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). T