The Tda2–Aim21 complex binds actin-capping protein and regulates its recruitment to CME sites. (A) GST pulldown assay was performed with GST-Aim21 (lanes 1 and 2), GST-Tda2 (lane 3), and GST alone (lane 4). Each GST fusion protein was incubated with purified actin-capping protein carrying a polyhistidine tag in its Cap1 subunit (His-Cap1/Cap2; lanes 1–4). One GST-Aim21 sample was incubated with His-Tda2 (to reconstitute the Tda2–Aim21 complex) in addition to His-Cap1/Cap2 (lane 2). The bound proteins were analyzed by Coomassie staining (top) and anti-His immunoblotting (IB; bottom). His-Cap1/Cap2 bound robustly to the GST-Aim21–His-Tda2 complex but not to GST-Aim21 alone or GST-Tda2 alone. (B, left) Live-cell fluorescence microscopy of actin-capping protein (Cap1-GFP) showing reduced recruitment to endocytic sites in tda2Δ cells relative to WT cells (TDA2). (Right) Quantification of the Cap1-GFP peak fluorescence intensity at endocytic patches in control (TDA2) and tda2Δ cells. (C, left) Live-cell fluorescence microscopy of actin-capping protein (Cap1-GFP) showing reduced recruitment to endocytic sites in aim21Δ cells relative to WT cells (AIM21). (Right) Quantification of the Cap1-GFP peak fluorescence intensity at endocytic patches in control (AIM21) and aim21Δ cells. (D, left) Live-cell fluorescence microscopy of Tda2-GFP showing increased recruitment to endocytic sites in cap1Δ cells relative to WT cells (CAP1). (Right) Quantification of the Tda2-GFP peak fluorescence intensity at endocytic patches in control (CAP1) and cap1Δ cells. Bars, 1 µm. Error bars indicate means ± SEM. *, P < 0.05; ***, P < 0.001.