gestion that the cSMAC might be involved in signal termination (Lee et al., 2003). Computer models suggested that signals in response to weaker ligands might survive in the cSMAC due to a less aggressive attack by ubiquitin ligases. As discussed below this preservation of signaling in the cSMAC may also depend upon a reorganization of F-actin to fill in the normally F-actin depleted central area (Cemerski et al., 2008; Lee et al., 2003). The enigma with peripheral signaling in the immunological synapse was that TCR was not clearly associated with the signaling. This issue was resolved with TIRFM, which greatly increased the contrast of imaging of the cell-planar bilayer interface, as described above, but the discovery of the late submicron TCR microclusters that formed in the dSMAC and moved to the pSMAC over ~ 2 minutes.
The movement of microcluster was interesting for what it said about actin dynamics, but the signaling behavior was even more interesting. Varma repeated the Valittuti experiment of blocking 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 in sustained signaling even after internalization (Barr et al., 2006; Bunnell et al., 2006) Nguyen et al, 2008). This behavior is strikingly similar to the observed movement of MHC-peptide based TCR microclusters, which occurs in the plane of the plasma membrane in the synapse (Varma et al., 2006). However, the anti-CD3ε driven TCR clusters become systematically F-actin independent after formation (Douglass and Vale, 2005). The inclusion of β1 integrin ligands like fibronectin on the surface with anti-CD3ε results in stabilization of the SLP-76 interaction with the TCR clusters and also appears to dampen F-actin dynamics (Nguyen et al., 2008). Integrin ligands generally enhance TCR signaling (Shimizu et al., 1990) and this study suggests that one component of this effect may be stabilization of TCR signaling complexes. There are clearly differences between the polyvalent anti-CD3ε interaction with the TCR complex, which contains two copies of the CD3ε chain (Call et al., 2002), and the monovalent interaction