CPs’ ability to cap barbed ends is required for its localization and function. (A) Top: Crystal structure of the chicken CP complex (PDB entry 3AA7) displayed with the α-subunit (Cap1) in dark blue and the β-subunit (Cap2) in light blue. The α-tentacle, which is critical for the capping function of CP, is shown in red. Bottom: Alignment of the S. cerevisiae α-tentacle sequence with homologs from Homo sapiens and Mus musculus. The mutations to Cap1 (Cap1*) that will be used throughout the figure are displayed in red. (B) Pyrene-labeled actin (1 µM, 20% labeled) was polymerized in the absence or presence of His-Cap1/2 (CP, 100 nM) or His-Cap1^239,240A/2 (CP¹*, 100 nM). CP¹* showed no ability to cap actin filaments. (C) Quantification of the relative polymerization rate from three independent pyrene actin polymerization assays in B. From left to right, mean relative polymerization rate = 1.00, 0.25, 0.97. Error bars, mean ± SD. ns = not significant. (D) Live-cell fluorescence microscopy showing reduced recruitment of Cap1-GFP to endocytic sites in cells with the α-tentacle of Cap1 mutated (SDY1582), the CPI motifs of Aim21 and Bsp1 mutated (SDY1604), or with the mutations combined (SDY1608). Scale bar, 1 µm. (E) Quantification of Cap1-GFP peak fluorescence intensity at endocytic patches in WT, cap1*, and aim21^CPI*/bsp1^CPI* cells. From left to right, mean peak patch/cytosol ratio = 32.08, 11.86, 5.75, and n = 50 for all groups. Error bars, mean with 95% CI. ****P ≤ 0.0001. (F) Live-cell fluorescence microscopy showing no localization of Cap1*-GFP with Abp1-mCherry at endocytic patches in cells with the CPI motifs of Aim21 and Bsp1 mutated (SDY1703). Endocytic patch used for construction of kymograph to the right is indicated by an arrowhead. Scale bar, 1 µm.