his F-actin flow is a consequence of the polymerization of F-actin at the protruding edge, which is pushed backwards because the outer edge advances more slowly than actin is added to the growing filament ends. TCR microclusters are transported at about 40% of the speed of the F-actin in the lamellipodium. This suggests that the microclusters bind and dissociate from the actin network. This notion is also supported by studies showing that TCR clusters can navigate around barriers placed in planar bilayers by nanofabrication methods (DeMond et al., 2008). The ability to move around the barriers appears to depend upon periods of transport punctuated by diffusive movement, which allows the microcluster to make progress around diagonal barriers. TCR microcluster transport, contractile oscillations and centripetal F-actin flow support the model that immunological synapses are based on maintaining a radially symmetrical lamellipodium-the same structure Kupfer described as a dSMAC (Freiberg et al., 2002). The dSMAC was reported as having a high concentration of both CD4 and CD45, but TIRFM imaging of these structures on bilayers does not consistently show this. It is likely that the impression of increased CD4 or CD45 in wide-field imaging is based on the lamellipodium having two plasma membrane layers separated by only 100–200 nm of cytoplasmic and thus appearing twice as bright as surrounding single membrane layers in the pSMAC. In fact, by TIRFM CD45 appears relatively uniform in the synapse except for discrete areas of exclusion around TCR microclusters (Varma et al., 2006). Thus, dSMAC is immunological shorthand for a radial lamellipodium.
The pSMAC must also maintain radial symmetry to have a stable synapse because this structure contains most of the integrins that generate traction for movement (Sims et al., 2007). If the symmetry of the pSMAC is broken the cell will migrate, even if a radially symmetrical dSMAC appears to be maintained. The precise mechanism by which symmetry is established or broken is poorly understood. 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 sug