Representative experimental images of aggregates containing different densities of Nck SH3 molecules. (A–E) To test the effect of decreasing Nck SH3 density on localized actin polymerization, Nck SH3 domains with dummy fusion proteins were cotransfected and aggregated, resulting in aggregates of varying density. In individual images, CD16-7–mCherry–Nck SH3–HA is red, CD16-7–mCerulean-HA is cyan, and YFP-actin is green. For the mCherry-Nck/YFP-actin merged images, brightness and contrast have been adjusted to allow for comparison of Nck SH3 density-dependent actin polymerization associated with Nck SH3 aggregates. Higher magnifications of clusters are shown in the insets. Bars, 10 µm. (A) Aggregates containing 80–100% dummy and 0–20% Nck SH3 (0–5,000 Nck SH3/µm²) induce no detectable actin polymerization. (B) Aggregates containing 60–80% dummy and 20–40% Nck SH3 (5,000–10,000 Nck SH3/µm²) induce minimal actin polymerization that results in the formation of sparse actin spots. (C) Aggregates containing 40–60% dummy and 40–60% Nck SH3 (10,000–15,000 Nck SH3/µm²) induce actin polymerization that results in the formation of dense actin spots. (D) Aggregates containing 20–40% dummy and 60–80% Nck SH3 (15,000–20,000 Nck SH3/µm²) induce actin polymerization that results in the formation of dense actin spots or short actin comet tails. (E) Aggregates containing 0–20% dummy and 80–100% Nck SH3 (20,000–25,000 Nck SH3/µm²) induce actin polymerization that results in the formation of a robust actin comet tail.