Dynamic F-actin foci at the DC immune synapse. (A) Schematic overview of PDMS confiner setup. (B) Left: Maximum intensity projection of Lifeact-eGFP–expressing mature WT DC interacting with TAMRA-stained T cells. Scale bar: 10 µm. Right: Z-stack of bright Lifeact-eGFP signal region on the left. Scale bar: 5 µm. (C) Examples of mature WT and hem1^−/− DC–T cell immune synapses and machine learning–based segmentation of synaptic DC Lifeact-eGFP signal. Scale bar: 2 µm. (D) Normalized area of synaptic DC Lifeact-eGFP signal, n = 100 synapses each, Mann-Whitney test, mean ± SD, three biological replicates. (E) Normalized synaptic DC Lifeact-eGFP signal, n = 100 synapses each, Mann-Whitney test, mean ± SD, three biological replicates. (F) Left: Time-lapse series of synaptic DC Lifeact-eGFP signal. Right: Stack through time-lapse series. (G) Kymograph of yellow line in F. (H) Frame-to-frame similarity of synaptic DC Lifeact-eGFP signal, n ≪ 6,000 frame comparisons each, Mann-Whitney test, mean ± min/max, two biological replicates. ****, P ≤ 0.0001.