The mechanisms by which organs and their component cells acquire differentiated functions and morphology are critical to normal human development. This functional and morphologic regionality of cells is referred to as polarity. Epithelial cells, and in particular hepatocytes, serve as a useful model to study the development and maintenance of cell polarity since hepatocytes display morphologic polarity in monolayer culture. In addition, they are developmently regulated to perform many metabolic functions and display biochemical markers for human development (e.g. alpha fetoprotein). To examine the mechanisms whereby epithelial cells develop and maintain polarity, human hepatoma-derived cell lines will be established in monolayer culture on suspended Millipore filters. The formation of an intact, polarized monolayer will be assessed by electron microscopy and determination of electrical resistance across the monolayer. Using this culture system, the organization of intracellular organelles and cell-cell junctions will be assessed by electron microscopy. The localization of cell nutrient and growth factor entry will be determined by radio-ligand binding assay and by localization of the growth factor receptors using immunofluorescence. The intracellular traffic of nutrients and growth factors as well as their receptors will be examined using the asialoglycoprotein (ASGP) receptor system as a model. The fate of both the ligand (ASGP) and the receptor will be determined biochemically. The development of cell-cell communication and cell adhesion will be addressed by determining the localization of cell adhesion molecules in human hepatoma-derived cell lines. In addition, the role of tight junctions in maintenance of surface membrane polarity will be assessed by determining the fate of polarized integral membrane proteins after disruption of tight junctions. The development of specialized membrane domains will be examined in this system by determining the steady state localization of individual integral membrane proteins as well as their time of appearance at the cell surface following biosynthesis. Finally, the rates and sites of secretion for a host of secretory proteins will be determined. Thus, this system will allow for the direct examination of several aspects of cell polarity which will aid in understanding normal morphogenesis, embryogenesis, and fetal development.
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