CAR microcluster dynamics. (A) TIRF imaging of CAR-mEmerald is shown with spots and tracks made in Imaris. Tracks analysis shows that HA CAR microclusters on low HER2 (6.25 ng/well) are not mobile, and on high HER2 (625 ng/well) only LA monomer is mobile. Scale bars = 3 μm. (B) A random 10 tracks are shown in flower plots for each condition. Top: Limited mobility is apparent for dimeric CARs on high-HER2 loaded bilayers. Bottom: On low-HER2 bilayers, LA CAR microclusters show increased mobility. Plots of low affinity dimer from Fig. 5 C are shown again here for comparison. (C) Flower plots are shown including all tracks for each cell. (D) The normalized dot product of V(rad) and V(i) was calculated for all tracks, where V(rad) = vector from synapse center to track start position and V(i) = vector from track start to track final position. Using this calculation, movements directly towards the center equal −1 and movements directly away from center equal 1. The average dot product for 3 cells were calculated (30–294 tracks per cell). No significant differences are identified in the direction of CAR microcluster movement across conditions. All n.s. by Tukey’s multiple comparisons test.