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