Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing

Abstract

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.

Figure 1.

Time-lapse video microscopy reveals the sequence of events during wound healing of a monolayer of T84 cells. Times above the panels refer to the number of minutes elapsed after wounding. The first image of the wound, approximately 0.018 mm², was taken at 2 minutes. Cell bodies were randomly chosen on the right side of the denuded area and outlined in red. At 10 minutes postwound, retraction reached its fullest extent and the denuded area was now approximately 0.021 mm². The refractile ring, greatly resembling an actin purse string, is indicated by arrows. By 40 minutes, the wound closed to 0.015 mm², a 30% reduction from its largest size at 10 minutes. The outline of the injury at 10 minutes is transposed onto the image taken at 40 minutes. Cell bodies, also outlined, were randomly chosen on the right side of the denuded area for comparison with those at 2 minutes. Similar comparisons of 10 randomly chosen cells, followed from 16 minutes to 40 minutes, showed that during this period of wound contraction, cell surface area increased on the average twofold (from 54 ± 3 μm to 116 ± 7 μm², P < 0.01). By 60 minutes, lamellae, indicated by arrows, were obvious. At 90 minutes, many of the cells adjacent to the wound protruded lamellae. Lamellae are highlighted in red, and the margin of the cell bodies is outlined in black. The area between these two outlines is considered to be the amount of lamella surface area contributing to wound sealing up to this point in time, a measurement used in Figure 5 ▶ . By 120 minutes, the denuded area was nearly completely covered by lamellae. Scale bar, 10 μm.

Figure 2.

Rhodamine-phalloidin fluorescent staining of recently wounded T84 monolayers reveals actin purse string formation. T84 monolayers were wounded by pipet aspiration and fixed 30 minutes later. A: Monolayers grown on porous supports were wounded. This en face view of the injury shows a continuous belt of actin or purse string, indicated by arrows, encircling the denuded area. The purse string is not visible in the upper half of the injury because the porous support at this point declines away from the plane of focus. B: An en face view of a larger injury reveals that it is not yet fully surrounded by an actin belt. The actin purse string (arrows) is in the process of forming. The asterisk marks cells, not included in the purse string, that were damaged and have lifted off the substrate. C: An optical cross-section taken at the point of the upper arrow in B reveals the actin purse string (arrow) forming in the basolateral compartment of the attached cells marginal to the wound. Cells not included in the purse string are detached from the substrate. Scale bars, 10 μm.

Figure 3.

Rhodamine-phalloidin staining demonstrates that cell elongation is accompanied by actin stress fiber rearrangement. A: T84 monolayers were fixed 3 hours after wounding. In this en face view taken at the basal surface, the arrow points to the actin purse string. Cells rearward of those producing the purse string exhibit stress fibers arrayed at right angles to the injury. B: An optical cross-section of an area similar to that shown in A shows how the monolayer flattens as it reaches the actin purse string, highlighted by an arrow. Scale bars, 10 μm.

Figure 4.

Rhodamine-phalloidin staining of lamellae formation. Monolayers were wounded and then fixed 7 hours later. A: An en face view shows lamellae protruding into the denuded area. The arrows, pointing to lamellae, indicate areas through which optical cross-sections were taken (B and C). Rearward of many of these lamellae lies a relatively actin-deficient area. One such area is denoted by an asterisk. B: A cross-section taken through the monolayer at the point indicated by the lower arrow in A shows fine actin suspension cables (asterisk) reaching down from the rearward, apical-lateral cell surface to the dense actin array at the base of the lamella. The dashed line, level with the substratum, is shown to help visualize the angle at which these cables descend. C: An optical cross-section taken through the monolayer at the point indicated by the upper arrow in A. In this area, where filamentous actin is visible rearward of the lamellae, actin cables are present but not as elongated as those in B. D: A wound surrounded by lamellae. These lamellae exhibit a dense actin array at their base and actin-rich lamellopodia at their leading edges, to which the arrows point. Areas behind the lamellae that appear actin-deficient are denoted by asterisks. Based on cross-sections in B and C, these areas correspond to cell bodies that contain fine actin suspension cables extending down to the bases of the lamellae. Scale bar, 10 μm.

Figure 5.

Antibody inhibition of T84 monolayer wound healing. a: Wounds were made in the presence of 20 μg/ml of nonspecific IgG or monoclonal antibodies against the α3 or α6 integrin subunit. The area of the denuded region measured immediately after wounding was compared to that value measured 2 hours later to determine the percentage of total closure. The mean percent closure of wounds treated with α3 and α6 integrin-specific antibodies was statistically significantly different from those treated with nonspecific IgG (P < 0.01). b: Wounds were allowed to close until lamellae appeared and then 20 μg/ml nonspecific IgG, antibody against the α3 subunit or antibody against the α6 integrin subunit was added. After 2 hours, the percent closure due to lamella surface area was determined by subtracting the area of the denuded region from the area bounded by the cell bodies of the marginal cells. (See the 90 minute panel in Figure 1 ▶ , where the red line encircles the denuded region and the black line traces the area bounded by the cell bodies of the marginal cells). The mean percent closure due to lamellar surface area of wounds treated with α3 and α6 integrin-specific antibodies was statistically significantly different from those treated with nonspecific IgG (P < 0.01).

Figure 6.

The α6β4 integrin resides in basal plaques that are disassembled during lamellae formation. T84 monolayers, either intact or injured, were fixed and then stained with rhodamine-phalloidin and by immunofluorescence for the β4 integrin subunit (A−C and E) or for the β4 integrin subunit alone (D) or by immunofluorescence for the β1 and α6 integrin subunit (F). A: An injured T84 monolayer was fixed during wound retraction and stained with rhodamine-phalloidin (red and blue) and for the β4 integrin subunit (green). An en face view of an actin purse string (blue, denoted by the white arrow) forming in a relatively apical plane is superimposed over the corresponding view of the basal compartment showing actin stress fibers (red, denoted by black arrow) running through α6β4 integrin (green). This image demonstrates that α6β4-containing plaques remain intact after wounding, including marginal cells that form the purse string during retraction. Scale bar, 5 μm. B: An intact T84 monolayer was fixed and permeabilized in F1 buffer. Treatment with F1 buffer allowed antibody access to the basal surface but left the actin cytoskeleton intact. This en face view of the basal plane was stained with rhodamine-phalloidin (red) and for β4 integrin subunit (green). The α6β4 integrin is found in ovoid plaques that are interrupted by actin stress fibers, three of which are indicated by arrows. The outline of a single cell demonstrates that almost the entire basal surface expresses α6β4 integrin. Scale bar, 5 μm. C: An en face view of the basal surface of an injured T84 monolayer fixed 6 hours after wounding and stained with Hoechst stain for nuclei (blue) as well as rhodamine-phalloidin (red) and for the β4 integrin subunit (green). (Not every cell in this image exhibits a nucleus because many nuclei lie above the basal plane.) Many of the cells adjacent to the wound extend lamellae (long arrow). The relatively actin-deficient areas that contain thin actin cables as described in Figure 4 ▶ are here seen to contain nuclei, one of which is denoted by the short arrow. This image demonstrates that the actin-deficient areas, which contain nuclei and therefore are the bodies of marginal cells, do not exhibit plaques. Rearward of the marginal cells lies a second tier of cells that surround the wound but are not directly adjacent to it. One of the nuclei of these cells is indicated by the dashed arrow. Cells in the second tier do exhibit plaques, some of which are circled. Scale bar, 10 μm. D: An enlargement of the basal surface of cells in the second tier stained for the β4 integrin subunit. The wound is out of view to the left of the image and the asterisks are placed in F-actin-deficient areas. This image shows plaques stretched toward the wound edge. Scale bar, 10 μm. E: Higher magnification reveals that the β4 subunit (green) is scattered over the surface of a lamella (contained within the box) but does not colocalize with actin filaments (red, arrows) radiating out from the base. Scale bar, 10 μm. F: Cells were double-stained for the β1 (green) and α6 (red) integrin subunits to detect α6β1 integrin (yellow). Yellow points of colocalization are dispersed across the lamella, contained within the lines. Scale bar, 10 μm.

Figure 7.

Emerging lamellae are characterized by intense α3β1 integrin staining at their leading edge. Wounded T84 monolayers were fixed and stained with rhodamine-phalloidin and by immunofluorescence for the β1 integrin subunit (A) or the α3 integrin subunit (B−E). A: In this en face image, small protrusions, indicated by arrows, are intensely labeled for the β1 integrin subunit (green) and emerge past the actin purse string (red) denoted by the asterisk. To the right of the purse string are broader extensions in which the actin purse string is no longer evident. B: An en face view of emerging lamellae showing that α3β1 integrin (green) is prevalent on the outermost edge of some of these protrusions, as indicated by the arrows. The asterisk denotes actin filaments (red) either disbanding from the purse string or assembling into the base of a forming lamella. C: An en face image of an emerging lamella shows that it is intensely labeled for the α3 integrin subunit (green) at its outer edge, as indicated by the arrow. D: An optical cross-section taken at the point of the arrow in C reveals that α3β1 integrin is intensely concentrated at the outermost edge of the lamella to which the arrow points. An actin filament (red) runs into this concentration of integrin. E: An en face image shows the α3 subunit (green) evenly scattered over a lamella, part of which is contained within the box. Scale bars, 10 μm.