from A.G. Scientific, Inc. Polyethyleneimine was purchased form Sigma- Aldrich. Protein concentrations were determined by the detergent-compatible protein assay (Bio-Rad Laboratories). DNA constructs -Actinin-4 WT and K255E cDNAs were provided by M. Pollak (Brigham and Women’s Hospital and Harvard Medical School, Boston, MA). The coding sequence of the -actinin-4 full-length actin-binding domain (1–270) and full-length K255E was subcloned into EcoR1 and Xho1 sites in the bacterial expression vector pET30a+ (EMD) for expression in Rosetta cells (EMD). Full-length -actinin-4 WT and K255E mutant was subcloned into EcoR1 and Xho1 sites of the G418-selectable mammalian expression vector pNTAPB (Agilent Technologies) with a streptavidin- binding peptide tag at the N terminus for expression in MDCK cells. shRNAs for -actinin-4 (5-ACACAGATAGAGAACATCGACGAGGACTT-3, 5-CTGTCAACCAGGAGAATGAACACCTCATG-3, and 5-AGGTCCTG- TTCCTCTGACTCGGTATCTAT-3) were synthesized and subcloned into blasticidin-selectable pGFP-B-RS vector (OriGene). The E-cadherin–-catenin fusion construct was provided by C. Gottardi (Northwestern Medical School, Chicago, IL). Protein purification For expression of recombinant 6-His–tagged -actinin-4, actin-binding domain, and K255E, Rosetta cells were induced with 500 µM isopropyl -d -1-thiogalactopyranoside for 8 h at 25°C. Cells were centrifuged at 6,000 g for 15 min and resuspended in 20 mM NaCl and 20 mM Hepes, pH 7.8, in the presence of 5 mg/ml lysozyme. After a freeze-thaw cycle, lysed cells were centrifuged at 20,000 g for 30 min. The supernatant was loaded onto a nickel column (QIAGEN). The column was washed with 20-bed columns of 500 mM NaCl, 25 mM imidazole, and 20 mM Hepes, pH 7.8. The recombinant proteins were eluted with 10-bed volumes of 500 mM NaCl, 500 mM imidazole, and 20 mM Hepes, pH 7.8. Full- length proteins were concentrated using Centricon (Millipore) and purified by gel filtration in 150 NaCl, 20 mM Hepes, and 10 mM -mercaptoetha- nol. Recombinant CapZ (Maun et al., 1996), actin (Brieher et al., 2004), aortic -actinin (Brieher et al., 2004), filamin (Brieher et al., 2004), and profilin were purified as previously described (Brieher et al., 2006). The profilin–actin complex was allowed to form overnight at a 2:1 profilin/ actin ratio. Actin and aorta -actinin were labeled with fluorophores as previously described (Brieher et al., 2004). In brief, F-actin was labeled on lysine residues using two N-hydroxysuccinimide–activated fluorophores for every actin molecule for 1 h at room temperature. Filaments were then pel- leted at 100,000 g for 30 min, resuspended in G buffer, and dialyzed ex- haustively against G buffer. This procedure typically labeled actin to 50–80%. -Actinin was labeled on cysteine using maleimide-activated fluorophores at a ratio of five fluorophores for every -actinin at room tempera- ture for 1 h. Labeled proteins were separated from free dyes by gel filtration using Superdex 200 (GE Healthcare). Cell culture MDCK cells were maintained in MEM/Earle’s balanced salt solution (EBSS) supplemented with 25 mM Hepes and 10% FBS. For transfection, MDCK cells were incubated in Opti-MEM (Invitrogen) with a 1:1 mixture of DNA/ polyethyleimine and selected for 10 d using G418 or blasticidin. In vitro and in vivo actin incorporation For in vitro assays, MDCK cells grown on Costar transwells (Corning) for 7 d were either treated with 10 µM latrunculin or DMSO in normal growth me- dia for 2 h before use. Cells were rinsed twice in EBSS with 20 mM Hepes, pH 7.5, at room temperature. Cells were permeabilized in the presence of 0.5 µM fluorescently labeled monomeric actin in saponin buffer (0.2 mg/ml saponin, 100 mM KCl, 2 mM MgCl2, 2 mM EGTA, and 20 mM Hepes, pH 7.8) at room temperature for 10 min. The cells were rinsed quickly in saponin buffer and fixed with 1% formaldehyde in saponin buffer at 4°C for 60 min and processed for immunofluorescence. For in vivo assays, MDCK cells grown on Costar transwells for 7 d were treated with 10 µM latrunculin in normal growth media for 2 h and then either processed for immunofluorescence or rinsed in fresh media in the presence or the absence of CK-548/CK-636 or SMIFH2 to initiate actin recovery. Cells were subsequently processed for immunofluorescence at various time points. Immunofluorescence of cells MDCK cells grown on Transwell-Clear (Corning) for 10 d were rinsed twice in EBSS and fixed in 1% formaldehyde/150 mM NaCl/20 mM Hepes, pH 7.8, at 4°C for 90 min. The reaction was quenched with 50 mM Tris in staining buffer (0.1% Triton X-100/100 mM NaCl/20 mM Hepes, pH 7.8) for 1 h. After rinsing in staining buffer, the cells were incubated with primary antibodies in staining buffer overnight. After rinsing in stain- ing buffer three times, the cells were incubated in secondary antibodies for 90 min. The cells were rinsed again three times and poststain fixed with 1% formaldehyde in staining buffer. Finally, the cells were incubated with fluorescently labeled phalloidin for 60 min. Transwell filters were excised and mounted on glass slides using ProLong Gold antifade (Invitrogen). Optical z slices in 200-nm steps were collected with a microscope (1X71; Olympus) attached to a 1K × 1K charge-coupled device camera using a 60× objective (NA 1.42) with a 1.6× auxiliary magnification. All images were deconvolved using DeltaVision software (Applied Precision). Z stack projections were generated from deconvolved slices using the max- imum intensity criteria. Composite images were generated using ImageJ software (National Institutes of Health). Quantitation of actin puncta inten- sity was performed in ImageJ using unprocessed original images. A de- fined area (120 × 120 pixels) was used to compare the signal intensity of actin (phalloidin), K255E (immunofluorescence using S-tag antibodies), and -actinin-4 (immunofluorescence using N terminus -actinin-4 antibodies). The measured intensities were subtracted from background (cytoplasm) be- fore being used for calculating the intensity ratios. For figure generation, images were cropped, contrasted, and scaled using Photoshop software (Adobe) before importing into Illustrator (Adobe). Actin assembly and reconstitution assays Actin assembly assays were performed in actin assembly buffer (50 mM KCl, 2 mM EGTA, 2 mM MgCl2, and 100 mM Hepes, pH 7.8) supple- mented with 2 mM buffered ATP, pH 8. In brief, a 20-µL reaction consists of 15 µg of total proteins from membrane fraction and 0.5 µM fluores- cently labeled monomeric actin. For assay with filamentous actin, fluores- cently labeled monomeric actins were allowed to polymerize overnight before use. For reconstitution assays, purified membranes were stripped 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. Stripped membranes were collected by centrifugation at 10,000 g for 10 min. The supernatant was dialyzed against actin assembly buffer and used for reconstitution assay. A 20-µL re- constitution assay consists of 8 µg of total protein from stripped mem- branes, 0.5 µM fluorescently labeled actin, and test fractions with protein concentrations ranging from 0.1 µg to several micrograms. The reactions were imaged using an Axio Imager with the Colibri illumination system (Carl Zeiss) using a 63× objective (NA 1.4) attached to a 1K × 1K charge- coupled device camera (ORCA-ER; Hamamatsu Photonics). Immunofluorescence of membranes Actin assembly reactions were allowed to carry out for 3 h before incuba- tion with primary antibodies in the presence of 0.1% Triton X-100. The membranes were spun through a 20% sucrose cushion and resuspended in 0.1% Triton X-100 in assay buffer. The membranes were incubated with secondary antibodies for 2 h, spun through a 20% sucrose cushion, and resuspended in 0.1% Triton X-100 in assay buffer. For immunofluorescence of -actinin-4 on membranes, the WT and K255E proteins were allowed to prebind to stripped membranes for 1 h on ice and were processed for immunofluorescence as previously described. Stained membranes were imaged using the Axio Imager as previously described. Quantitating actin assembly on membranes Actin incorporation on membranes was measured by fluorometry. The actin assembly reactions were initiated by the addition of ATP and rhodamine- labeled monomeric actin. The reactions were stopped at varies time points by centrifuging the membranes through a 20% sucrose cushion. Membrane pellets were solubilized in 1% SDS, and fluorescence was measured in a fluorometer with excitation at 542 nm and emission at 568 nm. The read- ings were subtracted from a background reading of membrane and ATP without actin. The amount of actin incorporation was calculated from the reading of a known quantity of rhodamine-labeled actin. For F-actin binding, G-actin was prepolymerized for 2 h before use. The readings were subtracted from the background reading in the presence of latrunculin. For drug inhibition assays, cytochalasin D and latrunculin B were added to the membranes before initiation of actin assembly reactions. For profilin–actin assay, preformed pro- filin–rhodamine-labeled actin complex (at a ratio of two profilins to one actin) was added instead of actin. For CapZ experiments, CapZ was added to the membranes before the initiation of actin assembly reactions. For CapZ pre- binding, 100 nM CapZ was added to the membranes for 30 min before spin- ning through 20% sucrose. The CapZ-treated membranes were resuspended in assembly buffer, and the actin assembly reactions were initiated as previ- ously described. Reconstituting actin assembly at adherens junction • Tang and Brieher 127