PRIMARY HEPATOCYTES · Primary mouse hepatocytes as a model system to study polarized endocytic trafficking
FP7 — People (Marie Curie Actions)
- Duration
- 2010-01-01 → 2011-12-31
- EU contribution
- €146,051
- Participants
- 1
- Scheme
- MC-IEF
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Results in brief
Primary mouse hepatocytes as a model system to study polarized endocytic trafficking
During the second year of my Marie Curie Fellowship I focused on dissecting the phenotype of Rab5 knockdown in mouse liver in vivo and primary hepatocytes in vitro. Knockdown of Rab5, the master regulator of the endosomal machinery, led to a dramatic reduction in early endosomes, late endosomes and lysosomes in the liver. I found that this was accompanied by reduced kinetics of LDL endocytosis in primary hepatocytes in vitro: an assay, which I developed during the first funding year. This result was in agreement with an increase in LDL in the serum of the Rab5KD animals, further strengthening the observed block of endocytosis. Using my newly established interferin knockdown technique in primary cultures, I was able to show that the loss of the endosomal organelles was not due to the reduction of membrane flux, since blocking membrane input by knocking down dynamin-2 had no affect on the number of endosomes. In the liver, I demonstrated that loss of endosomes also caused failure to deliver apical proteins, such as the bile acid transporter BSEP, to the bile canaliculi, suggesting a requirement of early endosomes for polarised cargo sorting. Interestingly, failure to deliver BSEP to the bile canaliculi resulted in impairment in bile acid metabolism, which I confirmed by an increase in total bile acids in the serum of these animals. In addition, knockdown of Rab5 caused induction of hypoglycaemia, which I defined as a complete inhibition of major gluconeogenic genes, glucose-6-phosphatase and PEPCK. I am currently further characterising this phenotype and hope to reveal new connection between glucose metabolism and endocytosis. Therefore, I expect my work to give rise to significant contributions of our understanding how endocytosis contributes to the regulation of cell polarity and liver physiology
Data: CORDIS, © European Union
Project objective
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.
Original text from CORDIS.
Participants
- MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV · MUNCHENCoordinatorGermany
Links
Data: CORDIS, © European Union