nderstood. We know that on the supported planar bilayer system WASP is required to maintain a stable synapse and PKC-θ contributes to symmetry breaking (Sims et al., 2007). Symmetry breaking in the contact plane leads to polarity and in a migrating cells this process is based two opposing actin-myosin networks-the lamellipodium dominated by Rac and a high F-actin:myosin II ratio and the uropod dominated by Rho and a low F-actin:myosin II ratio (Xu et al., 2003). Thus, we have speculated that myosin II may be a target in symmetry breaking. PKC-θ phosphorylates WASP interacting protein (WIP) and regulates its interaction with myosin II (Krzewski et al., 2006). WASP also interacts with this complex. One hypothesis is that myosin II activation via WIP phosphorylation leads to contraction of the pSMAC actin network leading to local thinning and breakdown, whereas WASP may either antagonize this myosin II activation or induced ventral actin polymerization to repair the pSMAC and restore symmetry after its been broken. PKC-θ mediated synapse breaking seems like a negative feedback loop because the other major activity of PKC-θ in T cells is activation of NFkB and other transcription factors. Thus, the functional consequences of synapse stabilization as a result of PKC-θ inhibition can only be studied in pre-armed effector cells, which don’t need to activate transcription for function.
The cSMAC was initially shown to contain PKC-θ and Lck, two kinases that are important for T cell activation (Monks et al., 1998). This suggested that the cSMAC might be involved in sustained signaling. Our early studies on planar bilayers demonstrated that TCR signaling was initiated before the cSMAC was formed based on cytoplasmic Ca2+ elevation at a time point where TCR-MHC-peptide interactions were focused in the periphery (Grakoui et al., 1999). This was more explicitly demonstrated with a specific phopho-Lck antibody, which revealed early TCR signaling in the periphery of the nascent synapse, but not at the cSMAC. This led to the suggestion 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