The long-term goal of this research is to learn how epithelial cells establish and maintain their polarized phenotype, a essential feature to their successful functioning as selective barriers throughout the body. Past research in this project has studied the composition of the plasma membrane (PM) domains of polarized hepatocytes in vivo and identified th routes by which new PM proteins reach the apical and basolateral surfaces. The future focus will be to study th molecular basis of vesicle targeting to and docking at these distinct PM domains. This will be accomplished b comparing and contrasting how polarized and non-polarized cells package, target and deliver the same membrane an secretory cargo. In vivo rat hepatocytes and in vitro rat-human hybrid WIFB cells (developed by this lab) are the polarize cell models; in vitro rat Fao cells (a parent of the WIFB's) are the non-polarized cells. A comparison of these cell provides a relevant test of two alternative hypotheses regarding vesicle formation/targeting/docking. The first hypothesi asserts that non-polarized cells move their soluble and membrane cargo from the trans-Golgi network to the PM b default; in contrast, polarized epithelial cells accomplish this task in a signal-mediated manner. The alternative hypothesi is that polarized and non-polarized hepatocytes share identical machinery for vesicle traffic/targeting of cargo that destined for the basolateral surface but differ functionally in that polarized cells have unique apical targeting/docking machinery. Parts of this machinery are either absent from non-polarized cells or modified in amount or organization. Test of the two hypotheses include: i.) Studies of the hepatocyte syntaxins in polarized versus non-polarized cells. This protein family seems to be a part of the PM docking/fusion machinery in yeast and neurons. The number of distinct P syntaxins in any epithelial cell, their distributions and roles are not yet defined. Specific polyclonal antibodies to hepatocyte syntaxins (already prepared) will be used to localize the syntaxins (by immunofluorescence (IMF) an subcellular fractionation) and determine their functions (by microinjection combined with biochemical/morphological assays). 2.) Analysis of the dynamics of selected apical membrane proteins in polarized versus non-polarized cells. Non polarized cells by definition lack the discrete membrane domains of polarized, epithelial cells; nonetheless they express many membrane proteins that are present in distinct surfaces of polarized epithelial cells. What is the behavior (T- 1/2, recycling, etc) of these proteins in both cell types? Approaches will include: pulse-chase metabolic and cell surface labelling; use of antibodies bound to their cognate PM protein on live cells to trace the protein's itinerary (by IMF an EM). 3. Search for additional (possibly unique) components of the apical targeting/docking machine in polarized hepatocytes. Fusion proteins will be used as bait to fish out and study molecules that interact with the hepatocyte syntaxins. Fao, WIF-B and liver cell extracts are the sources. Likely candidates are: a cytosolic 68kDa protein (N-sec1 that binds to a subset of syntaxins prior to vesicle docking; and a 25kDa fatty acylated membrane protein (SNAP-25) that binds to neuronal syntaxins in vitro. Additional components (of the PM cytoskeleton?) will be sought using a hybridoma approach. Selection of antibodies is based on IMF patterns in each of the three hepatic cell types.
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