Actin displays transient lamellal accumulation.

<p><b>(A, B)</b> Phalloidin stained 5C.C7 T cell:CH27 APC conjugates imaged by STED 1-2min (A) and >2min (B) after cell conjugate formation are shown in a rainbow-like color scale (scale bars = 2μm)(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#sec010" target="_blank">methods</a> for precise time point definition). (<b>C</b>) Actin amounts relative to maximum as a function of the distance from the interface in the periphery (outer 25% of the interface diameter) and center (middle 50% of the interface) measured from fixed Phalloidin stained 5C.C7 T cell:CH27 APC conjugates at an early (1-2min, number of cell couples analyzed across multiple independent experiments, n = 26) and late (2-5min, n = 16) time point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#sec010" target="_blank">methods</a> for precise time point definition) by STED and live cell conjugates expressing GFP-actin (n = 15, same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#pone.0133299.g001" target="_blank">Fig 1G</a>) or F-tractin-GFP (n = 19) (both measured at 1min) are given. (<b>D</b>) A representative interaction of a 5C.C7 T cell expressing F-tractin-GFP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#pone.0133299.s006" target="_blank">S3 Video</a>) is given similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#pone.0133299.g001" target="_blank">Fig 1B</a>. In addition, an en face view of the T cell is shown at the bottom. (<b>E</b>) The pattern classification graph is given for F-tractin-GFP similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#pone.0133299.g001" target="_blank">Fig 1E</a> (n = 51). <b>(F)</b> The depth to width ratio of resolved phalloidin stained F-actin structures (>135% above cellular background) in early (n = 12) and late (n = 12) STED imaged 5C.C7 T cell:CH27 APC conjugates is given (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133299#pone.0133299.s001" target="_blank">S1 Fig</a> for analysis details). <b>(G)</b> F-actin structure depth and width from F are given with dotted lines connecting paired measurements from the same structure for early and late cell conjugates. (<b>H</b>) The average intensity to area ratio of F-actin structures from F is given. Error bars are s.e.m. Significance was determined by Student’s t-test (*p<0.05, **p<0.001, ***p<0.0001).