Structural and functional roles of cytoskeletal proteins during repair of native guinea pig intestinal epithelium

Abstract

The cytoskeletal events that assist restitution of the native intestinal epithelium are poorly understood. To enhance our understanding of repair mechanisms in the native intestinal epithelium we assessed the functional role of actin and the temporal and spatial alterations in actin and villin that occur in native enterocytes migrating in response to injury. Using a well-characterized in vitro Ussing chamber model of native intestine epithelial restitution, the actin inhibitor cytochalasin D (CD) was applied to determine the functional importance of actin to restitution as assessed by sensitive electrophysiological means and structural techniques. Additionally we used phalloidin and indirect immunohistochemistry to localize and semiquantitate F-actin and villin in migrating cells during restitution. We report new data that shows that when cytoskeletal changes were impaired with CD, the epithelial monolayer was re-established in fewer than 20% of CD-treated villi, cells bordering the epithelial defect did not assume the characteristic phenotype associated with migrating cells, and transepithelial resistance did not return to pre-injury levels. F-actin and villin were present at the leading edge of the migrating cells, basolateral F-actin was decreased, and cytoplasmic villin was increased as determined by phalloidin and immunohistochemical methods. We conclude that in vitro repair of the native intestinal epithelium is functionally and structurally dependent on major changes in the cytoskeleton of cells involved in re-establishing the epithelial monolayer over a complex extracellular matrix.