The actin cytoskeleton is important for the structure and integrity of signaling microclusters. (A) Hel-specific A20 B cells expressing GFP-Syk were settled on planar lipid bilayers containing antigen (Ag) and imaged by TIRFM. Cells were allowed to spread and form microclusters before 2 µM Latrunculin A (LatA) was injected into the imaging chamber. Cells were imaged every 10 s for 20 min. Antigen and GFP-Syk clusters before treatment (yellow outline), 300 s after treatment (blue outline), and 600 s after treatment (red outline) are shown. Overlaid images of microclusters before Latrunculin A treatment (filled yellow) and 600 s after treatment (filled red) are shown in the right column. (B) Mean intensity ± SEM of antigen microclusters (red line) was quantified over time after injection of 2 µM Latrunculin A (indicated by black arrow). Background control (black line) indicates negligible photobleaching over the image acquisition. (C) Antigen microcluster area was measured before (circles) and 600 s after 2 µM Latrunculin A (squares) with the mean ± SEM indicated in red. *, P = 0.02. Data are representative of at least two independent experiments.