Fig. 1. Submarginal cells in a migrating epithelial cell sheet extend cryptic lamellipodia in the direction of the wound margin. MDCK cell monolayers composed of GFP-actin expressing cells (green) grown with nontransfected cells in a 1:10 ratio were fixed 6 hours after wounding, permeabilized, stained for F-actin with TRITC-phalloidin (red) and imaged by confocal microscopy. The initial width of the rectangular wounds was 700 µm. In all images, the orthogonal z-stack profiles (0.20-0.25-µm slices) along the indicated lines in the x or y dimension of the central xy image are shown in the rectangular windows at the bottom (xz) and to the left (yz) of the central image. (A,B) GFP-actin-expressing and nontransfected cells located behind the wound edge extend lamellipodial protrusions basally underneath cells in front of them in the direction of the wound margin (toward the right side in both images). This is especially clear in the enlarged copy of the relevant portion of the xz orthogonal profile at the bottom of A, demonstrating a membrane protrusion (arrow) from a GFP-actin-expressing cell (green) extending under the nontransfected cell in front of it (red). An enlarged copy of the relevant portion of the yz profile to the left of B is also shown, again with a basal protrusion (arrow). Notice that in all the orthogonal profiles the apical side of the monolayer is directly adjacent to the central xy image, while the basal side is on the outside. Because the submarginal protrusions are hidden under other cells, which remain in contact with one another throughout wound closure, they are not visible by phase-contrast microscopy (see supplementary material, Movie 1). (C) GFP-actin-expressing cells and nontransfected cells pictured between 20 and 30 cell rows behind the wound margin display just a small amount of spreading (similar to the unwounded monolayer), with the xz enlargement showing some wedging (arrowhead) of nontransfected cells under the edges of GFP-actin-expressing cells. Images are representative of three independent experiments. Bars, 25 µm.