FIGURE 1:

Membrane clustering of Nck SH3 and VCA induces formation of dissimilar actin structures. (A, C) Schematic of the CD16/7-mCherry-Nck-SH3 and CD16/7-mCherry-VCA transmembrane fusion proteins. Both were aggregated by sequential application of primary mouse anti-CD16 and secondary anti-mouse antibodies. N-WASP recruitment to Nck SH3 required WIP (not shown in the schematic). (B) Aggregation of the Nck SH3 domains induces formation of actin comet tails. Staining: CD16/7-mCherry-Nck (red), actin (green). (D) Aggregation of the VCA domain of N-WASP induces formation of blob-like actin structures. Staining: CD16/7-mCherry-VCA (red), actin (green). Scale bars, 5 μm. (E) Morphology comparison of Nck- and VCA-induced actin structures. The bars represent the average percentage of particles per cell with circularity <0.6. Nck: three cells, 540 aggregates; VCA: three cells, 336 aggregates. Error bars are mean ± SEM. *p < 0.01. (F) Velocity comparison of Nck- and VCA-induced actin structures and vaccinia actin comet tails. Distributions of mean velocities (μm/s) are shown with boxplots. On each box, the red mark is the median, the black mark is the mean, and the lower edge is the 25th and the upper edge the 75th percentile. The whiskers are the most extreme data points, and outliers are shown by red plus signs. Nck: seven cells, 654 aggregates; VCA: seven cells, 945 aggregates.