Rapid resealing of superficial wounds in the alimentary tract, vital for maintaining a physiologically intact barrier, occurs by migration of epithelial cells and is referred to as """"""""restitution"""""""". Presumably the cytoskeleton provides the basic machinery required for profound change in columnar cell shape and motion that characterizes restitution. Motility factors, such as Scatter Factor (SF) and its homolog Hepatocyte Growth Factor (HGF) likely enhance restitution by influencing the cell-cell adhesion proteins and/or cytoskeleton, thus permitting easy separation of cells. The overall aim of this proposal is to study restitution of intestinal epithelial wounds. I propose to investigate mechanisms underlying movement of these calls and a) analyze functional importance of cytoskeletal proteins, b) determine dynamics of the cell adhesion protein E-cadherin and c) study the influence of motility factors, Scatter Factor (SF) and its homolog Hepatocyte growth factor (HGF). The human intestinal epithelial cell line, T84, was utilized to develop an In vitro model of restitution. T84 cells adjoining a wound, spread, extend out lamellipodia and filopodia to migrate and reseal the defect. Actin related mechanisms required for lamellipodial protrusion and migration of epithelial cells will be investigated. Present studies localizing cytoskeletal proteins (to date actin, villin, myosin 1 & 2) in migrating T84 cells will be extended to include other actin binding proteins. Villin, an epithelial specific actin bundling and severing protein redistributes to lamellipodia of motile T84 cells and is likely to be important in actin restructuring. Contribution of villin and its related actin severing protein gelsolin to actin remodelling will be analyzed by determination of: a) relative expression of villin and gelsolin in intact and wounded T84 monolayers, b) migratory patterns of T84 cells microinjected with functionally characterized antibodies, and c) effects of overexpressing gelsolin by transfection experiments. Role of force transducing actin based mechanoproteins, myosin 1 & 2 in migration of these cells will be studied by microinjection of functionally characterized antibodies. With wound closure and reestablishment of intercellular contacts, E-cadherin is probably mobilized to the cell surface. During resealing, dynamics of E-cadherin with respect to epithelial plasma membrane will be explored. The effects of SF/HCF on E- cadherin and cytoskeletal redistribution will be studied.
| Nusrat, A; von Eichel-Streiber, C; Turner, J R et al. (2001) Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 69:1329-36 |
| Nusrat, A; Parkos, C A; Verkade, P et al. (2000) Tight junctions are membrane microdomains. J Cell Sci 113 ( Pt 10):1771-81 |
| Lotz, M M; Nusrat, A; Madara, J L et al. (1997) Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium. Am J Pathol 150:747-60 |
