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First published online 7 December 2004
doi: 10.1242/jcs.01577

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Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement

Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA

View larger version (108K): [in a new window]   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.

View larger version (128K): [in a new window]   Fig. 2. Transmission electron micrographs of wounded MDCK cell monolayers sectioned along the apicobasal axis perpendicularly to the wound margin. (A-D) Submarginal cells 5-7 cell rows away from the wound margin at 12 hours post-wounding extend basal membrane protrusions under cells in front of them in the direction of the margin (located outside of the images to the right). The initial width of the rectangular wounds was 700 µm. Images B and D are of the same regions as in A and C, respectively, but at higher magnification. (E) Cells >100 rows away from the wound edge display a more cuboidal morphology with a small amount of wedging of cells underneath each other but no long protrusions. In all images, the apical surface is on the top – with microvilli visible – and the basal side is on the bottom. Long submarginal protrusions are indicated by arrows, whereas spreading in an unwounded portion of monolayer is indicated by an arrowhead. Notice the different close cell-cell contacts apical to the looser areas around the basal protrusions, allowing sheet continuity to be maintained simultaneously with the extension of lamellipodia. All of the MDCK cells in this and subsequent figures are nontransfected, unless otherwise noted. Bars, 1 µm.

View larger version (89K): [in a new window]   Fig. 3. Submarginal membrane protrusions form shortly after wounding and display typical lamellipodial morphology and dynamics. Panels from a time-lapse sequence (see supplementary material, Movie 2) of a basal focal plane of a wounded MDCK cell monolayer (1:10 ratio of GFP-actin-expressing:nontransfected cells) immediately after wounding to 4 hours post-wounding, imaged by two-photon microscopy. The initial width of the rectangular wounds was 700 µm. The visible GFP-actin-expressing submarginal cells are 4-5 cells rows from the wound margin. The dark area to the right of the visible cells is composed of nontransfected submarginal cells. The wound margin is located outside of the images to the right in the direction of the arrow.

View larger version (72K): [in a new window]   Fig. 5. Inhibition of Rho-kinase or nonmuscle myosin II results in decreased marginal actin bundle and stress fiber numbers and staining intensity. MDCK cell monolayers (1:10 ratio of GFP-actin expressing cells and nontransfected cells) were fixed, permeabilized and stained for F-actin with TRITC-phalloidin 6 hours after wounding following treatment with (A) 0.2% DMSO carrier solvent, (B) 50 µM H1152, a Rho-kinase inhibitor, or (C) 100 µM (±)-blebbistatin, a nonmuscle and skeletal muscle myosin II inhibitor. The initial width of the rectangular wounds was 700 µm. Similar results were obtained with Y-27632, another Rho-kinase inhibitor, and ML-7, a myosin light chain kinase inhibitor. Marginal actin bundles are indicated by asterisks, whereas membrane protrusions are indicated by arrows. Submarginal protrusions are also discernable in these images. Images are representative of at least three independent experiments in each case. Bar, 25 µm.

View larger version (23K): [in a new window]   Fig. 4. The rate of cell migration is inversely proportional to the distance from the wound edge, and cells move along related, though still partly independent, paths toward the wound area. The row number refers to rough distance from the wound margin in terms of cell rows, with row 1 being cells at the margin, and rows 2-5 and 10 being submarginal cells progressively further from the edge of the cell sheet. Centroids from individual cells in the wounded MDCK cell monolayer were tracked for 12 hours after wounding. The initial width of the rectangular wounds was 700 µm. Each graph represents the paths of three cells, all in the same cell row at a similar distance from the margin: a reference cell (black), a directly neighboring cell (red) and another more distant cell (blue). Marks in each trace represent the centroid positions of a single cell at 10-minute intervals. Cell paths were tracked along x and y coordinates (the axes perpendicular and parallel to the wound margin, respectively). Traces are representative of three independent experiments. The statistical rates of displacement along the x axis toward the wound area over the period of 0 to 12 hours for each row of cells are indicated at the top of each graph. Rates are mean ± s.e.m. in µm/minute calculated from traces shown and others not shown for a total of n=9 cells for each row position from three separate wounds.

View larger version (28K): [in a new window]   Fig. 6. Inhibition of Rho-kinase or nonmuscle myosin II slightly increases protrusive activity and slightly decreases the regularity of wound closure. Progress of wound closure in MDCK cell monolayers in the presence or absence of H1152 (A) or (±)-blebbistatin (B). The initial diameter of the circular wounds was 500 µm. Lamellipodial density at 6 hours after wounding (number of lamellipodia at the wound margin and dividing by margin perimeter length) in the presence or absence of H1152 (C) or (±)-blebbistatin (D). Regularity of wound closure (percent increase from 0 to 6 hours after wounding in the quotient of margin perimeter length divided by area) in the presence or absence of H1152 (E) or (±)-blebbistatin (F). Data are mean and s.e.m. for n=12 individual wounds in separate wells on multiwell tissue culture plates from three independent experiments for each treatment at each indicated concentration.

View larger version (54K): [in a new window]   Fig. 7. Wounding triggers delayed cell proliferation but inhibition of proliferation does not affect wound closure. (A) Fluorescence and corresponding phase-contrast images showing BrdU incorporation over the course of wound closure in fixed MDCK cell monolayers. The initial width of the rectangular wounds in A-C was 200 µm. Arrows indicate location of wound margin at 0 and 6 hours, edge-edge contact at 12 hours or former wound area at 24 hours. Bar, 50 µm. In other experiments, wider wounds were made that closed by 24-36 hours, and similar results were obtained, with BrdU incorporation peaking 24 hours (data not shown). (B) Percentage of BrdU-positive nuclei for wounded monolayers as a function of time after wounding, as well as parallel unwounded monolayers and subconfluent cultures (mean and s.e.m.; n=15 fields of view from three independent experiments). (C) The DNA-alkylating agent mitomycin C (MMC, 1.5 µM) blocks MDCK cell proliferation based on BrdU incorporation as in B, as well as the tetrazolium salt assay, with no evidence of general toxicity as assessed by Trypan Blue dye exclusion (data not shown). (D) Wound closure in the presence and absence of MMC added 48 hours before wounding (mean and s.e.m. for n=24 wounds). The initial diameter of the circular wounds in this experiment was 500 µm.