he inability to amplify signals in the absence of F-actin. It is not known how the TCR microclusters continue to move to the center of the synapse when the F-actin conveyor belt stops 1–2 μm from the center. This could be a diffusive process or might involve some alternative transport system that remains to be described. For example, the zone of F-actin depletion is observed in cytotoxic T cell synapses and allows close approach of the centrosome to the plasma membrane for efficient directed secretion (Stinchcombe et al., 2006).
It is not clear why F-actin is so important for TCR signal transduction because anti-CD3 stimulation to Ca2+ mobilization and cytokine production doesn’t require intact F-actin (Valitutti et al., 1995a). Similarly, B cells require F-actin to recognize ligands on a surface, but not aggregating ligands in solution. Stimulation by surface presented ligands requires F-actin, Rap1 and CD19 (Depoil et al., 2008; Lin et al., 2008). The model for stimulation of activation of BCR by monovalent ligands in supported planar bilayers is based on diffusion trapping of receptors and ligands in microdomains with multiple BCR and ligands. This mode of clustering is likely driven by F-actin dependent membrane fluctuations and adhesion, perhaps accounting for the role of F-actin in enhancing B cell recognition of solid phase ligands (Carrasco et al., 2004; Tolar et al., 2008). For T cells, the F-actin requirement seems to involve both forming microclusters and then formation of signaling complexes at the TCR clusters. This is based on the observation that many TCR microclusters are stable after F-actin depolymerization, but signaling is still terminated even though the receptor clusters persist. All antigen receptors engage in signaling that activates actin polymerization via the Rac and Cdc42 small G-proteins leading to activation of the WAVE2 complexes and WASp (Barda-Saad et al., 2005; Nolz et al., 2006). In the absence of WAVE2 the lamellipodium at the periphery of T cell contacts with anti-CD3 coated surfaces is lost (Nolz et al., 2006). This does not alter that ability of these cells to initiate early TCR signaling in response to solid phase anti-CD3, but more work is needed to determine if this alters sensitivity to MHC-peptide ligands as expected.
The recent focus on microclusters raises questions about cSMAC-associated signals. Is the cSMAC just for signal termination or is it a site of TCR or other relevant signaling? Is the cSMAC even one compartment? Kupfer defined the cSMAC based on accumulation of TCR and PKC-θ. Dynamic studies using fluorescence recovery after photobleaching (FRAP) revealed that TCR-MHC-peptide interactions are particularly stable in the cSMAC (Grakoui et al., 1999) and that the TCR microclusters that converge to form the cSMAC appear to fuse together into one stable structure on planar bilayers (Varma et al., 2006). This structure is partly shed by the T cell if it breaks the synapse and migrates or wanes over 10’s of minutes in stable synapses. Is this highly stabilized structure in which TCR seem to be shed or degraded all there is to the cSMAC? Analysis of CD28-CD80 interactions in the synapse suggests that there is a second, more dynamic component to the cSMAC that is typically not well resolved, but represents a very distinct sub-cellular compartment (Tseng et al., 2008; Yokosuka et al., 2008). CD28-CD80 interactions colocalize with TCR-MHC-peptide interactions in microclusters. When these mixed microclusters reach the cSMAC, mostof the TCR are incorporated into a stable central cluster, but all of the CD28 remains in an intermediate compartments which is very dynamic and appears to be critical for sustained PKC-θ signaling (Yokosuka et al., 2008). This annular structure sits at the inside edge of the F-actin rich lamella and thus the small amounts of TCR in this compartment may continue to signal to maintain the dynamic CD28-CD80 interactions. Similar segregation of TCR and CD28 is observed in T cell-DC interfaces, although it was not possible to observe the precursor microclu