r results were obtained in live cell-cell systems in which TCR in small clusters moved to the center to form the cSMAC (Krummel et al., 2000). However, these authors eventually rejected the hypothesis that myosin II is required for this contraction and transport process because knockdown of myosin IIA did not appear to eliminate the ability to form a synapse with an APC (Jacobelli et al., 2004). More recent studies in the planar bilayer model actually supported the hypothesis that myosin IIA is important for microcluster transport and signal amplification in that system and for signal amplification in cell-cell models (Ilani et al., 2009). It is possible that there are alternative mechanisms to form a cSMAC in cell-cell systems that involve directed vesicular transport, which would be myosin II independent (Das et al., 2004). Nonetheless, the extension of an APC embracing lamellipodium is observed in cell-cell systems (Antón et al., 2002; Tskvitaria-Fuller et al., 2003). In Jurkat T leukemia cells, spreading on anti-CD3 coated surfaces form a well organized F-actin ring at early time points, and that this ring appear to break up after a contractile phase (Bunnell et al., 2001). These studies suggest that the initial contact expansion is mediated by F-actin driven protrusion, which then shift to a more dynamic extension-retraction process during sustained signaling. In fact the pivoting process observed with solid phase MHC-peptide complexes suggest that the stop was based on loss of persistent polarity. What could account for the ability of cells to rapidly transition from migration to arrest?
Quantitative analysis of TCR dynamics and cell spreading provided insight into how the stable and dynamic characteristics of the synapse are reconciled. Observations of contact areas on planar bilayers with total internal reflection fluorescence microscopy (TIRFM) reveal that the dSMAC is a radial lamellipodium. First, TCR in cells form synapses over a period of 30 minutes and even after cSMAC formation, sub-micron TCR clusters 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