Figure 5. NZO-3 colocalizes with ZO-1 around cell bor- ders during TJ assembly af- ter calcium switch. (A) Pa- rental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch and then costained with anti–ZO-1 and anti-VSVG (NZO- 3) antibodies at 6, 12, 24, and 48 h after the re-addition of calcium. Parental MDCK cells stained with anti-VSVG antibodies show nonspecific nuclear staining. In MDCK/NZO-3 cells, NZO-3 is present at cell borders of small groups of cells where it colocalizes with ZO-1. These groups of cells showing NZO-3 and ZO-1 around their peripheries enlarge over time, with complete localization at cell borders by 48 h. Boxed areas are enlarged in B. (B) NZO-3 colocalizes with ZO-1 along ex- tended cell peripheries, but not in intracellular aggregates or initial planes of cell–cell contact. Shown is a cell with mature TJ staining (asterisk) where ZO-1 and NZO-3 colocalize. However, NZO-3 does not colocalize with ZO-1 at initial cell–cell contact planes (arrow) or in large intracel- lular aggregates (arrowheads). Bars: (A) 30 mm; (B) 10 mm. Fanning et al., 1998; Wittchen et al., 1999). Disruption of the actin cytoskeleton is associated with impaired barrier function of the TJ (Madara et al., 1986; Madara and Pap- penheimer, 1987; Stevenson and Begg, 1994), whereas the fence function appears to be unaffected (Takakuwa et al., 2000). Actin localization during TJ assembly in the cal- cium switch system was studied by rhodamine-phalloidin staining of actin filaments (Fig. 6). In parental MDCK cells, actin recruitment to a ring-like structure at the apical aspect of the lateral cell surface occurs rapidly after re- addition of calcium; as early as 6 h all parental cells show a circumferential actin ring. This ring thickens and con- denses as the cells become more tightly packed and uni- form in size in the 12–48-h time period. However, exoge- nous expression of NZO-3 delays recruitment of actin to the apical ring in a manner similar to that observed with ZO-1. Development of an apical actin ring at points of cell–cell contact in MDCK/NZO-3 cells occurred only in isolated patches of cells at 6 h, with the final localization not complete until 48 h. In addition, at the earlier time points (6, 12, and 24 h), actin also localized to large aggre- gates in cells which show underdeveloped actin rings. The delayed association of actin filaments with the junctional complex in MDCK/NZO-3 cells is likely a factor in the de- layed TJ barrier formation in these cells. As the prevalent model of TJ formation involves coordi- nated assembly with the AJ, we were interested to deter- mine if E-cadherin was delayed in its movement to the junc- tional complex in MDCK/NZO-3 cells in a calcium switch. In parental cells, E-cadherin appears at all cell–cell contacts 6 h after re-addition of calcium (Fig. 7). This time course of E-cadherin recruitment to the AJ mirrors that of TJ assem- bly and recruitment of ZO-1 to the TJ (Fig. 4 A). Similar to ZO-1, E-cadherin localization during the calcium switch was also delayed in NZO-3–expressing cells (Fig. 7). At 6 h, E-cadherin is localized to cell–cell contacts in small, isolated clusters of cells; these cell clusters also show ZO-1 localiza- tion to the junctional complex. An increasing number of cells show junctional E-cadherin staining at 12 and 24 h, but it is not until 48 h after calcium re-addition that E-cadherin is completely recruited to all cell borders in MDCK/NZO-3 cells. In addition, we identified cells where junctional re- cruitment of E-cadherin precedes that of ZO-1. The disrup- tion of E-cadherin localization in MDCK/NZO-3 cells after calcium switch indicates that adherens and TJs are both af- fected by exogenous expression of the NZO-3 construct. We corroborated our E-cadherin findings by assessing the localization of b-catenin, an AJ protein peripherally associ- ated with the membrane (Fig. 8). In parental cells, b-catenin appears at cell–cell contacts 6 h after calcium re-addition in a manner identical to the localization of E-cadherin after calcium switch (data not shown). However, in a distribution similar to E-cadherin and ZO-1, b-catenin recruitment to the lateral cell surface during junctional complex assembly was delayed in MDCK/NZO-3 cells. Protein Biochemistry In addition to the altered localization of junctional com- plex proteins and disruption of the actin cytoskeleton, it is possible that the perturbation of junctional assembly in NZO-3–expressing cells is also due to changes in the expression levels of component proteins. Equivalent pro- tein loadings of whole cell lysates harvested from paren- tal and MDCK/NZO-3 cells at 6, 12, 24, and 48 h after re- The Journal of Cell Biology, Volume 151, 2000 830