Figure 6. -Actinin is an essential factor in actin assembly at E-cadherin junctional complexes. (a) Actin assembly on stripped membranes can be rescued by from Superdex 200 (S200) fraction B13, -actinin purified from aorta, or bacterially expressed recombinant -actinin-4 (A4). (b) However, filamin (FIL) or the actin-binding domain alone (A4 ABD) cannot support actin assembly. Error bars represent SEM. n = 3. (c) -Actinin-4 is recruited to E-cadherin foci on stripped membranes. (d) Prebinding -actinin-4 to membranes rescued actin assembly. (e) -Actinin-4 can rescue actin assembly on cofilin-treated membranes. (f) Negative-stain EM showing F-actin associated with a stripped membrane fragments after incubation with -actinin-4 and G-actin. (g) -Actinin-4 is recruited to cell–cell interactions in CHO cells expressing E-cadherin–-catenin fusion protein. A projection of five deconvolved optical z slices spanning 1 µm from the apical region of the cells is shown. Bars: (a–c, e, and g) 10 µm; (d) 5 µm; (f) 200 nm. Actin assembly at adhesion complex requires a salt-extractable factor As we have an in vitro assay, we can begin to dissect the actin assembly reaction using biochemical reconstitution. Stripping the membranes with 500 mM KCl severely com- promised its ability to assemble actin (Fig. 5, a and b). Add- ing this fraction back to stripped membranes rescued actin assembly (Fig. 5 c). Therefore, an extractable factor can re- versibly interact with the membranes to support actin assem- bly. We used the reconstitution assay to track the extractable factor through six different chromatographic separations, re- sulting in a 300-fold enrichment of the activity (see Materi- als and methods; Fig. 5, d and f). The final fraction ran as a symmetrical peak on gel filtration that overlapped with ac- tivity (Fig. 5, d and e). The peak activity showed a major band at 100 kD on SDS-PAGE, which was identified by mass spectroscopy as -actinin-4 as the major component and -actinin-1 as the minor component (Fig. 5 f). -Actinin-4 can rescue actin assembly on stripped membranes To verify that -actinin is indeed the salt-extractable activity, we compared the ability of three different preparations of -actinin to rescue actin assembly on stripped membranes (Fig. 6 a). We found that -actinin from liver (which expresses -actinin- 1 and -4), aorta (which expresses smooth muscle -actinin-2), and bacterially expressed recombinant -actinin-4 all can rescue actin assembly, suggesting that there is no preference for a specific isoform of -actinin. -Actinin is an actin-bundling protein that exists as a dimer and belongs to the calponin homology domain family of actin-binding proteins (Otey and Carpen, 2004). We tested Reconstituting actin assembly at adherens junction • Tang and Brieher 121