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Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing
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UMass Chan Affiliations
Department of Cancer BiologyDocument Type
Journal ArticlePublication Date
2000-03-07Keywords
ActinsAntibodies, Monoclonal
Antigens, Surface
Cell Movement
Cytoskeleton
Epithelial Cells
Humans
Immunoglobulin G
Integrin alpha3beta1
Integrin alpha6beta1
Integrin alpha6beta4
Integrins
Intestinal Mucosa
Microscopy, Confocal
Microscopy, Fluorescence
Microscopy, Video
Receptors, Laminin
Tumor Cells, Cultured
Wound Healing
Cancer Biology
Neoplasms
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Show full item recordAbstract
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.Source
Am J Pathol. 2000 Mar;156(3):985-96.
DOI
10.1016/S0002-9440(10)64966-8Permanent Link to this Item
http://hdl.handle.net/20.500.14038/26241PubMed ID
10702414Related Resources
ae974a485f413a2113503eed53cd6c53
10.1016/S0002-9440(10)64966-8
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Integrin signaling in leukocytes: lessons from the alpha6beta1 integrinWei, Jueyang; Shaw, Leslie M.; Mercurio, Arthur M. (1997-04-01)The adhesive interactions of leukocytes with basement membrane components and other extracellular matrix (ECM) proteins are mediated largely by the integrin family of receptors. These interactions can be regulated by various effector molecules including chemokines, growth factors, and other cell surface proteins by a mechanism termed inside-out signaling of integrin function. On engagement by their ligands, integrins activate a wide range of signaling pathways that regulate a broad array of leukocyte functions such as chemotaxis, cytokine responsiveness, phagocytosis, and gene expression. This review highlights recent advances in the understanding of integrin signaling in leukocytes with an emphasis on our own work on the alpha6beta1 integrin, the major laminin receptor expressed by these cells.
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Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophagesShaw, Leslie M.; Mercurio, Arthur M. (1994-06-01)Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.