Figure 5. Purification of high-salt–extractable actin assembly activity via biochemical re- constitution. (a) Purified membranes were incubated in high salt to obtain a stripped membrane fraction and a high-salt fraction. (b) Stripped membranes cannot assemble actin. (c) Dialyzed high-salt (HS) extract res- cued actin assembly on stripped membranes. (b and c) Bars, 10 µm. (d) The actin assembly activity from high-salt extract binds to Q HP and source Q columns and subsequently puri- fied on Superdex 200. Bars on traces show fractions with activity. (e) Purification table for actin assembly activity. AU, arbitrary unit. (f) Coomassie blue–stained gel for the purifi- cation of actin assembly activity. Lane 1, ho- mogenate; lane 2, purified membrane; lane 3, high-salt stripped membrane; lane 4, high-salt extract; lane 5, S and Blue flowthrough; lane 6, heparin flowthrough; lane 7, Q HP fractions with peak activity; lane 8, source Q fractions with peak activity; lane 9, Superdex 200 frac- tion B13 with peak activity. Q PE, quaternary ammonium polystyrene/divinylbenzene; SHP, sulfopropyl high performance. published potency of these inhibitors (Nolen et al., 2009). Thus, both liver and MDCK membranes have similar properties for actin assembly, indicating that liver membranes would be use- ful for further biochemical investigation (Fig. 3 i). Others had reported that cellular membranes have the ca- pacity to bind actin monomers (Tranter et al., 1991; Cao et al., 1993). To rule out the possibility that actin was simply recruited to the membrane as monomers, we performed the assembly reac- tion in the presence of latrunculin, an actin monomer–sequestering drug that blocks polymerization (Morton et al., 2000). We found that actin incorporation on membranes was inhibited down to 20% by latrunculin (Fig. 3 j). The residual 20% incorporation represents diffuse fluorescence across the entire membrane but not bona fide assembly events. Although unlikely, it is possible that monomers are add- ing to the pointed ends instead of the normally growing barbed ends. Therefore, we repeated the assay using preformed pro- filin–actin complexes that can only add to the barbed end (Pring et al., 1992; Kang et al., 1999). Profilin–actin was able to sup- port membrane–actin puncta formation at a rate slightly slower than that of actin alone (Fig. 3 k). This is in agreement with published work that profilin–actin polymerizes at 85% of the rate of free actin (Pasic et al., 2008). If membrane puncta formation is a result of monomer addition at the barbed end, barbed-end cappers such as cyto- chalasin D and CapZ should block actin assembly (Flanagan and Lin, 1980; Yamashita et al., 2003). Indeed, both cytochalasin D (Fig. 3 l) and CapZ (Fig. 3 m) inhibited actin incorporation in a dose-dependent manner. However, monomer growth from the barbed end can either result from nucleation or addition to pre- existing filaments. To distinguish between these two possibilities, we blocked preexisting barbed ends with CapZ before testing for actin assembly. We found that actin incorporation was almost the same with or without CapZ pretreatment, suggesting that actin assembly is supported by de novo nucleation rather than growth from preexisting free barbed ends (Fig. 3 m). Titration of actin concentrations revealed that the inten- sities of actin incorporated are proportional to the amounts of actin added (Fig. 4 a). However, the number and sizes of the actin foci are the same in all three actin concentrations, sug- gesting that the formation of foci is predetermined by factors on the membrane. Cadherin–catenin–Arp2/3 complexes are sites of actin incorporation Our earlier in vivo MDCK experiments indicated that discrete membrane adhesion complexes serve as sites for Arp2/3- dependent actin assembly. We found that junctional proteins and Arp2/3 colocalized with sites of actin incorporation on liver membranes, indicating that junctional foci are preserved in our biochemical purification procedure (Fig. 4 b). Western blot analysis confirmed that the purified liver membrane frac- tion contains Arp2/3 and was enriched in cadherin and - and -catenins (Fig. 4 c). 120 JCB • VOLUME 196 • NUMBER 1 • 2012