Actin assembly and protrusion formation at junctions. (A) Low-magnification view of a wild-type (A) or αEcat KO (B) Caco2 cell sheet that is engaging in wound-healing movement. Stained for actin. (B and C) Costaining for E-cadherin and actin in wild-type (B) and αEcat KO (C) Caco2 cells. Fluorescence signals in the boxed regions, which are enlarged at the right, were scanned along the white bar. Asterisks indicate split actin cables. White arrows indicate examples of E-cadherin–positive LCs. Green arrowheads point to closed junction. (D) Montage of Video 3. Time-lapse images of the boxed region are shown. Arrows indicate emergence of protrusions. (E) LLSM images of a marginal wild-type Caco2 cell expressing LifeAct-RFP are displayed in 3D and viewed from two distinct angles. These were picked out from the images of Video 4, which had been edited to observe from desired angles. Arrows indicate protrusions that crawl under the cell shown here, and arrowheads point at another form of protrusions that grow upward. Numbers indicate identical structures at the left and right images. AJ shows the position of this junction, which is 1.5–2.0 µm high above the bottom of the cell. Dotted arrow shows an approximate direction for the vertical observation at the right panel. lam, lamellipodium; n, the position of nucleus; asterisk, the position where two bicellular junctions merge. The images were cropped three-dimensionally for better visualization of vertical protrusions. (F) Oscillation of the junctional space in αEcat KO cells. Montage of Video 9. Time-lapse images of the boxed region are shown. Arrows and green arrowheads indicate the open and closed stages of the junction, respectively. At 60, 120, and 180 min, the junction is only slightly open. Magnification is shown in the partly enlarged images. Scale bars, 100 µm for A; 10 µm for B–F.