Actin assembly on epithelial membranes requires Arp2/3 nucleation coupled to barbed-end growth. (a–f) Fluorescently labeled actin is incorporated into membrane puncta in MDCK cell homogenate (a) but not in the presence of Arp2/3 inhibitors CK-636 and CK-548 (b) or in the absence of ATP (c). Fluorescently labeled actin is incorporated into membrane puncta in T84 intestinal epithelial cell homogenate (d), rat liver homogenate (e), and purified rat liver membranes (f). (g) Quantitation of actin incorporation. (h) Actin assembly on membranes can be supported by G-actin but not F-actin. (i) Actin incorporation to liver membranes is blocked by Arp2/3 inhibitor CK-548 or CK-636. (j) Latrunculin B inhibits actin assembly. (k) The profilin–actin complex supports actin assembly. (l) Cytochalasin D (Cyto D) inhibits actin assembly. (m) CapZ only inhibits actin assembly when present continuously. (g–m) Actin assembly reactions were performed in duplicates (g) and triplicates (h–m). Error bars represent SDs. Bars, 10 µm.