2007;Nguyen et al 2008;Ilani et al 2009).…”

“…Microclusters play a predominant role in the generation of sustained signals. Current models also indicate that cluster centralization plays an important role in signal termination and that the cSMAC is a site both for TCR down-regulation yet also for amplification of weak signals (Lee et al 2003;Mossman et al 2005;Varma et al 2006;Balagopalan et al 2007;Cemerski et al 2008;Nguyen et al 2008). A recent study using a photoactivatable agonist in a lipid bilayer system has provided even greater precision in the temporal resolution of these events and demonstrated that adapters are recruited to microclusters within four seconds (Huse et al 2007).…”

“…Fine spatial imaging studies indicated that signaling protein clusters may actually represent discrete interdigitating domains (Douglass and Vale 2005;Nguyen et al 2008). These structures resembled the distinct protein and lipid domains observed by transmission electron microscopy (TEM) (Wilson et al 2001;Lillemeier et al 2006).…”

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

“…GFP or LAT.GFP were gifts of L. Samelson (National Institutes of Health) (42). J14 cells stably expressing SLP-76.YFP (J14SLP-76.YFP) were provided by S. Bunnell (Tufts University) (43,44). For transfection, 1 3 10 7 Jurkat T lymphocytes were electroporated using 25-50 mg plasmid DNA (850 mF, 250 V; Bio-Rad Genepulser) and analyzed 24 h posttransfection.…”

“…J14SLP-76.YFP cells (Jurkat T lymphocytes stably expressing SLP-76 fused to YFP) were used for these analyses as established model of SLP-76 MC formation (43,44). Whereas control cells displayed a large number of SLP-76-positive MCs across the cover glass-cell interface, including the cell periphery, only few such MCs could be detected in cells expressing Nef with most cells virtually lacking detectable SLP-76 MCs (Fig.…”