ters continue to form in the periphery (Varma et al., 2006). These small TCR microclusters forming later during stimulation are invisible to wide-field imaging on bilayers or cell-cell system. In fact, the continual formation of small microclusters to sustain signaling has only recently been observed in cell-cell systems using a combination of laser tweezers and spinning disk confocal microscopy (Oddos et al., 2008). This sustained movement of small TCR clusters suggested that some centripetal F-actin flow continued in the stable synapse.
How can one define a lamellipodium quantitatively? The Sheetz and Wiggins labs developed methods based on loading cells with a fluorescent dye that fills the cytoplasm and then use TIRFM to image the dynamic footprints of the cell. Edge tracking algorithms quantify the advances and retraction of the edges and modify this to compensate for rapid movement of lymphocytes forming kinapses. This data can then be subjected to graphic and correlation analysis to compare the contact area dynamics between cell types (Dobereiner et al., 2006; Sims et al., 2007). Our collaborative studies revealed that the outer edge of the immunological synapse displayed an evolutionarily conserved dynamic pattern of movement referred to as contractile oscillations (Fig 1). This is thought to emerge from cycles of F-actin polymerization and myosin II dependent contraction that periodically pull the membrane protrusion up and back. This is an excellent way to test the mechanical properties of the surface on which cells are interacting. This protrusion and retraction cycle then propagates as a wave around the entire periphery of the cell. These experiments provided strong quantitative evidence the periphery of primary mouse T cells forming synapses or kinapses on planar bilayers with MHC-pepide complexes, ICAM-1 and CD80 form a radial lamellipodium. More recently, Vale and my lab used speckle microscopy to explicitly demonstrate centripetal actin flow in the dSMAC and pSMAC regions (Kaizuka et al., 2007). This F-actin flow is a consequence of the polymerization of F-actin at the protruding edge, which is pushed backwards because the outer edge advances more slowly than actin is added to the growing filament ends. TCR microclusters are transported at about 40% of the speed of the F-actin in the lamellipodium. This suggests that the microclusters bind and dissociate from the actin network. This notion is also supported by studies showing that TCR clusters can navigate around barriers placed in planar bilayers by nanofabrication methods (DeMond et al., 2008). The ability to move around the barriers appears to depend upon periods of transport punctuated by diffusive movement, which allows the microcluster to make progress around diagonal barriers. TCR microcluster transport, contractile oscillations and centripetal F-actin flow support the model that immunological synapses are based on maintaining a radially symmetrical lamellipodium-the same structure Kupfer described as a dSMAC (Freiberg et al., 2002). The dSMAC was reported as having a high concentration of both CD4 and CD45, but TIRFM imaging of these structures on bilayers does not consistently show this. It is likely that the impression of increased CD4 or CD45 in wide-field imaging is based on the lamellipodium having two plasma membrane layers separated by only 100–200 nm of cytoplasmic and thus appearing twice as bright as surrounding single membrane layers in the pSMAC. In fact, by TIRFM CD45 appears relatively uniform in the synapse except for discrete areas of exclusion around TCR microclusters (Varma et al., 2006). Thus, dSMAC is immunological shorthand for a radial lamellipodium.
The pSMAC must also maintain radial symmetry to have a stable synapse because this structure contains most of the integrins that generate traction for movement (Sims et al., 2007). If the symmetry of the pSMAC is broken the cell will migrate, even if a radially symmetrical dSMAC appears to be maintained. The precise mechanism by which symmetry is established or broken is poorly u