Figure 3. Actin assembly on epithelial membranes requires Arp2/3 nucleation coupled to barbed-end growth. (a–f) Fluorescently labeled actin is incorpo- rated 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. localization at these apical puncta (Fig. 2 e), implicating a role of Arp2/3 nucleation in actin assembly at the apical junction. Indeed, actin assembly (Fig. 2 f) was greatly retarded after latrun- culin washout in the presence of Arp2/3 inhibitors CK-548 and CK-636 (Fig. 2 g; Nolen et al., 2009). Another actin nucleator, formin-1, has previously been shown to play a role in actin polymerization at nascent adherens junctions formed by keratino- cytes (Kobielak et al., 2004). However, the addition of a broad- spectrum formin inhibitor, SMIFH2 (Rizvi et al., 2009), during latrunculin washout did not prevent actin recovery (Fig. 2 h). We found that Arp2/3 and formin inhibition showed dif- ferential effects on the overall actin cytoskeleton of polarized 118 JCB • VOLUME 196 • NUMBER 1 • 2012