Fig. 8.

Cdc42 acts downstream of Eya1–aPKCζ–Notch1 signaling to induce TJ protein trafficking during TJ assembly. (A–J) Double staining of ZO-1 and either trafficking protein Sec8 or LAMP1 shows that knocking down Eya1 or Notch1 disrupted the continuous cell border distribution of ZO-1, and decreased the colocalization between ZO-1 and the exocyst Sec8 in MLE15 cells (C,G; arrowheads), compared to control cells (A; arrowheads). Colocalization between ZO-1 and the endosomal marker LAMP1 increased in these cells (D,H; arrowheads), compared to control cells (B; arrowheads). (E,I) Treatment of Eya1siRNA- or Notch1siRNA-transfected cells with 200 nM of the Cdc42 activator bradykinin for 5 minutes prior to fixation restored colocalization of ZO-1 with both Sec8 and LAMP1 (arrowheads), and TJ assembly. Arrowheads in A–J indicate colocalization puncta. Scale bars: 50 µm. (K,L) Quantification of the colocalization shown in A–J (n = 4). Error bars represent the s.d. *Significantly different from control (ANOVA, Dunnett's test; *P<0.05 versus control cells).