urfaces 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 microclusters (Tseng et al., 2008). Blocking TCR signaling with anti-MHC-peptide antibodies rapidly eliminated the bright CD28-CD80 foci, consistent with a continual role for TCR signaling in maintaining this component of the cSMAC. These studies demonstrate that the cSMAC consists of two compartments: one a stable structure enriched in non-signaling TCR destined for shedding or degradation (a) and one highly dynamic structure rich in F-actin, CD28 and PKC-θ (b) (table 1). These compartments are difficult to resolve in cSMACs formedin cell-cell interfaces. The mechanisms by which CD28 and TCR are segregated into these two cSMAC compartments is not known.
The mechanism of TCR triggering has been an enduring problem. The prevailing model has been based on TCR dimerization or clustering as a triggering modality (Germain, 1997; Weiss and Littman, 1994). This is largely based on models from receptor tyrosine kinases and observations that antibody crosslinking triggers signaling. However, the is an adapter that recruits non-receptor tyrosine kinases to 10 docking sites, the immunotyrosine based activation motifs (ITAMs), in response to ligand binding. With all these docking sites, it is not clear that dimerizing this receptor would increase its activity. Recent studies have provided indirect evidence that monovalent ligands can trigger TCR microclusters and signaling events without directly cross-linking the receptor to form dimers e.g.(Varma et al., 2006). It is interesting at this point to think about how the TCR would sense such a monovalent engagement event? Recent data from the BCR and TCR systems provide insights into accessibility changes in cytoplasmic domains of the receptor during triggering and provide raw material for a draft model that incorporates an early role for F-actin.
The BCR has two signal transduction subunits with one ITAM in each. BCR with a FRET donor at the C-terminus of transmembrane immunoglobulin (sIg) and a FRET acceptor on the C-terminus of Igα have a high FRET signal in the basal, non-signaling 