The understanding of the sorting out mechanisms involved in the specific delivery of proteins from the site of synthesis to the site of function constitutes a central question in Cell Biology. One aspect of this is the asymmetric distribution of plasma membrane proteins between apical and basolateral surfaces in epithelial cells. To study the mechanisms responsible for this polarity, we have developed a model system consisting of kidney epithelial cells (MDCK) infected with enveloped viruses. Using this model system we plan: (a) to localize the intracellular site of sorting of apical and basolateral glycoproteins and to identify """"""""carrier vesicles"""""""" that transport them to the respective plasma membrane domain by immunoelectron microscopy on monolayers double infected with temperature sensitive mutants of apically (influenza) and basolaterally (vesicular stomatitis virus -VSV) budding viruses. We will attempt to identify apical receptors involved in the polarized exocytosis of influenza hemagglutinin (HA) to the apical surface using monoclonal antibodies raised against the surface of MDCK cells; (b) to characterize structurally and functionally the apical and basolateral endosomal populations of MDCK cells and to investigate whether the exocytic route of HA involves the endosomal compartment; (c) to search for pharmacologic (anticytoskeletal agents; calmodulin inhibitors, etc.) and physiologic (transmonolayer electric gradient) conditions that may specifically affect the apical and basolateral exocytic pathways; (d) to isolate cellular mutants in the apical pathway; (e) to develop an """"""""in vitro"""""""" assay for the delivery of viral glycoproteins to the cell surface; (f) to study the polarity properties of """"""""inverted epithelia"""""""" (choroid plexus, eye pigmentary epithelium); (g) to investigate the relationship between development of functional tight junctions and development of polarity. These studies should help to elucidate the biological basis of polarized epithelial cell function, central to the function of kidney, liver, intestine, and exocrine glands. Furthermore, since epithelia are usually the first barriers to viruses, they may also provide insights on how viral disease spreads in the organism.
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