PRIMARY HEPATOCYTES · Primary mouse hepatocytes as a model system to study polarized endocytic trafficking

Hepatocytes as polarized cells consist of two distinct membranes, the apical (towards the bile) and the basolateral (towards the blood and each other). Discrete endosomal networks and machineries are responsible for their proper maintenance and function. Therefore, internalized cargo is delivered specifically via different populations of endosomes to the apical and basolateral membranes ensuring their specificity in trafficking and signaling. Isolated mouse hepatocytes maintain polarity and in vivo functionality when cultured using a 3D collagen sandwich system and provide therefore a good model system to study polarized trafficking. By adapting functional transport assays we can quantitatively measure the flow through the endosomal system using immunofluorescence microscopy. The aim of our project is to develop a mathematical model that can describe and predict the behavior of the endocytic pathway in hepatocytes with respect to cargo transport as well as signaling. It is clear in fact from resent studies that a complete understanding of the signaling machinery will not be achieved without taken into account the endocytic trafficking of signaling molecules. To pursue this goal, we set out to characterize in a quantitative fashion the endosomal distribution in the apical versus basolateral area by using confocal imaging analysis. The acquired data are fit into modeling software, to model mathematically the endosomal system. These results are compared with data we obtained in non-polarized HeLa cells. This system has been used to identify regulators of endocytosis in a functional genomics screen. The genes identified therein will be tested in isolated primary mouse hepatocytes. This will help us to understand the function of the endosomal system in hepatocytes and how it differs compared to a non-polarized cell.