 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