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 bon