Epithelial morphogenesis in vitro and the cytoskeletal association of proteins

Epithelial morphogenesis is the development of an epithelial phenotype. Epithelial morphogenesis in vitro has been studied in cultures soon after cell-cell contact at confluency when several actin-associated proteins (fodrin, adherens junction molecules E-cadherin and catenins) localize to specific subcellular domains and become resistant to extraction with non-ionic detergents. Here we demonstrate that early confluency is followed by a long postconfluent period of several weeks during which these proteins and actin itself become progressively enriched in the detergent-resistant fraction of MDCK epithelial cells. Cultures from another tissue (human retinal pigment epithelium) which produces weakly epithelialized monolayers in culture do not exhibit similar late-stage increases in protein insolubility. After confluency some cells in MDCK cultures undergo additional morphogenetic changes giving rise to cord-like structures, and the MDCK adherens junction becomes more stable to disrupting agents. Using the same analytic methods for known cytoskeletally associated proteins, we also show that a subset of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a traditionally soluble glycolytic enzyme, also becomes progressively partitioned to a detergent resistant fraction with time postconfluency in MDCK cells. The retention of GAPDH in the insoluble fraction of postconfluent cells is sensitive to ATP depletion, a result not found for several known cytoskeletally associated proteins or for other glycolytic enzymes. Morphogenetic changes in the postconfluent MDCK monolayer result in cells with differing phenotype, and the apparent amount as well as distribution of insoluble GAPDH varies with cell shape. Insoluble GAPDH is found in the nuclei of a small subset of cells, and insoluble GAPDH in the cytoplasm displays various patterns including flocculent, punctate, and filamentous distributions. Disruptors of cytoskeletal filaments were relatively ineffective in the highly stabilized postconfluent MDCK monolayers, but use of these effectors implicates actin as a candidate filament to tether GAPDH in the insoluble fraction of the cytoplasm.; These results indicate that in vitro morphogenesis is not restricted to early confluency in MDCK epithelial cells but rather molecular stabilization and dynamic changes in cell shape occur over a protracted postconfluent interval. During late-stage morphogenesis, epithelial cells develop stable subcellular compartments due to progressive association of both cell surface and cytoplasmic proteins with a progressively more stable cytoskeleton.