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

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 (alpha 6 beta 4, alpha 6 beta 1, and alpha 3 beta 1) to mounding, 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 heating begins with retraction of the monolayer, alpha 6 beta 4 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 mound 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 alpha 6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha 6 subunit inhibit lamellae formation, indicating that redistribution of the alpha 6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha 3 beta 1 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 alpha 6 integrins, particularly alpha 6 beta 4, help contain retraction and both the alpha 6 integrins and alpha 3 beta 1 integrin contribute to lamellae formation.