Figure 4. E-cadherin–catenin–Arp2/3 puncta marked sites of actin assembly on purified membranes. (a) Puri- fied membranes were incubated with increasing con- centrations of rhodamine-labeled G-actin. Although the total amounts of actin incorporated increased with actin concentration (unscaled), the sizes and numbers of mem- brane actin puncta were the same (scaled). Bar, 10 µm. (b) Immunofluorescence staining showing colocalization of actin puncta with E-cadherin, -catenin, -catenin, and Arp2/3 but not PI3K p110-. Bar, 5 µm. (c) Western blot of total liver homogenate and purified membrane fraction (see Materials and methods). MDCK cells (Fig. S2). Arp2/3 inhibition by CK-548 greatly decreased the apical pool of actin without affecting the basal actin bundles (Fig. S2, c and d). On the other hand, formin inhi- bition by SMIFH2 had little effect on the apical actin population but eliminated most of the basal actin bundles (Fig. S2, e and f). In addition, formin-1 does not show strong colocalization with -catenin and actin in polarized MDCK cells (Fig. S3). There- fore, formin-1 is unlikely to have primary roles in actin nucle- ation at the cadherin-based apical junction in polarized cells. On the other hand, the potent inhibition of actin recovery by Arp2/3 inhibitors and localization of Arp3 at latrunculin-stable foci strongly implicate an essential role of Arp2/3-dependent nucleation in actin assembly at the apical junction. However, it is difficult to distinguish whether actin nucleation occurs directly at junctional puncta or originates in the cytoplasm that was subsequently captured by the membrane complexes. Actin assembly on epithelial cell membranes The best way to distinguish between capturing versus polymer- ization at the membrane is to reconstitute the reaction in vitro. We started by adding fluorescently labeled monomeric actin and ATP to whole–MDCK cell homogenate. This resulted in labeling of membranes with actin puncta (Fig. 3 a). The label- ing was inhibited by Arp2/3 inhibitors CK-548 and CK-636 (Fig. 3 b) and required ATP (Fig. 3 c). Homogenates of T84 intestinal epithelial cells and whole livers would also label membrane foci (Fig. 3, d and e), suggesting that actin incorpora- tion is an universal property of epithelial membranes. Although total cell homogenates can support actin assem- bly, we do not know whether the activity comes from the cyto- sol or the membrane itself. Therefore, we purified the membranes away from the cytosol and assayed for their actin incorporation activity. Combining differential centrifugation and equilibrium density gradients, eight membrane fractions were purified, and two fractions were capable of incorporating actin into puncta (Fig. S4). The majority of actin-incorporating activities reside in a major fraction consisting of large membranes, which will be used for further biochemical characterization (Fig. 3 f). Quantitation of actin incorporation showed that the as- sembly reaction starts almost immediately upon monomer addition and continues linearly until it plateaus around 20 min (Fig. 3 g). In contrast, addition of filamentous actin to mem- branes did not support puncta formation (Fig. 3 h). Therefore, it is unlikely that filaments are formed de novo in solution and subsequently captured at specific membrane sites. Rather, actin incorporation into puncta requires direct polymerization at the membrane. Moreover, actin incorporation to membranes can be inhibited by CK-548 and CK-636 to the extent similar to the Reconstituting actin assembly at adherens junction • Tang and Brieher 119