Despite the essential role of liver for the organism, very little is known about the molecular requirements for its specific microarchitecture, especially about the generation of cell polarity as a prerequisite for organ structure and function. Moreover, hepatocytes, unlike the classical apico-basally polarized cells (e.g. cells lining the intestine or cultures MDCK cells), possess spatially restricted apical membrane regions forming a narrow belt on their surface. Evidence on how this peculiar structure is created is scarce, demanding for in-depth analysis to pinpoint the exact pathways involved in this process that dictates the normal functioning of the liver. The aim of this project is to identify key proteins involved in the initiation and establishment of hepatocyte polarity. The experimental strategy consists of functional characterization of genes differentially expressed with respect to hepatocyte cell polarity using RNA-interference, confocal microscopy with image analysis on mouse primary hepatocytes in vitro and in vivo. Given the essential role of actin machinery in cell polarity, I will prioritize on the spatiotemporal activation of regulators of actin dynamics (e.g. Rho GTPases) in context of hepatocyte polarization and address their role in shaping hepatocyte apical membranes. Remarkable advantage of the experimental approach is the possibility to silence genes in hepatocytes both in vitro and in vivo. Extensive expertise of the host laboratory in membrane trafficking in combination with my background in macromolecule delivery presents advantages to refine the siRNA-delivery approaches to systematically assess cytoskeletal and membrane transport machinery for hepatocyte polarity. The results from this project will help to characterize and determine the spatiotemporal order of the major components involved in hepatocyte polarization and perhaps also shed light on how to reverse the pathological non- or mis-polarized conditions in diseased liver tissue.