signaling with anti-MHC while observing TCR microclusters and the cSMAC. He found that the TCR microclusters stopped forming and the last microclusters reached the cSMAC about 2 minutes after addition of the antibody, the exact time course with which Ca2+ signaling was eliminated (Varma et al., 2006). Thus, the cSMAC was unable to sustain TCR signaling beyond the 2 minute lifetime of the microclusters, although cSMAC interactions persisted for many minutes. These experiments implicated TCR microclusters in signaling by way of kinetics and complemented experiments that were performed subsequently demonstrating co-localization of TCR microclusters with signaling molecules including ZAP-70, LAT and SLP-76 in live cells and activated ZAP-70, LAT and Lck in fixed cells (Campi et al., 2005; Yokosuka et al., 2005). The movement of these microclusters was a clue to the cellular context of the activation process.
How does the Jurkat T cell line model using solid phase anti-CD3 relate to work in primary cells using bona fide MHC-peptide ligands? Signaling of T cells in response to anti-CD3ε is not blocked by depolymerizatin of F-actin (Valitutti et al., 1995a). Similarly, knock-down of the WAVE2 complex completely eliminates lamellipodial F-actin, but doesn’t inhibit early T cell signaling (Nolz et al., 2006). All but the largest TCR microclusters formed with MHC-peptide ligands are dissolved by depolymerization of F-actin (Varma et al., 2006). Nonetheless, the spreading of Jurkat cells on surfaces coated by anti-CD3ε is accompanied by the formation of a dramatic lamellipodial F-actin ring (Bunnell et al., 2001) and the formation of many discrete TCR microclusters throughout the interface that require F-actin to form in the normal manner (Bunnell et al., 2002). These structures recruit ZAP-70 and SLP-76 and, interestingly, the SLP-76 foci dissociate from the ZAP-70 rich TCR clusters and stream toward the center of the interface based on a mixture of F-actin and microtubule dependent transport processes and appear to engage