Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells

DESCRIPTION (provided by applicant): Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells Columnar epithelia (e.g. kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. They differ morphologically in that columnar epithelia establish their apical domain (AP) and basolateral domains (BL) at opposing poles whereas hepatocytes establish their AP domain, the bile caliculi (BC) in the midst of their BL domains. They also differ drastically in how they establish and maintain their surface domains. Whereas columnar epithelia target their plasma membrane (PM) proteins predominantly from the trans Golgi network (TGN) to the AP domain, hepatocytes target both AP and BL PM proteins from the TGN to the BL surface, from where AP proteins are sorted to BC by transcytosis. Although important progress has been made in understanding the trafficking routes, sorting compartments, and targeting mechanisms of columnar cells, particularly the kidney epithelial cell line MDCK, the trafficking routes, and sorting compartments and mechanisms of hepatocytes remain poorly understood. This is in large part due to the fact that hepatic cell lines are not amenable to trafficking studies with the classical biotin based targeting assays utilized in MDCK cells and are distinctly more difficult to transfect. We propose innovative experimental approaches to identify the trafficking itinerary, subcellular compartments and molecular machinery involved in the trafficking of model AP PM proteins in hepatic WIFB cells, comparatively with MDCK cells. Our hypotheses are: (i) novel imaging technologies and (ii) analysis of the post-exocytic fate of AP proteins provide enough resolution to understand the different sorting and trafficking strategies of hepatocytes and columnar cells and (iii) the serine/threonine kinase Par1b acts as a molecular switch between the direct and transcytotic pathways. We propose the following Specific Aims: Aim 1: To quantitatively assess the extent of vectorial and transcytotic delivery of model apical PM proteins in WIFB and MDCK cells. Aim 2: To define comparatively the site of exocytic sorting of AP and BL markers in WIFB and MDCK cells. Aim 3: To identify the machinery used by AP PM proteins to exit the TGN in WIFB and MDCK cells. With our novel approaches we will set the framework for a long overdue comparative molecular model of protein targeting in hepatic versus columnar cells. This is a question of high priority for epithelial polarity that has remained unanswered for more than 25 years.