 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 the 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 s