Cell homogenization and liver membrane purification MDCK and T84 cell homogenates were prepared as follows. Cells were rinsed and hypoosmotically shocked on dish in 10 mM Hepes, pH 8.5, for 60 min at 4°C. Cells were scraped and homogenized on ice through a 25-guage needle in 10 mM Hepes, pH 8.5, in the presence of 10 mM DTT and protease inhibitors (10 µg/ml leupeptin, 1 mg/ml Pefabloc, 10 µg/ml E-64, 2 µg/ml antipain, 2 µg/ml aprotinin, 50 µg/ml bestatin, 20 µg/ml calpain inhibitor I, and 10 µg/ml calpain inhibitor II). Liver homogenate was prepared as follows. Frozen rat livers (Pel-Freez Biologicals) were thawed in 2 vol of 10 mM Hepes, pH 8.5/10 mM DTT. Pro- tease inhibitors (see above) were added, and the livers were briefly blended in a blender (5 × 15 s; Waring). The liver slush was filtered through four layers of cheesecloth to obtain the total liver homogenate. Liver membrane fraction- ation was performed using sequential centrifugation and equilibrium density sucrose gradient (Fig. S3). Altogether, eight different membrane fractions were isolated, and only two have actin assembly activity under the assay conditions used. The majority of actin assembly activity was in a single membrane fraction. This membrane fraction was prepared as follows. Total liver homogenate was centrifuged at 1,000 g for 30 min. The pellet was homogenized in 10 mM Hepes, pH 8.5/10 mM DTT in a Dounce homogenizer and centrifuged at 100 g for 30 min. The supernatant was collected and centrifuged at 1,000 g for 30 min. This membrane pellet contains the majority of actin assembly activity and is used as the purified membrane fraction in all experiments. Fractionation of high-salt–extractable factor Reconstitution assays (see Actin assembly and reconstitution assays section) were used to track the fractions that could rescue actin assembly on stripped membranes. High-salt extracts were obtained by stripping membranes with 500 mM NaCl, 2 mM MgCl2, 2 mM EGTA, 20 mM Hepes, pH 7.8, and 10 mM DTT on ice for 1 h. The membranes were removed by centrifugation at 10,000 g for 30 min. The supernatant was dialyzed against 10 mM Hepes, pH 7.8, and 10 mM -mercaptoethanol overnight at 4°C and subse- quently centrifuged at 10,000 g for 30 min. This clarified supernatant was used for purification of actin assembly activity using a fast protein liquid chromatography system (AKTA; GE Healthcare). The supernatant was loaded on to a HiTrap S HP column (GE Healthcare). The S flowthrough con- tained most of the actin assembly activity and was loaded onto a HiTrap Blue HP column. The Blue flowthrough contained most of the activity and was loaded onto a HiTrap Heparin HP column. The HP flowthrough contained most of the activity and was loaded onto a HiTrap Q HP column. Most of the activity was bound to Q HP and was eluted with a linear gradient of 0–500 mM NaCl, 10 mM Hepes, pH 7.8, and 10 mM -mercaptoethanol. The fractions were desalted against the actin assembly buffer using Sephadex G-25 (GE Healthcare) spin columns before testing in the reconstitution assay. Peak activity was found between 27.8 and 31.5 mS/cm. The fractions were pooled, dialyzed against 10 mM Hepes, pH 7.8, and 10 mM -mercapto- ethanol overnight at 4°C and subsequently centrifuged at 10,000 g for 30 min. The supernatant was loaded on to a Tricorn Source Q column (GE Healthcare) and eluted with a linear gradient of 0–500 mM NaCl, 10 mM Hepes, pH 7.8, and 10 mM -mercaptoethanol. The fractions were desalted using Sephadex G-25 spin columns before testing in the reconstitution assay. Fractions eluted at 24.2–25.1 mS had peak actin assembly activity and were concentrated using a Centricon filter unit. The concentrate was centri- fuged at 10,000 g for 30 min and loaded onto a Superdex 200 column. Two major and several minor symmetrical peaks were resolved in the Super- dex column, and only one peak has the actin assembly activity. EM MDCK cells grown on Transwell-Clear were processed for EM as previously described (Tang, 2006). In brief, cells/transwells were chilled at 4°C for 6 h before fixation with 3.75% glutaraldehyde, 150 mM NaCl, and 20 mM Hepes, pH 7.5, at 4°C for 18 h. The fixation reaction was quenched with 50 mM glycine and 150 mM Hepes, pH 7.5, on ice for 1 h. Cells/transwells were rinsed in ice-cold distilled water three times, secondary fixed with 1% osmium tetroxide/1.5% potassium ferrocyanide for 2 h on ice, rinsed four times in ice-cold distilled water, en bloc stained with freshly prepared and fil- tered 2% uranyl acetate in distilled water on ice for 2 h, and rinsed four times in ice-cold distilled water. Cells/transwells were dehydrated with sequential 5-min incubations in 50, 75, 95, 100, 100, and 100% ethanol at room temperature. Epon-Araldite (EMbed 812) was added to transwells and al- lowed to polymerize at 60°C for 48 h. Ultrathin sections were cut using a microtome (UltraCut S; Reichert), layered onto carbon-coated copper grids, and stained with freshly made/filtered 2% lead citrate. Images were col- lected with a microscope (1200EX; JOEL, Ltd.) at 60 kV. For actin-negative stain, recombinant -actinin-4 and K255E proteins were copolymerized with actin for 2 h in actin assembly buffer. The samples were put onto 128 JCB • VOLUME 196 • NUMBER 1 • 2012 glow-discharged carbon-coated grids for 5 min, washed three times with as- sembly buffer, and stained with 2% uranyl acetate. For membrane-negative stain, stripped membranes were prebound with recombinant -actinin-4 for 60 min before initiation of actin assembly by adding ATP and monomeric actin. The reaction was allowed to continue for 60 min before put onto on glow-discharged carbon-coated grids for 5 min, washed three times with as- sembly buffer, and stained with 2% uranyl acetate. Images were collected with a microscope (JEOL 1200EX) at 200 kV using a 2K × 2K charge-coupled device camera (UltraScan; Gatan, Inc.). Online supplemental material Fig. S1 shows incorporation of fluorescently labeled actin at apical, lateral, and basal regions of saponin-permeabilized cells. Fig. S2 shows relative contribution of actin nucleators, Arp2/3, and formin in MDCK cells. Fig. S3 shows immunofluorescence staining of -catenin and formin-1. Fig. S4 shows fractionation of liver membranes. Fig. S5 shows that actin incorpo- ration at the membrane is saturable. Online supplemental material is avail- able at http://www.jcb.org/cgi/content/full/jcb.201103116/DC1. We thank Dr. Barry Gumbiner for - and -catenin antibodies. We thank Dr. Martin Pollak for -actinin-4 WT and K255E cDNAs. We thank Dr. Cara Gottardi for cDNA of human E-cadherin–-catenin fusion protein. We thank Krista Angileri for assistance with molecular biology. EM was carried out in part in the University of Illinois Frederick Seitz Materials Research Laboratory central facilities. This work is funded by a grant from the March of Dimes. Submitted: 22 March 2011 Accepted: 6 December 2011 References Abe, K., and M. Takeichi. 2008. EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl. Acad. Sci. USA. 105:13–19. http://dx.doi.org/10.1073/pnas.0710504105 Adams, C.L., Y.T. Chen, S.J. Smith, and W.J. Nelson. 1998. Mechanisms of epi- thelial cell–cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin–green fluorescent protein. J. Cell Biol. 142:1105– 1119. http://dx.doi.org/10.1083/jcb.142.4.1105 Angres, B., A. Barth, and W.J. Nelson. 1996. Mechanism for transition from ini- tial to stable cell-cell adhesion: Kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. J. Cell Biol. 134:549–557. http://dx.doi.org/10.1083/jcb.134.2.549 Bershadsky, A. 2004. Magic touch: How does cell-cell adhesion trigger actin assembly? Trends Cell Biol. 14:589–593. http://dx.doi.org/10.1016/j.tcb .2004.09.009 Brieher, W.M., M. Coughlin, and T.J. Mitchison. 2004. Fascin-mediated propul- sion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex. J. Cell Biol. 165:233–242. http://dx.doi.org/10.1083/ jcb.200311040 Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. Rapid actin monomer–insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1. J. Cell Biol. 175:315–324. http://dx.doi.org/ 10.1083/jcb.200603149 Cao, L.G., D.J. Fishkind, and Y.L. Wang. 1993. Localization and dynamics of nonfilamentous actin in cultured cells. J. Cell Biol. 123:173–181. http:// dx.doi.org/10.1083/jcb.123.1.173 Carramusa,L.,C.Ballestrem,Y.Zilberman,andA.D.Bershadsky.2007.Mammalian diaphanous-related formin Dia1 controls the organization of E-cadherin- mediated cell-cell junctions. J. Cell Sci. 120:3870–3882. http://dx.doi .org/10.1242/jcs.014365 Catimel, B., J. Rothacker, J. Catimel, M. Faux, J. Ross, L. Connolly, A. Clippingdale, A.W. Burgess, and E. Nice. 2005. Biosensor-based micro- affinity purification for the proteomic analysis of protein complexes. J. Proteome Res. 4:1646–1656. http://dx.doi.org/10.1021/pr050132x Cavey, M., M. Rauzi, P.F. Lenne, and T. Lecuit. 2008. A two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature. 453:751–756. http://dx.doi.org/10.1038/nature06953 Charras, G.T., C.K. Hu, M. Coughlin, and T.J. Mitchison. 2006. Reassembly of contractile actin cortex in cell blebs. J. Cell Biol. 175:477–490. http://dx.doi .org/10.1083/jcb.200602085 Chu, Y.S., W.A. Thomas, O. Eder, F. Pincet, E. Perez, J.P. Thiery, and S. Dufour. 2004. Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J. Cell Biol. 167:1183–1194. http://dx.doi.org/ 10.1083/jcb.200403043