a fide MHC-peptide ligands? Signaling of T cells in response to anti-CD3ε is not blocked by depolymerizatin of F-actin (Valitutti et al., 1995a). Similarly, knock-down of the WAVE2 complex completely eliminates lamellipodial F-actin, but doesn’t inhibit early T cell signaling (Nolz et al., 2006). All but the largest TCR microclusters formed with MHC-peptide ligands are dissolved by depolymerization of F-actin (Varma et al., 2006). Nonetheless, the spreading of Jurkat cells on surfaces coated by anti-CD3ε is accompanied by the formation of a dramatic lamellipodial F-actin ring (Bunnell et al., 2001) and the formation of many discrete TCR microclusters throughout the interface that require F-actin to form in the normal manner (Bunnell et al., 2002). These structures recruit ZAP-70 and SLP-76 and, interestingly, the SLP-76 foci dissociate from the ZAP-70 rich TCR clusters and stream toward the center of the interface based on a mixture of F-actin and microtubule dependent transport processes and appear to engage in sustained signaling even after internalization (Barr et al., 2006; Bunnell et al., 2006) Nguyen et al, 2008). This behavior is strikingly similar to the observed movement of MHC-peptide based TCR microclusters, which occurs in the plane of the plasma membrane in the synapse (Varma et al., 2006). However, the anti-CD3ε driven TCR clusters become systematically F-actin independent after formation (Douglass and Vale, 2005). The inclusion of β1 integrin ligands like fibronectin on the surface with anti-CD3ε results in stabilization of the SLP-76 interaction with the TCR clusters and also appears to dampen F-actin dynamics (Nguyen et al., 2008). Integrin ligands generally enhance TCR signaling (Shimizu et al., 1990) and this study suggests that one component of this effect may be stabilization of TCR signaling complexes. There are clearly differences between the polyvalent anti-CD3ε interaction with the TCR complex, which contains two copies of the CD3ε chain (Call et al., 2002), and the monovalent interaction of MHC-peptide ligands with TCR that change requirements for signaling. The transport process in the planar bilayers also creates a very useful lateral segregation of TCR based structures by age with the earliest TCR microclusters in the periphery and the oldest TCR clusters in the cSMAC. Recently, monovalent biotinylation of anti-CD3εhas created a means to achieve such tempero-spatial segregation of TCR signaling complexes by attaching anti-CD3 to planar bilayers with lipid anchored ICAM-1 to form classical synapses with Jurkat cells or polyclonal T cell populations (Kaizuka et al., 2007). This method has contributed to our understanding of the synapse dynamics. We have two quite different ways to study microclusters that are appropriate for different questions and have different degrees of difficulty. It is impossible to say that one is better than the other at this point-it depends upon the experiment. Further study of microclusters in T cell-DC interfaces using advanced confocal methodologies and even in vivo will be needed to drill deeper into T cell signaling (Oddos et al., 2008).
The relationship between TCR microclusters and F-actin suggests that the TCR clusters are formed actively rather than by diffusion trapping by ligand alone. The formation of TCR microclusters is F-actin dependent whether the ligand is a physiological MHC-peptide complex or a CD3 antibody (Bunnell et al., 2001; Campi et al., 2005; Douglass and Vale, 2005; Varma et al., 2006). This also extends to signaling because depolymerization of actin in an established synapse results in rapid cessation of signaling, but does not disrupt larger TCR microclusters or the cSMAC (Varma et al., 2006). The synapse contains a zone of F-actin depletion at the center, corresponding to the cSMAC regardless of whether this structure is filled with TCR (Kaizuka et al., 2007). When TCR clusters reach the actin free zone in the center the signaling process appears to be terminated. The mechanism of signal termination is not known, but may be as simple as t